| 兼松 隆（かねまつ たかし） | データ更新日：2023.06.23 |
教授 ／
歯学研究院
歯学部門
口腔常態制御学講座
| 1. | Li, Rongzhi; Sano, Tomomi; Mizokami, Akiko; Fukuda, Takao; Shinjo, Takanori; Iwashita, Misaki; Yamashita, Akiko; Sanui, Terukazu; Nakatsu, Yusuke; Sotomaru, Yusuke; Asano, Tomoichiro; Kanematsu, Takashi; Nishimura, Fusanori, miR-582-5p targets Skp1 and regulates NF-Kappa B signaling-mediated inflammation, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 10.1016/j.abb.2022.109501, 734, 2023.01. |
| 2. | Hayashi, Chikako; Fukuda, Takao; Kawakami, Kentaro; Toyoda, Masaaki; Nakao, Yuki; Watanabe, Yukari; Shinjo, Takanori; Sano, Tomomi; Iwashita, Misaki; Yotsumoto, Karen; Shida, Miyu; Taketomi, Takaharu; Sanui, Terukazu; Uchiumi, Takeshi; Kanematsu, Takashi; Nishimura, Fusanori, miR-1260b inhibits periodontal bone loss by targeting ATF6 beta mediated regulation of ER stress, FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, 10.3389/icell.2022.1061216, 10, 2022.11. |
| 3. | Yamawaki Yosuke, So Hiroki, Oue Kana, Asano Satoshi, Furusho Hisako, Miyauchi Mutsumi, Tanimoto Kotaro, Kanematsu Takashi, Imipramine prevents Porphyromonas gingivalis lipopolysaccharide- induced microglial neurotoxicity, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2022.09.109, 634, 92-99, 2022.12. |
| 4. | Asano Satoshi, Maetani Yuka, Ago Yukio, Kanematsu Takashi, Phospholipase C-related catalytically inactive protein enhances cisplatin-induced apoptotic cell death, EUROPEAN JOURNAL OF PHARMACOLOGY, 10.1016/j.ejphar.2022.175273, 933, 2022.10. |
| 5. | Kayo Mori, Akiko Mizokami, Tomomi Sano, Satoru Mukai, Fumitaka Hiura, Yasunori Ayukawa, Kiyoshi Koyano, Takashi Kanematsu, Eijiro Jimi, RANKL elevation activates the NIK/NF-κB pathway, inducing obesity in ovariectomized mice, Journal of Endocrinology, https://doi.org/10.1530/JOE-21-0424, 254, 1, 27-36, 2022.05, Menopausal women are susceptible to visceral obesity, which increases the risk of metabolic disorders. However, the mechanisms of menopause-induced visceral fat accumulation are not fully understood. Circulating levels of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) are elevated in an animal model of menopause. RANKL, a multifunctional cytokine, activates the NF-κB pathway, which serves as a pivotal mediator of inflammatory responses. Here, we investigated whether RANKL-induced non-canonical NF-κB pathway activation induces inflammation and lipid accumulation in adipose tissues. RANKL induced Tnfa expression via the non-canonical NF-κB pathway in bone marrow cells. We therefore analyzed aly/aly mice, in which the non-canonical NF-κB pathway is not activated, owing to an inactive form of NF-κB-inducing kinase. A postmenopausal obesity model was generated by ovariectomy and subsequent high-fat and high-sucrose diet feeding. In aly/aly mice with postmenopausal obesity, serum RANKL levels were elevated, and hepatic lipid accumulation and adipocyte hypertrophy were suppressed, resulting in reduced macrophage infiltration and inflammatory cytokine mRNA expression in visceral adipose tissue. Furthermore, aly/aly mice showed protection from glucose intolerance and insulin resistance, which were observed in ovariectomized WT obese mice. These findings indicate that non-canonical NF-κB pathway activation via serum RANKL elevation contributes to postmenopausal obesity.. |
| 6. | Chiori Onizuka, Masahiro Irifune, Akari Mukai, Yoshitaka Shimizu, Mitsuru Doi, Kana Oue, Mitsuhiro Yoshida, Takahiro Kochi, Eiji Imado, Takashi Kanematsu, Yoki Nakamura, Norimitsu Morioka, Yoshihiro Nakata, Norio Sakai, Pentobarbital may protect against neurogenic inflammation after surgery via inhibition of substance P release from peripheral nerves of rats, Neuroscience Letters, https://doi.org/10.1016/j.neulet.2022.136467, 771, 136467, 2022.02, The inflammatory response related to surgery is considered surgical inflammation. Most anesthetic agents directly or indirectly suppress the immune response. However, the intravenous anesthetics pentobarbital and ketamine were reported to inhibit the lipopolysaccharide-induced inflammatory response such as cytokines formation. Neurogenic inflammation is inflammation originating from the local release of inflammatory mediators, such as substance P (SP), by primary afferent neurons after noxious stimuli like surgery. Thus, in this study, we examined whether pentobarbital and ketamine suppress SP release from cultured dorsal root ganglion (DRG) neurons. DRG cells were dissected from male Wistar rats. Released SP was measured by radioimmunoassay. We demonstrated that higher concentrations of pentobarbital (100-1,000 μM) significantly inhibited capsaicin (100 nM)-induced, but not high K+ (50 mM)-induced, SP release from DRG cells, although a high concentration of ketamine (1 mM) did not. This study revealed that pentobarbital functions between the activation of vanilloid receptor subtype 1 (TRPV1) receptors, to which capsaicin selectively binds, and the opening of voltage-operated Ca2+ channels (VOCC) in the nerve endings. Therefore, the anti-inflammatory action of pentobarbital is mediated through different mechanisms than those of ketamine. Thus, the inhibitory effect of pentobarbital on SP release from peripheral terminals may protect against neurogenic inflammation after surgery.. |
| 7. | Jing Gao, Akiko Mizokami, Hiroshi Takeuchi, Aonan Li, Fei Huang, Haruki Nagano, Takashi Kanematsu, Eijiro Jimi, Masato Hirata, Phospholipase C-related but catalytically inactive protein acts as a positive regulator of insulin signalling in adipocytes, Journal of Cell Science, https://doi.org/10.1242/jcs.258584, 135, 1, jcs258584, 2022.01, Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We have previously reported that phospholipase C-related but catalytically inactive protein (PRIP; used here to refer to both PRIP-1 and PRIP-2, also known as PLCL1 and PLCL2, respectively) interacts with Akt1, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling, including insulin-stimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt, and glucose uptake were impaired in adipocytes from PRIP double-knockout (PRIP-KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell surface was decreased in PRIP-KO adipocytes. Immunoprecipitation assays showed that PRIP interacted with IR. The reduced cell surface IR in PRIP-KO adipocytes was comparable with that in WT cells when Rab5 (Rab5a, -5b and -5c) expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which have been reported to be involved in the internalisation of IR, was impaired in cells from PRIP-KO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes.. |
| 8. | Kouichi Hashimoto, Yosuke Yamawaki, Kenji Yamaoka, Takayuki Yoshida, Kana Okada, Wanqin Tan, Miwako Yamasaki, Yoshiko Matsumoto-Makidono, Reika Kubo, Hisako Nakayama, Tsutomu Kataoka, Takashi Kanematsu, Masahiko Watanabe, Yasumasa Okamoto, Shigeru Morinobu, Hidenori Aizawa Shigeto Yamawaki, Spike firing attenuation of serotonin neurons in learned helplessness rats is reversed by ketamine, Brain Communications, https://doi.org/10.1093/braincomms/fcab285, 3, 4, fcab285, 2021.12, Animals suffering from uncontrollable stress sometimes show low effort to escape stress (learned helplessness). Changes in serotonin (5-hydroxytryptamine) signalling are thought to underlie this behaviour. Although the release of 5-hydroxytryptamine is triggered by the action potential firing of dorsal raphe nuclei 5-hydroxytryptamine neurons, the electrophysiological changes induced by uncontrollable stress are largely unclear. Herein, we examined electrophysiological differences among 5-hydroxytryptamine neurons in naïve rats, learned helplessness rats and rats resistant to inescapable stress (non-learned helplessness). Five-week-old male Sprague Dawley rats were exposed to inescapable foot shocks. After an avoidance test session, rats were classified as learned helplessness or non-learned helplessness. Activity-dependent 5-hydroxytryptamine release induced by the administration of high-potassium solution was slower in free-moving learned helplessness rats. Subthreshold electrophysiological properties of 5-hydroxytryptamine neurons were identical among the three rat groups, but the depolarization-induced spike firing was significantly attenuated in learned helplessness rats. To clarify the underlying mechanisms, potassium (K+) channels regulating the spike firing were initially examined using naïve rats. K+ channels sensitive to 500 μM tetraethylammonium caused rapid repolarization of the action potential and the small conductance calcium-activated K+ channels produced afterhyperpolarization. Additionally, dendrotoxin-I, a blocker of Kv1.1 (encoded by Kcna1), Kv1.2 (encoded by Kcna2) and Kv1.6 (encoded by Kcna6) voltage-dependent K+ channels, weakly enhanced the spike firing frequency during depolarizing current injections without changes in individual spike waveforms in naïve rats. We found that dendrotoxin-I significantly enhanced the spike firing of 5-hydroxytryptamine neurons in learned helplessness rats. Consequently, the difference in spike firing among the three rat groups was abolished in the presence of dendrotoxin-I. These results suggest that the upregulation of dendrotoxin-I-sensitive Kv1 channels underlies the firing attenuation of 5-hydroxytryptamine neurons in learned helplessness rats. We also found that the antidepressant ketamine facilitated the spike firing of 5-hydroxytryptamine neurons and abolished the firing difference between learned helplessness and non-learned helplessness by suppressing dendrotoxin-I-sensitive Kv1 channels. The dendrotoxin-I-sensitive Kv1 channel may be a potential target for developing drugs to control activity of 5-hydroxytryptamine neurons.. |
| 9. | Yuka Maetani, Satoshi Asano, Akiko Mizokami, Yosuke Yamawaki, Tomomi Sano, Masato Hirata, Masahiro Irifune, Takashi Kanematsu, Expression of PRIP, a phosphatidylinositol 4,5-bisphosphate binding protein, attenuates PI3K/AKT signaling and suppresses tumor growth in a xenograft mouse model, Biochemical and biophysical research communications, https://doi.org/10.1016/j.bbrc.2021.03.045, 552, 106-113, 2021.05, Cancer is characterized by uncontrolled proliferation resulting from aberrant cell cycle progression. The activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling, a regulatory pathway for the cell cycle, stabilizes cyclin D1 in the G1 phase by inhibiting the activity of glycogen synthase kinase 3β (GSK3β) via phosphorylation. We previously reported that phospholipase C-related catalytically inactive protein (PRIP), a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding protein, regulates PI3K/AKT signaling by competitively inhibiting substrate recognition by PI3K. Therefore, in this study, we investigated whether PRIP is involved in cell cycle progression. PRIP silencing in MCF-7 cells, a human breast cancer cell line, demonstrated PI(3,4,5)P3 signals accumulated at the cell periphery compared to that of the control. This suggests that PRIP reduction enhances PI(3,4,5)P3-mediated signaling. Consistently, PRIP silencing in MCF-7 cells exhibited increased phosphorylation of AKT and GSK3β which resulted in cyclin D1 accumulation. In contrast, the exogenous expression of PRIP in MCF-7 cells evidenced stronger downregulation of AKT and GSK3β phosphorylation, reduced accumulation of cyclin D1, and diminished cell proliferation in comparison to control cells. Flow cytometry analysis indicated that MCF-7 cells stably expressing PRIP attenuate cell cycle progression. Importantly, tumor growth of MCF-7 cells stably expressing PRIP was considerably prevented in an in vivo xenograft mouse model. In conclusion, PRIP expression downregulates PI3K/AKT/GSK3β-mediated cell cycle progression and suppresses tumor growth. Therefore, we propose that PRIP is a new therapeutic target for anticancer therapy.. |
| 10. | Satoru Mukai, Akiko Mizokami, Takahito Otani, Tomomi Sano, Miho Matsuda, Sakura Chishaki, Jing Gao, Tomoyo Kawakubo-Yasukochi, Ronghao Tang, Takashi Kanematsu, Hiroshi Takeuchi, Eijiro Jimi, Masato Hirata, Adipocyte-specific GPRC6A ablation promotes diet-induced obesity by inhibiting lipolysis, The Journal of Biological Chemistry, https://doi.org/10.1016/j.jbc.2021.100274, 296, 100274, 2021.01. |
| 11. | Taiki Sanada, Tomomi Sano, Yusuke Sotomaru, Rehab Alshargabi, Yosuke Yamawaki, Akiko Yamashita, Hiroaki Matsunaga, Misaki Iwashita, Takanori Shinjo, Takashi Kanematsu, Tomoichiro Asano, Fusanori Nishimura, Anti-inflammatory effects of miRNA-146a induced in adipose and periodontal tissues, Biochemistry and Biophysics Reports, https://doi.org/10.1016/j.bbrep.2020.100757, 22, 100757-100757, 2020.07, MicroRNA (miRNA) plays an important role in diverse cellular biological processes such as inflammatory response, differentiation and proliferation, and carcinogenesis. miR-146a has been suggested as a negative regulator of the inflammatory reaction. Although, it has been reported as expressed in inflamed adipose and periodontal tissues, however, miR-146a's inhibitory effects against inflammatory response in both the tissues, are not well understood. Therefore, in this study, the inhibitory effects of miR-146a on both adipose and periodontal inflammation, was investigated. In vitro study has revealed that miR-146a transfection into either adipocytes or gingival fibroblasts, has resulted in a reduced cytokine gene expression, observed on co-culturing the cells with macrophages in the presence of lipopolysaccharides (LPS), in comparison to the control miRNA transfected. Similarly, miR-146a transfection into macrophages resulted in a reduced expression of TNF-α gene and protein in response to LPS stimulation. In vivo study revealed that a continuous intravenous miR-146a administration into mice via tail vein, protected the mice from developing high-fat diet-induced obesity and the inflammatory cytokine gene expression was down-regulated in both adipose and periodontal tissues. miR-146a appeared to be induced by macrophage-derived inflammatory signals such as TNF-α by negative feed-back mechanism, and it suppressed inflammatory reaction in both adipose and periodontal tissues. Therefore, miR-146a could be suggested as a potential therapeutic molecule and as a common inflammatory regulator for both obesity-induced diabetes and related periodontal diseases.. |
| 12. | Yuya Ogawa, Masahiro Irifune, Akari Mukai, Yoshitaka Shimizu, Mitsuru Doi, Kana Oue, Mitsuhiro Yoshida, Takashi Kanematsu, Norimitsu Morioka, Yoshihiro Nakata, Norio Sakai, The indirect γ-aminobutyric acid (GABA) receptor agonist gabaculine-induced loss of the righting reflex may inhibit the descending analgesic pathway, Pharmacology, biochemistry, and behavior, 10.1016/j.pbb.2020.173034., 198, 173034, 2020.11, In the spinal cord, γ-aminobutyric acid (GABA) interneurons play an essential role in antinociception. However, not all actions of GABA favor antinociception at the supraspinal level. We previously reported that gabaculine, which increases endogenous GABA in the synaptic clefts, induces loss of the righting reflex (LORR) that is one indicator of hypnosis, but not immobility in response to noxious stimulus. A slow pain is transmitted to the spinal cord via C fibers and evokes substance P (SP) release from their terminals. However, the antinociceptive effects of gabaculine are still unknown. Our study examined whether the analgesic effects of the opioid morphine or the α2-adrenoceptor agonist dexmedetomidine, whose actions are mediated through facilitation of the descending analgesic pathway, are affected by gabaculine-induced LORR. We also explored the effects of GABA receptor agonists on SP release from cultured dorsal root ganglion (DRG) neurons. All drugs were administered systemically to mice. To assess antinociception, loss of nociceptive response (analgesia) and immobility were evaluated. DRG cells were dissected from rats. Gabaculine produced no analgesia. Either morphine or dexmedetomidine in combination with gabaculine induced immobility; however, the doses of each drug required to induce immobility were much higher than those required to induce analgesia. Capsaicin significantly increased SP release from DRG cells, but a high concentration (1 mM) of the GABA receptor agonist muscimol, propofol, gaboxadol, or baclofen did not inhibit the capsaicin-induced SP release, suggesting that their antinociceptive effects were not through this mechanism. Thus, the gabaculine-induced LORR may inhibit the descending analgesic pathway.. |
| 13. | Daisuke Furutama, Shinji Matsuda, Yosuke Yamawaki, Saki Hatano, Ai Okanobu, Takumi Memida, Hiroshi Oue, Tsuyoshi Fujita, Kazuhisa Ouhara, Mikihito Kajiya, Noriyoshi Mizuno, Takashi Kanematsu, Kazuhiro Tsuga, Hidemi Kurihara, IL-6 Induced by Periodontal Inflammation Causes Neuroinflammation and Disrupts the Blood-Brain Barrier, Brain sciences, 10.3390/brainsci10100679., 10, 10, 679, 2020.09, Background: Periodontal disease (PD) is a risk factor for systemic diseases, including neurodegenerative diseases. The role of the local and systemic inflammation induced by PD in neuroinflammation currently remains unclear. The present study investigated the involvement of periodontal inflammation in neuroinflammation and blood-brain barrier (BBB) disruption. Methods: To induce PD in mice (c57/BL6), a ligature was placed around the second maxillary molar. Periodontal, systemic, and neuroinflammation were assessed based on the inflammatory cytokine mRNA or protein levels using qPCR and ELISA. The BBB permeability was evaluated by the mRNA levels and protein levels of tight junction-related proteins in the hippocampus using qPCR and immunofluorescence. Dextran tracing in the hippocampus was also conducted to examine the role of periodontal inflammation in BBB disruption. Results: The TNF-α, IL-1β, and IL-6 levels markedly increased in gingival tissue 1 week after ligation. The IL-6 serum levels were also increased by ligature-induced PD. In the hippocampus, the IL-1β mRNA expression levels were significantly increased by ligature-induced PD through serum IL-6. The ligature-induced PD decreased the claudin 5 expression levels in the hippocampus, and the neutralization of IL-6 restored its levels. The extravascular 3-kDa dextran levels were increased by ligature-induced PD. Conclusions: These results suggest that the periodontal inflammation-induced expression of IL-6 is related to neuroinflammation and BBB disruption in the hippocampus, ultimately leading to cognitive impairment. Periodontal therapy may protect against neurodegenerative diseases.. |
| 14. | Satoshi Asano, Yasuka Ikura, Mitsuki Nishimoto, Yosuke Yamawaki, Kozue Hamao, Keiju Kamijo, Masato Hirata, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein regulates cytokinesis by protecting phosphatidylinositol 4,5-bisphosphate from metabolism in the cleavage furrow, Scientific reports, https://doi.org/10.1038/s41598-019-49156-3, 9, 1, 2019.12, [URL], Cytokinesis is initiated by the formation and ingression of the cleavage furrow. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P<sub>2</sub>] accumulation followed by RhoA translocation to the cleavage furrow are prerequisites for cytokinesis progression. Here, we investigated whether phospholipase C (PLC)-related catalytically inactive protein (PRIP), a metabolic modulator of PI(4,5)P<sub>2</sub>, regulates PI(4,5)P<sub>2</sub>-mediated cytokinesis. We found that PRIP localised to the cleavage furrow during cytokinesis. Moreover, HeLa cells with silenced PRIP displayed abnormal cytokinesis. Importantly, PI(4,5)P<sub>2</sub> accumulation at the cleavage furrow, as well as the localisation of RhoA and phospho-myosin II regulatory light chain to the cleavage furrow, were reduced in PRIP-silenced cells. The overexpression of oculocerebrorenal syndrome of Lowe-1 (OCRL1), a phosphatidylinositol-5-phosphatase, in cells decreased PI(4,5)P<sub>2</sub> levels during early cytokinesis and resulted in cytokinesis abnormalities. However, these abnormal cytokinesis phenotypes were ameliorated by the co-expression of PRIP but not by co-expression of a PI(4,5)P<sub>2</sub>-unbound PRIP mutant. Collectively, our results indicate that PRIP is a component at the cleavage furrow that maintains PI(4,5)P<sub>2</sub> metabolism and regulates RhoA-dependent progression of cytokinesis. Thus, we propose that PRIP regulates phosphoinositide metabolism correctively and mediates normal cytokinesis progression.. |
| 15. | Yosuke Yamawaki, Satomi Shirawachi, Akiko Mizokami, Kanako Nozaki, Hikaru Ito, Satoshi Asano, Kana Oue, Hidenori Aizawa, Shigeto Yamawaki, Masato Hirata, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein regulates lipopolysaccharide-induced hypothalamic inflammation-mediated anorexia in mice, Neurochemistry International, 131, 2019.12, [URL], Peripheral lipopolysaccharide (LPS) injection induces systemic inflammation through the activation of the inhibitor of nuclear factor kappa B (NF-κB) kinase (IKK)/NF-κB signaling pathway, which promotes brain dysfunction resulting in conditions including anorexia. LPS-mediated reduction of food intake is associated with activation of NF-κB signaling and phosphorylation of the transcription factor signal transducer and activator of transcription 3 (STAT3) in the hypothalamus. We recently reported phospholipase C-related catalytically inactive protein (PRIP) as a new negative regulator of phosphatidylinositol 3-kinase/AKT signaling. AKT regulates the IKK/NF-κB signaling pathway; therefore, this study aimed to investigate the role of PRIP/AKT signaling in LPS-mediated neuroinflammation-induced anorexia. PRIP gene (Prip1 and Prip2) knockout (Prip-KO) mice intraperitoneally (ip) administered with LPS exhibited increased anorexia responses compared with wild-type (WT) controls. Although few differences were observed between WT and Prip-KO mice in LPS-elicited plasma pro-inflammatory cytokine elevation, hypothalamic pro-inflammatory cytokines were significantly upregulated in Prip-KO rather than WT mice. Hypothalamic AKT and IKK phosphorylation and IκB degradation were significantly increased in Prip-KO rather than WT mice, indicating further promotion of AKT-mediated NF-κB signaling. Consistently, hypothalamic STAT3 was further phosphorylated in Prip-KO rather than WT mice. Furthermore, suppressor of cytokine signaling 3 (Socs3), a negative feedback regulator for STAT3 signaling, and cyclooxogenase-2 (Cox2), a candidate molecule in LPS-induced anorexigenic responses, were upregulated in the hypothalamus in Prip-KO rather than WT mice. Pro-inflammatory cytokines were upregulated in hypothalamic microglia isolated from Prip-KO rather than WT mice. Together, these findings indicate that PRIP negatively regulates LPS-induced anorexia caused by pro-inflammatory cytokine expression in the hypothalamus, which is mediated by AKT-activated NF-κB signaling. Importantly, hypothalamic microglia participate in this PRIP-mediated process. Elucidation of PRIP-mediated neuroinflammatory responses may provide novel insights into the pathophysiology of many brain dysfunctions.. |
| 16. | Tomonori Furukawa, Yoshikazu Nikaido, Shuji Shimoyama, Yoshiki Ogata, Tetsuya Kushikata, Kazuyoshi Hirota, Takashi Kanematsu, Masato Hirata, Shinya Ueno, Phospholipase C-related inactive protein type-1 deficiency affects anesthetic electroencephalogram activity induced by propofol and etomidate in mice, Journal of Anesthesia, 10.1007/s00540-019-02663-z, 33, 4, 531-542, 2019.08, [URL], Purpose: The general anesthetics propofol and etomidate mainly exert their anesthetic actions via GABA A receptor (GABAA-R). The GABAA-R activity is influenced by phospholipase C-related inactive protein type-1 (PRIP-1), which is related to trafficking and subcellular localization of GABAA-R. PRIP-1 deficiency attenuates the behavioral reactions to propofol but not etomidate. However, the effect of these anesthetics and of PRIP-1 deficiency on brain activity of CNS are still unclear. In this study, we examined the effects of propofol and etomidate on the electroencephalogram (EEG). Methods: The cortical EEG activity was recorded in wild-type (WT) and PRIP-1 knockout (PRIP-1 KO) mice. All recorded EEG data were offline analyzed, and the power spectral density and 95% spectral edge frequency of EEG signals were compared between genotypes before and after injections of anesthetics. Results: PRIP-1 deficiency induced increases in EEG absolute powers, but did not markedly change the relative spectral powers during waking and sleep states in the absence of anesthesia. Propofol administration induced increases in low-frequency relative EEG activity and decreases in SEF95 values in WT but not in PRIP-1 KO mice. Following etomidate injection, low-frequency EEG power was increased in both genotype groups. At high frequency, the relative power in PRIP-1 KO mice was smaller than that in WT mice. Conclusions: The lack of PRIP-1 disrupted the EEG power distribution, but did not affect the depth of anesthesia after etomidate administration. Our analyses suggest that PRIP-1 is differentially involved in anesthetic EEG activity with the regulation of GABAA-R activity.. |
| 17. | Tomomi Sano, Taiki Sanada, Yusuke Sotomaru, Takanori Shinjo, Misaki Iwashita, Akiko Yamashita, Takao Fukuda, Terukazu Sanui, Tomoichiro Asano, Takashi Kanematsu, Fusanori Nishimura, Ccr7 null mice are protected against diet-induced obesity via Ucp1 upregulation and enhanced energy expenditure, Nutrition and Metabolism, 10.1186/s12986-019-0372-5, 16, 1, 2019.07, [URL], Background: The chemokine receptor CCR7, expressed on various immune cells, is associated with cell migration and lympho-node homing. Mice lacking Ccr7 are protected from diet-induced obesity and subsequent insulin resistance. We evaluated the mechanism underlying these protective effects from the standpoint of energy expenditure. Methods: Wild-type and Ccr7 null mice were fed a high-fat diet, and the regulation of energy metabolism and energy metabolism-related molecules, e.g., Ucp1, Cidea, and Pgc1α, were evaluated. Results: Food intake did not differ between groups. O2 consumption and CO2 production were higher in Ccr7 null mice than in wild-type mice, despite a similar respiratory quotient and glucose and lipid utilization, suggesting that energy expenditure increased in Ccr7 null mice via enhanced metabolism. In white adipose tissues of Ccr7 null mice, Prdm16, Cd137, Tmem26, Th, and Tbx1 expression increased. Similarly, in brown adipose tissues of Ccr7 null mice, Dio2, Pgc1α, Cidea, Sirt1, and Adiponectin expression increased. In both white and brown adipose tissues, Ucp1 gene and protein expression levels were higher in null mice than in wild-type mice. Conclusions: In Ccr7 null mice, browning of white adipocytes as well as the activation of brown adipocytes cause enhanced energy metabolism, resulting in protection against diet-induced obesity.. |
| 18. | Takashi Kanematsu, Kana Oue, Toshiya Okumura, Kae Harada, Yosuke Yamawaki, Satoshi Asano, Akiko Mizokami, Masahiro Irifune, Masato Hirata, Phospholipase C-related catalytically inactive protein A novel signaling molecule for modulating fat metabolism and energy expenditure, journal of oral biosciences, 10.1016/j.job.2019.04.002, 61, 2, 65-72, 2019.06, [URL], Background: Overweight and obesity are defined as excessive or abnormal fat accumulation in adipose tissues, and increase the risk of morbidity in many diseases, including hypertension, dyslipidemia, type 2 diabetes, coronary heart disease, and stroke, through pathophysiological mechanisms. There is strong evidence that weight loss reduces the risk of metabolic syndrome by limiting blood pressure and improving the levels of serum triglycerides, total cholesterol, low-density lipoprotein-cholesterol, and high-density lipoprotein-cholesterol. To date, several attempts have been made to develop effective anti-obesity medication or weight-loss drugs; however, satisfactory drugs for clinical use have not yet been developed. Therefore, elucidation of the molecular mechanisms driving fat metabolism (adipogenesis and lipolysis) represents the first step in developing clinically useful drugs and/or therapeutic treatments to control obesity. Highlight: In our previous study on intracellular signaling of phospholipase C-related catalytically inactive protein (PRIP), we generated and analyzed Prip-double knockout (Prip-DKO) mice. Prip-DKO mice showed tolerance against insulin resistance and a lean phenotype with low fat mass. Here, we therefore reviewed the involvement of PRIP in fat metabolism and energy expenditure. We conclude that PRIP, a protein phosphatase-binding protein, can modulate fat metabolism via phosphoregulation of adipose lipolysis-related molecules, and regulates non-shivering heat generation in brown adipocytes. Conclusion: We propose PRIP as a new therapeutic target for controlling obesity or developing novel anti-obesity drugs.. |
| 19. | Yusuke Nakatsu, Yasuka Matsunaga, Takeshi Yamamotoya, Koji Ueda, Masa ki Inoue, Yu Mizuno, Mikako Nakanishi, Tomomi Sano, Yosuke Yamawaki, Akifumi Kushiyama, Hideyuki Sakoda, Midori Fujishiro, Akihide Ryo, Hiraku Ono, Tohru Minamino, Shin Ichiro Takahashi, Haruya Ohno, Masayasu Yoneda, Kei Takahashi, Hisamitsu Ishihara, Hideki Katagiri, Fusanori Nishimura, Takashi Kanematsu, Tetsuya Yamada, Tomoichiro Asano, Prolyl Isomerase Pin1 Suppresses Thermogenic Programs in Adipocytes by Promoting Degradation of Transcriptional Co-activator PRDM16, Cell Reports, 10.1016/j.celrep.2019.02.066, 26, 12, 3221-3230.e3, 2019.03, [URL], Non-shivering thermogenesis in adipocytes provides defense against low temperatures and obesity development, but the underlying regulatory mechanism remains to be fully clarified. Based on both markedly increased Pin1 expression in states of excess nutrition and resistance to obesity development in Pin1 null mice, we speculated that adipocyte Pin1 may play a role in thermogenic programs. Adipose-specific Pin1 knockout (adPin1 KO) mice showed enhanced transcription of thermogenic genes and tolerance to hypothermia when exposed to cold. In addition, adPin1 KO mice were resistant to high-fat diet-induced obesity and glucose intolerance. A series of experiments revealed that Pin1 binds to PRDM16 and thereby promotes its degradation through the ubiquitin-proteasome system. Consistent with these results, Pin1 deletion in differentiated adipocytes showed enhancement of thermogenic programs in response to the β3 agonist CL316243 through the upregulation of PRDM16 proteins. These observations indicate that Pin1 is a negative regulator of non-shivering thermogenesis.. |
| 20. | Taka Fujita, Gentaro Kumagai, Xizhe Liu, Kanichiro Wada, Toshihiro Tanaka, Hitoshi Kudo, Toru Asari, Tatsuhiro Fukutoku, Ayako Sasaki, Yohshiro Nitobe, Yoshikazu Nikaido, Ken Ichi Furukawa, Masato Hirata, Takashi Kanematsu, Shinya Ueno, Yasuyuki Ishibashi, Poor Motor-Function Recovery after Spinal Cord Injury in Anxiety-Model Mice with Phospholipase C-Related Catalytically Inactive Protein Type 1 Knockout, Journal of Neurotrauma, 10.1089/neu.2017.5492, 35, 12, 1379-1386, 2018.06, [URL], Mice with a knockout of phospholipase C (PLC)-related inactive protein type 1 (PRIP1 -/- mice) display anxiety-like behavior and altered γ-aminobutyric acid (GABA) A -receptor pharmacology. Here, we examined associations between anxiety and motor-function recovery in PRIP1 -/- mice after a spinal cord injury (SCI) induced by a moderate contusion injury at the 10th thoracic level. Uninjured PRIP1 -/- mice showed less distance than wild-type (WT) mice in the center 25% in an open field test (OFT), indicating anxiety-like behavior. Anxiety behavior increased in both WT and PRIP1 -/- mice after SCI. WT and PRIP1 -/- mice were completely paralyzed on day 1 after SCI, but gradually recovered until reaching a plateau at ∼4 weeks. After SCI, the PRIP1 -/- mice had significantly greater motor dysfunction than the WT mice. In WT mice after SCI, the percentage of distance spent in the center 25% of the OFT was correlated with the OFT distance traveled and velocity, and with the reaction time in a plantar pressure-sensitivity mechanical test. In PRIP1 -/- mice after SCI, the percentage of distance spent in the center 25% of the OFT was correlated with the OFT distance traveled and with the latency to fall in the rotarod test. Six weeks after SCI, ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) expressions were elevated at the lesion epicenter in PRIP1 -/- mice, and spinal cord atrophy and demyelination were more severe than in WT mice. The axonal fiber development was also decreased in PRIP1 -/- mice, consistent with the poor motor-function recovery after SCI in these mice.. |
| 21. | Yosuke Yamawaki, Norika Yoshioka, Kanako Nozaki, Hikaru Ito, Keisuke Oda, Kana Harada, Satomi Shirawachi, Satoshi Asano, Hidenori Aizawa, Shigeto Yamawaki, Takashi Kanematsu, Hiroyuki Akagi, Sodium butyrate abolishes lipopolysaccharide-induced depression-like behaviors and hippocampal microglial activation in mice, Brain Research, 10.1016/j.brainres.2017.12.004, 1680, 13-38, 2018.02, [URL], Patients with major depressive disorder have elevated peripheral inflammation; the degree of this increase correlates with the severity of the disorder. Chronic psychological stress increases pro-inflammatory cytokines and promotes microglial activation, leading to stress vulnerability. Epigenetics, including DNA methylation and histone modification, are also related to the pathophysiology of major depressive disorder. Sodium butyrate (SB), a histone deacetylase inhibitor, exerts an antidepressant effect by altering gene expression in the hippocampus. In this study, we investigated whether lipopolysaccharide (LPS)-induced depressive-like behaviors in mice are affected by the repeated treatment with SB. Intraperitoneal injection of LPS (5 mg/kg) induced cytokines and ionized calcium-binding adaptor molecule 1(Iba1), a marker of microglial activation, in the hippocampus. It also increased the immobility time in a forced swim test, without changing locomotion. Repeated treatment with SB reduced LPS-induced alterations. These findings suggested that epigenetic regulation exist in hippocampal microglial activation, and is involved in depressive-like behaviors associated with neuro-inflammation. Further, using cDNA microarray analyses, we examined whether LPS and SB treatment affected the microglial gene profiles. Our results indicated 64 overlapping genes, between LPS-increased genes and SB-decreased genes. Among these genes, EF hand calcium binding domain 1 was a particularly distinct candidate gene. Altogether, our findings indicated that microglial activation mediated through epigenetic regulation may be involved in depressive-like behaviors. In addition, we demonstrated the effect of SB on gene information in hippocampal microglia under neuroinflammatory conditions.. |
| 22. | Satoshi Asano, Yuri Taniguchi, Yosuke Yamawaki, Jing Gao, Kae Harada, Hiroshi Takeuchi, Masato Hirata, Takashi Kanematsu, Suppression of cell migration by phospholipase C-related catalytically inactive protein-dependent modulation of PI3K signalling, Scientific reports, 10.1038/s41598-017-05908-7, 7, 1, 2017.12, [URL], The metabolic processes of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] into PI(3,4,5)P3 and the subsequent PI(3,4,5)P3 signalling are involved in cell migration. Dysfunctions in the control of this pathway can cause human cancer cell migration and metastatic growth. Here we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a PI(4,5)P2-binding protein, regulates cancer cell migration. PRIP overexpression in MCF-7 and BT-549 human breast cancer cells inhibited cell migration in vitro and metastasis development in vivo. Overexpression of the PRIP pleckstrin homology domain, a PI(4,5)P2 binding motif, in MCF-7 cells caused significant suppression of cell migration. Consistent with these results, in comparison with wild-type cells, Prip-deficient mouse embryonic fibroblasts exhibited increased cell migration, and this was significantly attenuated upon transfection with a siRNA targeting p110α, a catalytic subunit of class I phosphoinositide 3-kinases (PI3Ks). PI(3,4,5)P3 production was decreased in Prip-overexpressing MCF-7 and BT-549 cells. PI3K binding to PI(4,5)P2 was significantly inhibited by recombinant PRIP in vitro, and thus the activity of PI3K was downregulated. Collectively, PRIP regulates the production of PI(3,4,5)P3 from PI(4,5)P2 by PI3K, and the suppressor activity of PRIP in PI(4,5)P2 metabolism regulates the tumour migration, suggesting PRIP as a promising target for protection against metastatic progression.. |
| 23. | Masaki Hayashiuchi, Tomoya Kitayama, Katsuya Morita, Yosuke Yamawaki, Kana Oue, Taiga Yoshinaka, Satoshi Asano, Kae Harada, Youngnam Kang, Masato Hirata, Masahiro Irifune, Mitsugi Okada, Takashi Kanematsu, General anesthetic actions on GABA A receptors in vivo are reduced in phospholipase C-related catalytically inactive protein knockout mice, Journal of Anesthesia, 10.1007/s00540-017-2350-2, 31, 4, 531-538, 2017.08, [URL], Purpose: The aim of this study was to investigate the action of general anesthetics in phospholipase C-related catalytically inactive protein (PRIP)-knockout (KO) mice that alter GABA A receptor signaling. Methods: PRIP regulates the intracellular trafficking of β subunit-containing GABA A receptors in vitro. In this study, we examined the effects of intravenous anesthetics, propofol and etomidate that act via β subunit-containing GABA A receptors, in wild-type and Prip-KO mice. Mice were intraperitoneally injected with a drug, and a loss of righting reflex (LORR) assay and an electroencephalogram analysis were performed. Results: The cell surface expression of GABA A receptor β3 subunit detected by immunoblotting was decreased in Prip-knockout brain compared with that in wild-type brain without changing the expression of other GABA A receptor subunits. Propofol-treated Prip-KO mice exhibited significantly shorter duration of LORR and had lower total anesthetic score than wild-type mice in the LORR assay. The average duration of sleep time in an electroencephalogram analysis was shorter in propofol-treated Prip-KO mice than in wild-type mice. The hypnotic action of etomidate was also reduced in Prip-KO mice. However, ketamine, an NMDA receptor antagonist, had similar effects in the two genotypes. Conclusion: PRIP regulates the cell surface expression of the GABA A receptor β3 subunit and modulates general anesthetic action in vivo. Elucidation of the involved regulatory mechanisms of GABA A receptor-dependent signaling would inform the development of safer anesthetic therapies for clinical applications.. |
| 24. | Yoshikazu Nikaido, Tomonori Furukawa, Shuji Shimoyama, Junko Yamada, Keisuke Migita, Kohei Koga, Tetsuya Kushikata, Kazuyoshi Hirota, Takashi Kanematsu, Masato Hirata, Shinya Ueno, Propofol anesthesia is reduced in phospholipase c-related inactive protein type-1 knockout mices, Journal of Pharmacology and Experimental Therapeutics, 10.1124/jpet.116.239145, 361, 3, 367-374, 2017.06, [URL], The GABA type A receptor (GABAA-R) is a major target of intravenous anesthetics. Phospholipase C-related inactive protein type-1 (PRIP-1) is important in GABAA-R phosphorylation and membrane trafficking. In this study, we investigated the role of PRIP-1 in general anesthetic action. The anesthetic effects of propofol, etomidate, and pentobarbital were evaluated in wild-type and PRIP-1 knockout (PRIP-1 KO) mice by measuring the latency and duration of loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR). The effect of pretreatment with okadaic acid (OA), a protein phosphatase 1/2A inhibitor, on propofol- and etomidate-induced LORR was also examined. PRIP-1 deficiency provided the reduction of LORR and LTWR induced by propofol but not by etomidate or pentobarbital, indicating that PRIP-1 could determine the potency of the anesthetic action of propofol. Pre-treatment with OA recovered the anesthetic potency induced by propofol in PRIP-1 KO mice. OA injection enhanced phos-phorylation of cortical the GABAA-R β3 subunit in PRIP-1 KO mice. These results suggest that PRIP-1-mediated GABAA-R β3 subunit phosphorylation might be involved in the general anesthetic action induced by propofol but not by etomidate or pentobarbital.. |
| 25. | Kana Oue, Yosuke Yamawaki, Satoshi Asano, Akiko Mizokami, Masato Hirata, Masahiro Irifune, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein-knockout mice exhibit uncoupling protein 1 upregulation in adipose tissues following chronic cold exposure, journal of oral biosciences, 10.1016/j.job.2017.04.001, 59, 2, 108-112, 2017.05, [URL], Objectives We have previously demonstrated that phospholipase C-related catalytically inactive protein (PRIP) is involved in fat metabolism and energy consumption. However, whether PRIP participates in body energy metabolism in vivo remains to be determined. Therefore, we examined whether PRIP deficiency affects whole-body energy homeostasis, which is modulated by non-shivering thermogenesis in brown adipose tissue, using a cold exposure animal model. Methods Fasting plasma triacylglycerol levels were measured to evaluate fat metabolism in wild-type and Prip-KO mice. In addition, a glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed. To determine changes in energy consumption, mice were exposed to a cold environment for 7 days, and expression of uncoupling protein 1 (UCP1) in brown adipose tissue was analyzed via western blotting. Results Fasting plasma levels of triacylglycerols were significantly higher in Prip-KO mice than in wild-type mice. However, Prip-KO mice showed a healthy phenotype based on GTT and ITT. UCP1 expression was significantly upregulated in the brown and white adipose tissues of Prip-KO mice exposed to cold conditions. Conclusion Prip-KO mice exhibit greater ability to consume lipids as an energy source, indicating that PRIP modulates of systemic energy expenditure. Our findings provide increased understanding of PRIP-mediated non-shivering thermogenic mechanisms and offers important insights for the treatment and control of obesity.. |
| 26. | Tomomi Sano, S. Nagayasu, S. Suzuki, Misaki Iwashita, Akiko Yamashita, T. Shinjo, Terukazu Sanui, A. Kushiyama, Takashi Kanematsu, T. Asano, Fusanori Nishimura, Epicatechin downregulates adipose tissue CCL19 expression and thereby ameliorates diet-induced obesity and insulin resistance, Nutrition, Metabolism and Cardiovascular Diseases, 10.1016/j.numecd.2016.11.008, 27, 3, 249-259, 2017.03, [URL], Background and aims Epicatechin (EC) intake has been suggested to be beneficial for the prevention of cardiovascular disorders, and it is well known that adipose tissue inflammation is one of the major risk factors for coronary heart diseases. The purpose of the present study was to determine the in vitro and in vivo effects of EC on adipose tissue inflammation and obesity. Methods and results DNA microarray analysis was performed to evaluate the effects of EC on gene expression in adipocytes co-cultured with bacterial endotoxin-stimulated macrophages. To determine the in vivo effects of the catechin, C57BL/6 mice were fed either a high-fat diet (HFD) or HFD combined with EC, and metabolic changes were observed EC suppressed the expression of many inflammatory genes in the adipocytes co-cultured with endotoxin-stimulated macrophages. Specifically, EC markedly suppressed chemokine (C–C motif) ligand 19 (CCL19) expression. The target cell of EC appeared to macrophages. The in vivo study indicated that mice fed the EC-supplemented HFD were protected from diet-induced obesity and insulin resistance. Accordingly, the expression levels of genes associated with inflammation in adipose tissue and in the liver were downregulated in this group of mice. Conclusions EC exerts beneficial effects for the prevention of adipose tissue inflammation and insulin resistance. Since we previously reported that mice deficient in the CCL19 receptor were protected from diet-induced obesity and insulin resistance, it can be concluded that the beneficial effects of EC could be mediated, at least in part, by marked suppression of CCL19 expression.. |
| 27. | Yosuke Yamawaki, Kana Oue, Satomi Shirawachi, Satoshi Asano, Kae Harada, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein can regulate obesity, a state of peripheral inflammation, Japanese Dental Science Review, 10.1016/j.jdsr.2016.06.001, 53, 1, 18-24, 2017.02, [URL], Obesity is defined as abnormal or excessive fat accumulation. Chronic inflammation in fat influences the development of obesity-related diseases. Many reports state that obesity increases the risk of morbidity in many diseases, including hypertension, dyslipidemia, type 2 diabetes, coronary heart disease, stroke, sleep apnea, and breast, prostate and colon cancers, leading to increased mortality. Obesity is also associated with chronic neuropathologic conditions such as depression and Alzheimer's disease. However, there is strong evidence that weight loss reduces these risks, by limiting blood pressure and improving levels of serum triglycerides, total cholesterol, low-density lipoprotein (LDL)-cholesterol, and high-density lipoprotein (HDL)-cholesterol. Prevention and control of obesity is complex, and requires a multifaceted approach. The elucidation of molecular mechanisms driving fat metabolism (adipogenesis and lipolysis) aims at developing clinical treatments to control obesity. We recently reported a new regulatory mechanism in fat metabolism: a protein phosphatase binding protein, phospholipase C-related catalytically inactive protein (PRIP), regulates lipolysis in white adipocytes and heat production in brown adipocytes via phosphoregulation. Deficiency of PRIP in mice led to reduced fat accumulation and increased energy expenditure, resulting in a lean phenotype. Here, we evaluate PRIP as a new therapeutic target for the control of obesity.. |
| 28. | Kana Oue, Jun Zhang, Kae Harada-Hada, Satoshi Asano, Yosuke Yamawaki, Masaki Hayashiuchi, Hisako Furusho, Takashi Takata, Masahiro Irifune, Masato Hirata, Takashi Kanematsu, Phospholipase C-related Catalytically Inactive Protein Is a New Modulator of Thermogenesis Promoted by β-Adrenergic Receptors in Brown Adipocytes, Journal of Biological Chemistry, 10.1074/jbc.M115.705723, 291, 8, 4185-4196, 2016.02, [URL], Phospholipase C-related catalytically inactive protein (PRIP) was first identified as an inositol 1,4,5-trisphosphate-binding protein, and was later found to be involved in a variety of cellular events, particularly those related to protein phosphatases. We previously reported that Prip knock-out (KO) mice exhibit a lean phenotype with a small amount of white adipose tissue. In the present study, we examined whether PRIP is involved in energy metabolism, which could explain the lean phenotype, using high-fat diet (HFD)-fed mice. Prip-KO mice showed resistance to HFD-induced obesity, resulting in protection from glucose metabolism dysfunction and insulin resistance. Energy expenditure and body temperature at night were significantly higher in Prip-KO mice than in wild-type mice. Gene and protein expression of uncoupling protein 1 (UCP1), a thermogenic protein, was up-regulated in Prip-KO brown adipocytes in ther-moneutral or cold environments. These phenotypes were caused by the promotion of lipolysis in Prip-KO brown adipocytes, which is triggered by up-regulation of phosphorylation of the lipolysis-related proteins hormone-sensitive lipase and perilipin, followed by activation of UCP1 and/or up-regulation of thermogenesis-related genes (e.g. peroxisome proliferator-activated receptor-γ coactivator-1α). The results indicate that PRIP negatively regulates UCP1-mediated thermogenesis in brown adipocytes.. |
| 29. | Kana Oue, Kae Harada-Hada, Takashi Kanematsu, New molecular basis in the regulation of lipolysis via dephosphorylation, Folia Pharmacologica Japonica, 10.1254/fpj.146.93, 146, 2, 93-97, 2015.08, [URL]. |
| 30. | Hiroki Toyoda, Mitsuru Saito, Hajime Sato, Tsutomu Kawano, Shinpei Kawakami, Hirofumi Yatani, Takashi Kanematsu, Masato Hirata, Youngnam Kang, Enhanced lateral inhibition in the barrel cortex by deletion of phospholipase C-related catalytically inactive protein-1/2 in mice, Pflugers Archiv European Journal of Physiology, 10.1007/s00424-014-1592-1, 467, 7, 1445-1456, 2015.07, [URL], We previously demonstrated that the deletion of phospholipase C-related catalytically inactive protein-1/2 (PRIP-1/2) enhances the desensitization of GABAA receptors (GABAARs), while it facilitates their resensitization at the offset of GABA puff, causing a hump-like tail current (tail-I) in layer 3 (L3) pyramidal cells (PCs) of the barrel cortex. In the present study, we investigated whether inhibitory synaptic transmission in L3 PCs in the barrel cortex is altered in the PRIP-1/2 double-knockout (PRIP-DKO) mice, and if so, how the interaction between excitation and inhibition is subsequently modified. PRIP-1/2 deletion resulted in the prolongation of the decay phase of inhibitory postsynaptic currents/potentials (IPSCs/IPSPs) in L3 PCs evoked by stimulation of L3, leaving the overall features of miniature IPSCs unchanged. An optical imaging revealed that the spatiotemporal profile of a horizontal excitation spread across columns in L2/3 caused by L4 stimulation in the barrel cortex was more restricted in PRIP-DKO mice compared to the wild type, while those obtained in the presence of bicuculline were almost identical between the two genotypes. These findings suggest that PRIP-1/2 deletion enhances the lateral inhibition by prolonging inhibitory synaptic actions to limit the intercolumnar integration in the barrel cortex. Considering the present findings together with our previous study including a mathematical simulation, the prolongation of inhibitory synaptic actions is likely to result from an enhancement of desensitization followed by an enhanced resensitization in GABAARs.. |
| 31. | Nobuhito Kikuchi, Masahiro Irifune, Yoshitaka Shimizu, Keita Yoshida, Katsuya Morita, Takashi Kanematsu, Norimitsu Morioka, Yoshihiro Nakata, Norio Sakai, Selective blockade of N-methyl-D-aspartate channels in combination with dopamine receptor antagonism induces loss of the righting reflex in mice, but not immobility, Psychopharmacology, 10.1007/s00213-014-3634-y, 232, 1, 39-46, 2015.01, [URL], Rationale: The selective N-methyl-D-aspartate (NMDA) channel blocker MK-801 is known to induce no loss of the righting reflex (LORR) and to stimulate catecholaminergic (CAergic) neurons in rodents, playing a crucial role in arousal. Objectives: We examined whether MK-801 in combination with CA receptor ligands, which inhibit CAergic neuronal activities, could induce anesthesia including LORR. Methods: All drugs were administered systemically to mice. To assess anesthesia, three different behaviors were used: loss of nociceptive response (analgesia in the free-moving state without LORR), LORR, and loss of movement in response to noxious stimulation (immobility under LORR). Results: A very large dose of MK-801 (50 mg/kg) induced neither analgesia nor LORR. In contrast, MK-801 in combination with a small dose of the dopamine (DA) receptor antagonist haloperidol (0.2 mg/kg) dose-dependently produced LORR with a 50 % effective dose (ED50) of 1.6 (0.9-3.0; 95 % confidence limit) mg/kg, but not immobility. The α2-adrenoceptor agonist dexmedetomidine induced not only analgesia, but also immobility in animals treated with MK-801 (5 mg/kg) plus haloperidol (0.2 mg/kg), which then lost their righting reflex. The ED50 value of 0.26 (0.10-0.66) mg/kg (various doses of dexmedetomidine plus a fixed dose of MK-801 and haloperidol) for immobility was approximately three-fold larger than that of 0.09 (0.03-0.23) mg/kg (dexmedetomidine plus vehicle saline) for analgesia. This may occur, as LORR induced by MK-801 plus haloperidol inhibits the pain suppression system. The other ligands had little or no effect. Conclusions: The DAergic stimulant actions of MK-801 may mask its LORR effects by NMDA channel blockade.. |
| 32. | Satoshi Asano, Tomomi Nemoto, Tomoya Kitayama, Kae Harada, Jun Zhang, Kana Harada, Isei Tanida, Masato Hirata, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein (PRIP) controls KIF5B-mediated insulin secretion, Biology Open, 10.1242/bio.20147591, 3, 6, 463-474, 2014.06, [URL], We previously reported that phospholipase C-related catalytically inactive protein (PRIP)-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6) cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrinlabeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA -receptor-associated protein (GABARAP), a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms.. |
| 33. | Toshiya Okumura, Kae Harada, Kana Oue, Jun Zhang, Satoshi Asano, Masaki Hayashiuchi, Akiko Mizokami, Hiroto Tanaka, Masahiro Irifune, Nobuyuki Kamata, Masato Hirata, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein (PRIP) regulates lipolysis in adipose tissue by modulating the phosphorylation of hormone-sensitive lipase, PloS one, 10.1371/journal.pone.0100559, 9, 6, 2014.06, [URL], Phosphorylation of hormone-sensitive lipase (HSL) and perilipin by protein kinase A (PKA) promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A), is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO) mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP -KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis. Copyright:. |
| 34. | Kae Harada-Hada, Kana Harada, Fuminori Kato, Junzo Hisatsune, Isei Tanida, Michinaga Ogawa, Satoshi Asano, Motoyuki Sugai, Masato Hirata, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein participates in the autophagic elimination of Staphylococcus aureus infecting mouse embryonic fibroblasts, PloS one, 10.1371/journal.pone.0098285, 9, 5, 2014.05, [URL], Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule- associated protein 1 light chain 3 (LC3), a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP) that was originally identified as an inositol trisphosphatebinding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs). We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells. Copyright:. |
| 35. | Katsuya Morita, Seiji Shiraishi, Naoyo Motoyama, Tomoya Kitayama, Takashi Kanematsu, Yasuhito Uezono, Toshihiro Dohi, Palliation of bone cancer pain by antagonists of platelet-activating factor receptors, PloS one, 10.1371/journal.pone.0091746, 9, 3, 2014.03, [URL], Bone cancer pain is the most severe among cancer pain and is often resistant to current analgesics. Thus, the development of novel analgesics effective at treating bone cancer pain are desired. Platelet-activating factor (PAF) receptor antagonists were recently demonstrated to have effective pain relieving effects on neuropathic pain in several animal models. The present study examined the pain relieving effect of PAF receptor antagonists on bone cancer pain using the femur bone cancer (FBC) model in mice. Animals were injected with osteolytic NCTC2472 cells into the tibia, and subsequently the effects of PAF receptor antagonists on pain behaviors were evaluated. Chemical structurally different type of antagonists, TCV-309, BN 50739 and WEB 2086 ameliorated the allodynia and improved pain behaviors such as guarding behavior and limb-use abnormalities in FBC model mice. The pain relieving effects of these antagonists were achieved with low doses and were long lasting. Blockade of spinal PAF receptors by intrathecal injection of TCV-309 and WEB 2086 or knockdown of the expression of spinal PAF receptor protein by intrathecal transfer of PAF receptor siRNA also produced a pain relieving effect. The amount of an inducible PAF synthesis enzyme, lysophosphatidylcholine acyltransferase 2 (LPCAT2) protein significantly increased in the spinal cord after transplantation of NCTC 2472 tumor cells into mouse tibia. The combination of morphine with PAF receptor antagonists develops marked enhancement of the analgesic effect against bone cancer pain without affecting morphine-induced constipation. Repeated administration of TCV-309 suppressed the appearance of pain behaviors and prolonged survival of FBC mice. The present results suggest that PAF receptor antagonists in combination with, or without, opioids may represent a new strategy for the treatment of persistent bone cancer pain and improve the quality of life of patients. Copyright:. |
| 36. | Hiroki Toyoda, Mitsuru Saito, Hajime Sato, Takuma Tanaka, Takeo Ogawa, Hirofumi Yatani, Tsutomu Kawano, Takashi Kanematsu, Masato Hirata, Youngnam Kang, Enhanced desensitization followed by unusual resensitization in GABAA receptors in phospholipase C-related catalytically inactive protein-1/2 double-knockout mice, Pflugers Archiv European Journal of Physiology, 10.1007/s00424-014-1511-5, 467, 2, 267-284, 2014.01, [URL], Phospholipase C-related catalytically inactive proteins (PRIP-1/2) are previously reported to be involved in the membrane trafficking of GABAA receptor (GABAAR) and the regulation of intracellular Ca2+ stores. GABAAR-mediated currents can be regulated by the intracellular Ca2+. However, in PRIP-1/2 double-knockout (PRIP-DKO) mice, it remains unclear whether the kinetic properties of GABAARs are modulated by the altered regulation of intracellular Ca2+ stores. Here, we investigated whether GABAAR currents (IGABA) evoked by GABA puff in layer 3 (L3) pyramidal cells (PCs) of the barrel cortex are altered in PRIP-DKO mice. The deletion of PRIP-1/2 enhanced the desensitization of IGABA but induced a hump-like tail current (tail-I) at the GABA puff offset. IGABA and the hump-like tail-I were suppressed by GABAAR antagonists. The enhanced desensitization of IGABA and the hump-like tail-I in PRIP-DKO PCs were mediated by increases in the intracellular Ca2+ concentration and were largely abolished by a calcineurin inhibitor and ruthenium red. Calcium imaging revealed that Ca2+-induced Ca2+ release (CICR) and subsequent store-operated Ca2+ entry (SOCE) are more potent in PRIP-DKO PCs than in wild-type PCs. A mathematical model revealed that a slowdown of GABA-unbinding rate and an acceleration of fast desensitization rate by enhancing its GABA concentration dependency are involved in the generation of hump-like tail-Is. These results suggest that in L3 PCs of the barrel cortex in PRIP-DKO mice, the increased calcineurin activity due to the potentiated CICR and SOCE enhances the desensitization of GABAARs and slows the GABA-unbinding rate, resulting in their unusual resensitization following removal of GABA.. |
| 37. | Naoyo Motoyama, Katsuya Morita, Seiji Shiraishi, Tomoya Kitayama, Takashi Kanematsu, Yasuhito Uezono, Toshihiro Dohi, Relief of cancer pain by glycine transporter inhibitors, Anesthesia and Analgesia, 10.1213/ANE.0000000000000388, 119, 4, 988-995, 2014.01, [URL], Background: Recent studies have revealed the antinociceptive effects of glycine transporter (GlyT) inhibitors in neuropathic pain models such as sciatic nerve-injured and diabetic animals. Bone cancer can cause the most severe pain according to complex mechanisms in which a neuropathic element is included. Bone cancer modifies the analgesic action of opioids and limits their effectiveness, and thus novel medicament for bone cancer pain is desired. Methods: For the femur bone cancer model, NCTC 2472 tumor cells were injected into the medullary cavity of the distal femur of C3H/HeN mice. Effects of GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitors, ORG 25935, and knockdown of the expression of spinal GlyTs protein by GlyTs siRNA on pain-like behaviors, such as allodynia, withdrawal threshold, guarding behavior, and limb-use abnormality, were examined in the femur bone cancer model mice. Effects of morphine in combination with GlyT inhibitor were examined. Results: GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitor ORG 25935 by IV or oral administration or knockdown of the expression of spinal GlyTs protein improved painlike behaviors at 11 days after tumor transplantation. The pain-relief activity was potent and long lasting. Morphine at a dose with no analgesic activity combined with ORG 25543 further promoted the ORG 25543-induced pain-relief activity. Injection of ORG 25543 on the second day after tumor implantation caused 3 phases of pain responses; pain-like behaviors were initially accelerated (at 2-4 days) and subsequently almost disappeared (5-7 days) and then reappeared. Intrathecal injection of strychnine 1 day after injection of ORG 25543 transiently antagonized the pain-relief activity of ORG 25543. In control mice, strychnine improved pain-like behaviors 4 days after tumor implantation and aggravated the behaviors between 4 and 5 days. The evidence suggests that the different mechanisms are phase-dependently involved. Conclusions: GlyT inhibitors with or without morphine may be a new strategy for the treatment of bone cancer pain and lead to further investigations of the mechanisms underlying the development of bone cancer pain.. |
| 38. | N. Motoyama, K. Morita, T. Kitayama, S. Shiraishi, Y. Uezono, Fusanori Nishimura, Takashi Kanematsu, Toshihiro Dohi, Pain-releasing action of Platelet-activating factor (PAF) antagonists in neuropathic pain animal models and the mechanisms of action, European Journal of Pain (United Kingdom), 10.1002/j.1532-2149.2013.00289.x, 17, 8, 1156-1167, 2013.09, [URL], Background: Platelet-activating factor (PAF) has been implicated in the pathology of neuropathic pain. Previous studies reported that PAF receptor (PAF-R) antagonists have varied anti-allodynia effects by route of administration and nerve injury models in rats. Methods: The present study elucidated the effectiveness of PAF antagonists against neuropathic pain in four different models of peripheral nerve injury and provided insights into the mode of anti-allodynia action. Results: PAF antagonists, TCV-309, BN 50739 and WEB 2086 by intravenous (i.v.) and oral administration have potent and long-lasting anti-allodynia action in mice neuropathic pain models. Treatment with PAF antagonists before surgery delayed the initiation of allodynia until the effects of these treatments were abolished. Intrathecal (i.t.) injection of the PAF antagonists and siRNA against PAF receptor ameliorated allodynia. I.t. injection of the glycine receptor (GlyR)α3 siRNA reduced the anti-allodynia effect of PAF antagonists. This evidence suggests that the anti-allodynia effect of PAF antagonists is at least in part mediated by spinal relief of PAF-induced dysfunction of GlyRα3. An analysis of the mode of anti-allodynia action of TCV-309 in vivo revealed a competitive action against PAF shortly after the injection of TCV-309, converting to a non-competitive action later. Conclusions: The present results revealed the effectiveness in anti-allodynia of PAF antagonists in different nerve injury models, and the unique mode of action; long-lasting anti-allodynia effects mediated by spinal GlyRa3 with a competitive manner at the initial stage and the following non-competitive manner of inhibition.. |
| 39. | Tomoya Kitayama, Katsuya Morita, Rizia Sultana, Nami Kikushige, Keisuke Mgita, Shinya Ueno, Masato Hirata, Takashi Kanematsu, Phospholipase C-related but catalytically inactive protein modulates pain behavior in a neuropathic pain model in mice, Molecular Pain, 10.1186/1744-8069-9-23, 9, 1, 2013.05, [URL], Background: An inositol 1,4,5-trisphosphate binding protein, comprising 2 isoforms termed PRIP-1 and PRIP-2, was identified as a novel modulator for GABAA receptor trafficking. It has been reported that naive PRIP-1 knockout mice have hyperalgesic responses.Findings: To determine the involvement of PRIP in pain sensation, a hind paw withdrawal test was performed before and after partial sciatic nerve ligation (PSNL) in PRIP-1 and PRIP-2 double knockout (DKO) mice. We found that naive DKO mice exhibited normal pain sensitivity. However, DKO mice that underwent PSNL surgery showed increased ipsilateral paw withdrawal threshold. To further investigate the inverse phenotype in PRIP-1 KO and DKO mice, we produced mice with specific siRNA-mediated knockdown of PRIPs in the spinal cord. Consistent with the phenotypes of KO mice, PRIP-1 knockdown mice showed allodynia, while PRIP double knockdown (DKD) mice with PSNL showed decreased pain-related behavior. This indicates that reduced expression of both PRIPs in the spinal cord induces resistance towards a painful sensation. GABAA receptor subunit expression pattern was similar between PRIP-1 KO and DKO spinal cord, while expression of K+-Cl--cotransporter-2 (KCC2), which controls the balance of neuronal excitation and inhibition, was significantly upregulated in DKO mice. Furthermore, in the DKD PSNL model, an inhibitor-induced KCC2 inhibition exhibited an altered phenotype from painless to painful sensations.Conclusions: Suppressed expression of PRIPs induces an elevated expression of KCC2 in the spinal cord, resulting in inhibition of nociception and amelioration of neuropathic pain in DKO mice.. |
| 40. | Hisanori Umebayashi, Akiko Mizokami, Miho Matsuda, Kae Harada, Hiroshi Takeuchi, Isei Tanida, Masato Hirata, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein, a novel microtubule-associated protein 1 light chain 3-binding protein, negatively regulates autophagosome formation, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2013.01.119, 432, 2, 268-274, 2013.03, [URL], Upon starvation, cells undergo autophagy, an intracellular bulk-degradation process, to provide the required nutrients. Here, we observed that phospholipase C-related catalytically inactive protein (PRIP) binds to microtubule-associated protein 1 light chain 3 (LC3), a mammalian autophagy-related initiator that regulates the autophagy pathway. Then, we examined the involvement of PRIP in the nutrient depletion-induced autophagy pathway. Enhanced colocalization of PRIP with LC3 was clearly seen in nutrient-starved mouse embryonic fibroblasts under a fluorescent microscope, and interaction of the proteins was revealed by immunoprecipitation experiments with an anti-LC3 antibody. Under starvation conditions, there were more green fluorescent protein fused-LC3 dots in mouse embryonic fibroblasts from PRIP-deficient mice than in fibroblasts from wild type cells. The formation of new dots in a single cell increased, as assessed by time-lapse microscopy. Furthermore, the increase in autophagosome formation in PRIP-deficient cells was notably inhibited by exogenously overexpressed PRIP. Taken together, PRIP is a novel LC3-binding protein that acts as a negative modulator of autophagosome formation.. |
| 41. | Goro Sugiyama, Hiroshi Takeuchi, Takashi Kanematsu, Jing Gao, Miho Matsuda, Masato Hirata, Phospholipase C-related but catalytically inactive protein, PRIP as a scaffolding protein for phospho-regulation, Advances in Biological Regulation, 10.1016/j.jbior.2013.07.001, 53, 3, 331-340, 2013.01, [URL], PRIP, phospholipase C (PLC)-related but catalytically inactive protein is a protein with a domain organization similar to PLC-δ1. We have reported that PRIP interacts with the catalytic subunits of protein phosphatase 1 and 2A (PP1c and PP2Ac), depending on the phosphorylation of PRIP. We also found that Akt was precipitated along with PRIP by anti-PRIP antibody from neuronal cells. In this article, we summarize our current reach regarding the interaction of PRIP with Akt and protein phosphatases, in relation to the cellular phospho-regulations. PP1 and PP2A are major members of the protein serine/threonine phosphatase families. We have identified PP1 and PP2A as interacting partners of PRIP. We first investigated the interaction of PRIP with two phosphatases, using purified recombinant proteins. PRIP immobilized on beads pulled-down the catalytic subunits of both PP1 and PP2A, indicating that the interactions were in a direct manner, and the binding of PP1 and PP2A to PRIP were mutually exclusive. Site-directed mutagenesis experiments revealed that the binding sites for PP1 and PP2A on PRIP were not identical, but in close proximity. Phosphorylation of PRIP by protein kinase A (PKA) resulted in the reduced binding of PP1, but not PP2A. Rather, the dissociation of PP1 from PRIP by phosphorylation accompanied the increased binding of PP2A in invitro experiments. This binding regulation of PP1 and PP2A to PRIP by PKA-dependent phosphorylation was also observed in living cells treated with forskolin or isoproterenol. These results suggested that PRIP directly interacts with the catalytic subunits of two distinct phosphatases in a mutually exclusive manner and the interactions are regulated by phosphorylation, thus functioning as a scaffold to regulate the activities and subcellular localizations of both PP1 and PP2A in phospho-dependent cellular signaling.. |
| 42. | Gang Zhu, Shukuko Yoshida, Keisuke Migita, Junko Yamada, Fumiaki Mori, Masahiko Tomiyama, Koichi Wakabayashi, Takashi Kanematsu, Masato Hirata, Sunao Kaneko, Shinya Ueno, Motohiro Okada, Dysfunction of extrasynaptic GABAergic transmission in phospholipase C-related, but catalytically inactive protein 1 knockout mice is associated with an epilepsy phenotype, Journal of Pharmacology and Experimental Therapeutics, 10.1124/jpet.111.182386, 340, 3, 520-528, 2012.03, [URL], Phospholipase C-related, but catalytically inactive protein (PRIP) was first identified as a novel inositol 1,4,5-triphosphate binding protein. The PRIP-1 subtype is expressed predominantly in the central nervous system and binds directly to the GABA type A receptor (GABA A-R) β-subunit and several other proteins involved in the trafficking of GABA A-Rs to the plasma membrane. We found that the PRIP-1 knockout mouse showed an epileptic phenotype, confirmed by electroencephalogram. These ictal seizures were completely suppressed by diazepam (DZP), but the interictal discharges could not be abolished. We studied the electrophysiological properties of GABAergic transmission in hippocampal CA1 pyramidal neurons, using a slice patch-clamp technique. There was no difference in the effect of up to 1 μM DZP on the amplitude and frequency of miniature inhibitory postsynaptic currents between PRIP-1 knockout neurons versus wild-type neurons. In contrast, the amplitude of the tonic GABA current in PRIP-1 knockout neurons was markedly reduced compared with that in wild-type neurons. Consequently, the effect of DZP on PRIP-1 knockout mice was reduced. Dysfunction of extrasynaptic GABAergic transmission probably is involved in the epileptic phenotype of PRIP-1 knockout mice.. |
| 43. | Keisuke Migita, Masahiko Tomiyama, Junko Yamada, Masashi Fukuzawa, Takashi Kanematsu, Masato Hirata, Shinya Ueno, Phenotypes of pain behavior in phospholipase C-related but catalytically inactive protein type 1 knockout mice, Molecular Pain, 10.1186/1744-8069-7-79, 7, 2011.10, [URL], Phospholipase C-related inactive protein (PRIP) plays important roles in trafficking to the plasma membrane of GABAA receptor, which is involved in the dominant inhibitory neurotransmission in the spinal cord and plays an important role in nociceptive transmission. However, the role of PRIP in pain sensation remains unknown. In this study, we investigated the phenotypes of pain behaviors in PRIP type 1 knockout (PRIP-1 -/- ) mice. The mutant mice showed hyperalgesic responses in the second phase of the formalin test and the von Frey test as compared with those in wild-type mice. In situ hybridization studies of GABAA receptors revealed significantly decreased expression of γ2 subunit mRNA in the dorsal and ventral horns of the spinal cord in PRIP-1 -/- mice, but no difference in α1 subunit mRNA expression. β2 subunit mRNA expression was significantly higher in PRIP-1 -/- mice than in wild-type mice in all areas of the spinal cord. On the other hand, the slow decay time constant for the spontaneous inhibitory current was significantly increased by treatment with diazepam in wild-type mice, but not in PRIP-1 -/- mice. These results suggest that PRIP-1 -/- mice exhibit the changes of the function and subunits expression of GABAA receptor in the spinal cord, which may be responsible for abnormal pain sensation in these mice.. |
| 44. | Koshiro Tsutsumi, Miho Matsuda, Miho Kotani, Akiko Mizokami, Ayako Murakami, Ichiro Takahashi, Yoshihiro Terada, Takashi Kanematsu, Kiyoko Fukami, Tadaomi Takenawa, Eijiro Jimi, Masato Hirata, Involvement of PRIP, phospholipase C-related, but catalytically inactive protein, in bone formation, Journal of Biological Chemistry, 10.1074/jbc.M111.235903, 286, 35, 31032-31042, 2011.09, [URL], PRIP (phospholipase C-related, but catalytically inactive protein) is a novel protein isolated in this laboratory. PRIP-deficient mice showed increased serum gonadotropins, but decreased gonadal steroid hormones. This imbalance was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed mutant mice with special reference to the bone property. We first performed three-dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume were higher in mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number were up-regulated in the mutant, indicating that increased bone mass is caused by the enhancement of bone formation ability. We then cultured primary cells isolated from calvaria prepared from both genotypes. In mutant mice, osteoblast differentiation, as assessed by alkaline phosphatase activity and the expression of osteoblast differentiation marker genes, was enhanced. Moreover, we analyzed the phosphorylation of Smad1/5/8 in response to bone morphogenetic protein, with longer phosphorylation in the mutant. These results indicate that PRIP is implicated in the negative regulation of bone formation.. |
| 45. | Akiko Mizokami, Hiroto Tanaka, Hitoshi Ishibashi, Hisanori Umebayashi, Kiyoko Fukami, Tadaomi Takenawa, Keiichi I. Nakayama, Takeshi Yokoyama, Junichi Nabekura, Takashi Kanematsu, Masato Hirata, GABAA receptor subunit alteration-dependent diazepam insensitivity in the cerebellum of phospholipase C-related inactive protein knockout mice, Journal of Neurochemistry, 10.1111/j.1471-4159.2010.06754.x, 114, 1, 302-310, 2010.07, [URL], The GABAA receptor, a pentamer composed predominantly of α, β, and γ subunits, mediates fast inhibitory synaptic transmission. We have previously reported that phospholipase C-related inactive protein (PRIP) is a modulator of GABAA receptor trafficking and that knockout (KO) mice exhibit a diazepam-insensitive phenotype in the hippocampus. The α subunit affects diazepam sensitivity; α1, 2, 3, and 5 subunits assemble with any form of β and the γ2 subunits to produce diazepam-sensitive receptors, whereas α4 or α6/β/γ2 receptors are diazepam-insensitive. Here, we investigated how PRIP is implicated in the diazepam-insensitive phenotype using cerebellar granule cells in animals expressing predominantly the α6 subunit. The expression of α1/β/γ2 diazepam-sensitive receptors was decreased in the PRIP-1 and 2 double KO cerebellum without any change in the total number of benzodiazepine-binding sites as assessed by radioligand-binding assay. Since levels of the α6 subunit were increased, the α1/β/γ2 receptors might be replaced with α6 subunit-containing receptors. Then, we further performed autoradiographic and electrophysiologic analyses. These results suggest that the expression of α6/δ receptors was decreased in cerebellar granule neurons, while that of α6/γ2 receptors was increased. PRIP-1 and 2 double KO mice exhibit a diazepam-insensitive phenotype because of a decrease in diazepam-sensitive (α1/γ2) and increase in diazepam-insensitive (α6/γ2) GABAA receptors in the cerebellar granule cells.. |
| 46. | Kenji Seo, Hiroyuki Seino, Hiroyuki Yoshikawa, Andrey B. Petrenko, Hiroshi Baba, Naoshi Fujiwara, Genji Someya, Yoshiro Kawano, Takeyasu Maeda, Masato Matsuda, Takashi Kanematsu, Masato Hirata, Genetic reduction of GABAA receptor γ2 subunit expression potentiates the immobilizing action of isoflurane, Neuroscience Letters, 10.1016/j.neulet.2010.01.031, 472, 1, 1-4, 2010.03, [URL], Potentiation of inhibitory γ-aminobutyric acid subtype A (GABAA) receptor function is involved in the mechanisms of anesthetic action. The present study examined the immobilizing action of the volatile anesthetic isoflurane in mice with double knockout (DKO) of phospholipase C-related inactive protein (PRIP)-1 and -2. Both of these proteins play important roles in the expression of GABAA receptors containing the γ2 subunit on the neuronal cell surface. Immunohistochemistry for GABAA receptor subunits demonstrated reduced expression of γ2 subunits in the spinal cord of the DKO mice. Immunohistochemistry also revealed up-regulation of the α1 and β3 subunits even though there were no apparent differences in the immunoreactivities for the β2 subunits between wild-type and DKO mice. The tail-clamp method was used to evaluate the anesthetic/immobilizing effect of isoflurane and the minimum alveolar concentration (MAC) was significantly lower in DKO mice compared with wild-type controls (1.07 ± 0.01% versus 1.36 ± 0.04% atm), indicating an increased sensitivity to isoflurane in DKO mice. These immunohistochemical and pharmacological findings suggest that reduced expression of the GABAA receptor γ2 subunit affects the composition and function of spinal GABAA receptors and potentiates the immobilizing action of isoflurane.. |
| 47. | Makoto Fujii, Takashi Kanematsu, Hitoshi Ishibashi, Kiyoko Fukami, Tadaomi Takenawa, Keiichi I. Nakayama, Stephen J. Moss, Junichi Nabekura, Masato Hirata, Phospholipase C-related but catalytically inactive protein is required for insulin-induced cell surface expression of γ-aminobutyric acid type A receptors, Journal of Biological Chemistry, 10.1074/jbc.M109.070045, 285, 7, 4837-4846, 2010.02, [URL], The γ-aminobutyric acid type A (GABAA) receptors play a pivotal role in fast synaptic inhibition in the central nervous system. One of the key factors for determining synaptic strength is the number of receptors on the postsynaptic membrane, which is maintained by the balance between cell surface insertion and endocytosis of the receptors. In this study, we investigated whether phospholipase C-related but catalytically inactive protein (PRIP) is involved in insulin-induced GABAA receptor insertion. Insulin potentiated the GABA-induced Cl- current (IGABA) by about 30% in wild-type neurons, but not in PRIP1 and PRIP2 double-knock-out (DKO) neurons, suggesting that PRIP is involved in insulin-induced potentiation. The phosphorylation level of the GABAA receptor β-subunit was increased by about 30% in the wild-type neurons but not in the mutant neurons, which were similar to the changes observed in IGABA. We also revealed that PRIP recruited active Akt to the GABAA receptors by forming a ternary complex under insulin stimulation. The disruption of the binding between PRIP and the GABAA receptor β-subunit by PRIP interference peptide attenuated the insulin potentiation of IGABA. Taken together, these results suggest that PRIP is involved in insulin-induced GABAA receptor insertion by recruiting active Akt to the receptor complex.. |
| 48. | Katsunori Tomiyama, Liqiu Song, Masayuki Kobayashi, Anthony Kinsella, Takashi Kanematsu, Masato Hirata, Noriaki Koshikawa, John L. Waddington, Orofacial movements in phospholipase C-related catalytically inactive protein-1/2 double knockout mice Effect of the GABAergic agent diazepam and the D1 dopamine receptor agonist SKF 83959, Synapse, 10.1002/syn.20798, 64, 9, 714-720, 2010.01, [URL], Orofacial movements are regulated by D1-like dopamine receptors interacting with additional mechanisms. Phospholipase C-related catalytically inactive protein (PRIP) regulates cell surface expression of GABAA receptors containing a γ2 subunit. Mutant mice with double knockout of PRIP-1 and PRIP-2 were used to investigate aspects of GABAergic regulation of orofacial movements and interactions with D1 mechanisms. Vertical jaw movements, tongue protrusions and movements of the head and vibrissae were reduced in PRIP-1/2 double knockouts. The GABAAergic agent diazepam reduced movements of the head and vibrissae; these effects were unaltered in PRIP-1/2 double knockouts. The D1-like agonist SKF 83959 induced vertical jaw movements, incisor chattering, and movements of the head and vibrissae that were unaltered in PRIP-1/2 double knockouts. However, SKF 83959-induced tongue protrusions were reduced in PRIP-1/2 double knockouts. PRIP-mediated regulation of GABAAergic receptor mechanisms influences topographically distinct aspects of orofacial movement and interacts with D1 receptor systems.. |
| 49. | Takashi Kanematsu, Makoto Fujii, Hiroto Tanaka, Hisanori Umebayashi, Masato Hirata, Surface expression of gabaA receptors Roles for the binding partners, journal of oral biosciences, 10.2330/joralbiosci.52.322, 52, 4, 322-329, 2010.01, [URL], The transmission of sensory information to the central nervous system during oral functions such as mastication and swallowing is well regulated by neural networks, including 7gamma;-aminobutyric acid (GABA)-mediated inhibitory interneurons. Therefore, an understanding of the mechanism of GABAergic transmission might provide new insight into theclinical treatment of eating and swallowing disorders. Type A GABA receptors are ligand-gated Cl- channels composed pentamerically of two a(1-6), two y3(1-3), and one γ(1-3) subunit. One major issue regarding the efficacy of GABAergic transmissionis how to control receptor numbers on neuronal surfaces, determining synaptic strength. The number of receptors on the postsynaptic membrane is maintained by a balance between receptor insertion and endocytosis. Insulin increases the number of GABAA receptors by stimulating insertion into the surface membrane via Akt-mediated phosphorylation of the subunits. Conversely, brain-derived neurotrophic factor decreases the number by stimulating endocytosis. Here we show that PRIP (phospholipase C-related but catalytically inactive protein), isolated in our laboratory, actively participates in these two events by regulating the phosphorylation of/J subunits and controlling the functions of binding partners such as Akt and protein phosphatases.. |
| 50. | Miho Matsuda, Koushirou Tsutsumi, Takashi Kanematsu, Kiyoko Fukami, Yoshihiro Terada, Tadaomi Takenawa, Keiichi I. Nakayama, Masato Hirata, Involvement of phospholipase C-related inactive protein in the mouse reproductive system through the regulation of gonadotropin levels, Biology of Reproduction, 10.1095/biolreprod.109.076760, 81, 4, 681-689, 2009.12, [URL], Phospholipase C-related but catalytically inactive protein (comprising PRIP-1 and PRIP-2 [officially designated PLCL1 and PLCL2]) was first identified in our laboratory, but the biological functions have remained elusive. Therefore, we generated Plcll and Plcl2 double-knockout mice (Plcl1 tm1Mh; Plcl2tm1Tta) to gain insight into the biological function. Double-knockout mice apparently grew normally and became fertile; however, during animal maintenance, we noticed that mutant couples exhibited decreased litter events and litter size, indicating dysfunction of the reproductive system. Cross-mating experiments to discrim-inate whether males or females were defective indicated that the cause appeared to be on the female side. Mutant female mice had an apparently smaller uterus by gross anatomical observation and had more estrous days during the cycles. Levels of serum luteinizing hormone (LH) and follicle-stimulating hormone were measured for 5-6 consecutive days and were significantly higher in the mutant, which was also confirmed by examining the secretion of LH from the explant culture of anterior pituitary glands of wild-type and double-knockout mice. These results suggest that through gonadotropin secretion, PRIP plays an important role in female reproduction.. |
| 51. | Jing Gao, Hiroshi Takeuchi, Zhao Zhang, Makoto Fujii, Takashi Kanematsu, Masato Hirata, Binding of phospholipase C-related but catalytically inactive protein to phosphatidylinositol 4,5-bisphosphate via the PH domain, Cellular Signalling, 10.1016/j.cellsig.2009.03.008, 21, 7, 1180-1186, 2009.07, [URL], A well-known protein module regulating molecular interactions is the pleckstrin homology (PH) domain whose best-characterised ligand is phosphoinositide. In the present study, we analysed the PH domain from PRIP (phospholipase C-related but catalytically inactive protein, comprising types 1 and 2) regarding phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] binding employing a variety of binding assays. The PH domains prepared from PRIP-1 and -2 showed similar binding profiles to soluble ligands in vitro and showed similar plasma membrane localisation to that of PLC-δ1; however, the PH domain with the N-terminal extension of PRIP-1 but not PRIP-2 showed even distribution throughout the cytoplasm, indicating that the N-terminal extension of PRIP-1 inhibited binding to PtdIns(4,5)P2 present in the plasma membrane. A chimeric molecule of PLC-δ1 PH domain with the N-terminal extension of PRIP-1 exhibited similar localisation to PRIP-1 PH domain with the N-terminal extension. Binding assay to liposomes containing various concentrations of PtdIns(4,5)P2 revealed that the PH domain of PLC-δ1 bound steeply to the maximum, even at a concentration of 1.2 mol%, whereas the PH domains from PRIP-1 and -2 bound depending on the concentration up to 5 mol%. We also performed binding experiments using saponin-permeabilised PC12 cells. PH domains from PRIP increased the binding to cells preincubated with the brain cytosol extract in the presence of ATP, during which PtdIns(4,5)P2 were probably synthesised. The binding of PH domain with the following EF hand motifs showed Ca2+-dependent binding. These results indicate that the PH domain of PRIP binds to PtdIns(4,5)P2 present in the plasma membrane, depending on the concentrations of the lipid ligand and Ca2+, suggesting that PRIP might play physiological roles in events involved in the changes of these parameters, probably including Ins(1,4,5)P3.. |
| 52. | Takashi Kanematsu, Akiko Mizokami, Keiko Watanabe, Masato Hirata, Regulation of GABAA-receptor surface expression with special reference to the involvement of GABARAP (GABAA receptor-associated protein) and PRIP (phospholipase C-related, but catalytically inactive protein), Journal of Pharmacological Sciences, 10.1254/jphs.CP0070063, 104, 4, 285-292, 2007.09, [URL], GABAA receptors are heteropentameric ligand-gated chloride channels composed of a variety of subunits, including α1 - 6, β1 - 3, γ1 - 3, δ, ε, θ, and π, and play a key role in controlling inhibitory neuronal activity. Modification of the efficacy of the synaptic strength is produced by changes in both the number of neuronal surface receptors and pentameric molecular assembly, leading to differences of sensitivity to neurotransmitters and neuromimetic drugs. Therefore, it is important to understand the molecular mechanisms regulating the so-called "life cycle of GABAA receptors" including sequential pentameric assembly at the site synthesized, intracellular transport through the Golgi apparatus and the cytoplasm, insertion into the cell membrane, functional modulation at the cell surface, and finally internalization, followed by either recycling back to the surface membrane or lysosomal degradation. This review is focused on events related to the surface expression of the receptor containing the γ2 subunit and clathrin /AP2 complex-mediated phospho-regulated endocytosis of the receptor, with special reference to the function of novel GABAA receptor modulators, GABARAP (GABAA receptor-associated protein) and PRIP (phospholipase C-related, but catalytically inactive protein).. |
| 53. | Takashi Kanematsu, Makoto Fujii, Akiko Mizokami, Josef T. Kittler, Junichi Nabekura, Stephen J. Moss, Masato Hirata, Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABAA receptors mediated by clathrin and AP2 adaptor complex, Journal of Neurochemistry, 10.1111/j.1471-4159.2006.04399.x, 101, 4, 898-905, 2007.05, [URL], A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABAA receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABAA receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABAA receptors through association with receptor β subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABAA receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABAA receptor α/β/γ subunits and PRIP. PRIP was internalized together with GABAA receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to β subunits. The clathrin heavy chain, μ2 and β2 subunits of AP2 and PRIP-1, were complexed with GABAA receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-β2/3 GABAA receptor antibody or by pull-down assay using β subunits of GABAA receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABAA receptor that requires clathrin and AP2 protein complex.. |
| 54. | Hiroaki Wake, Miho Watanabe, Andrew J. Moorhouse, Takashi Kanematsu, Shoko Horibe, Noriyuki Matsukawa, Kiyofumi Asai, Kosei Ojika, Masato Hirata, Junichi Nabekura, Early changes in KCC2 phosphorylation in response to neuronal stress result in functional downregulation, Journal of Neuroscience, 10.1523/JNEUROSCI.3104-06.2007, 27, 7, 1642-1650, 2007.02, [URL], The K+ Cl- cotransporter KCC2 plays an important role in chloride homeostasis and in neuronal responses mediated by ionotropic GABA and glycine receptors. The expression levels of KCC2 in neurons determine whether neurotransmitter responses are inhibitory or excitatory. KCC2 expression is decreased in developing neurons, as well as in response to various models of neuronal injury and epilepsy. We investigated whether there is also direct modulation of KCC2 activity by changes in phosphorylation during such neuronal stressors. We examined tyrosine phosphorylation of KCC2 in rat hippocampal neurons under different conditions of in vitro neuronal stress and the functional consequences of changes in tyrosine phosphorylation. Oxidative stress (H2O2) and the induction of seizure activity (BDNF) and hyperexcitability (0 Mg2+) resulted in a rapid dephosphorylation of KCC2 that preceded the decreases in KCC2 protein or mRNA expression. Dephosphorylation of KCC2 is correlated with a reduction of transport activity and a decrease in [Cl-]i, as well as a reduction in KCC2 surface expression. Manipulation of KCC2 tyrosine phosphorylation resulted in altered neuronal viability in response to in vitro oxidative stress. During continued neuronal stress, a second phase of functional KCC2 downregulation occurs that corresponds to decreases in KCC2 protein expression levels. We propose that neuronal stress induces a rapid loss of tyrosine phosphorylation of KCC2 that results in translocation of the protein and functional loss of transport activity. Additional understanding of the mechanisms involved may provide means for manipulating the extent of irreversible injury resulting from different neuronal stressors.. |
| 55. | Akiko Mizokami, Takashi Kanematsu, Hitoshi Ishibashi, Taku Yamaguchi, Isei Tanida, Kei Takenaka, Keiichi I. Nakayama, Kiyoko Fukami, Tadaomi Takenawa, Eiki Kominami, Stephen J. Moss, Tsuneyuki Yamamoto, Junichi Nabekura, Masato Hirata, Phosholipase C-related inactive protein is involved in trafficking of γ2 subunit-containing GABAA receptors to the cell surface, Journal of Neuroscience, 10.1523/JNEUROSCI.3155-06.2007, 27, 7, 1692-1701, 2007.02, [URL], The subunit composition of GABAA receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of γ2 subunit-containing GABAA receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABAA receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of γ2 subunit-containing GABAA receptors. The surface numbers of diazepam binding sites (α/γ2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (α/β subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABAA receptors and GABAA receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABAA receptor β subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of γ2 subunit-containing GABAA receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of γ2 subunit-containing GABAA receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.. |
| 56. | Takashi Kanematsu, Akiko Mizokami, Miho Terunuma, Hiroshi Takeuchi, Masato Hirata, Identification of a Novel Signaling Molecule and Elucidation of Its Cellular Functions —Development of an Interface between Neuroscience and Oral Health Science—, journal of oral biosciences, 10.2330/joralbiosci.49.244, 49, 4, 244-258, 2007.01, [URL], The investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogs has led to the isolation of a novel protein with a molecular size of 130 kDa, characterized as a molecule with a domain organization similar to phospholipase C (PLC)-δ1 but lacking enzymatic activity. Two isoforms of the molecule were subsequently identified, the molecule has been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and-2. Regarding its ability to bind Ins (1,4,5)-P3 via the pleckstrin homology domain, the involvement of PRIP-1 in Ins(1,4,5)P3-mediated Ca2+ signaling was first examined. Yeast two-hybrid screening of a brain cDNA library identified GABARAP (GABAA receptor-associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible neurological involvement of PRIP, particularly in GABAA receptor signaling. PRIP-1 and-2 double knock-out (DKO) mice were analyzed for GABAA receptor function with special reference to the action of benzodiazepines whose target is the γ subunit of the receptors; sensitivity to benzodiazepine was reduced, as assessed by biochemical, electrophysiological, and behavioral analyses of DKO mice, suggesting the dysfunction of γ2 subunit-containing GABAA receptors. The mesencephalic trigeminal nucleus, which mediates perceptions from periodontal mechanoreceptors and jaw-closer muscle spindles, receives many synaptic inputs, including those from GABAA receptors, indicating that PRIP might indirectly be involved in rhythmical jaw movement. In the present article, we summarize our current research and the functional significance of PRIP.. |
| 57. | Akiko Mizokami, Takashi Kanematsu, Masato Hirata, Roles of PRIP in GABAA Receptor Signaling, journal of oral biosciences, 10.2330/joralbiosci.49.105, 49, 2, 105-112, 2007.01, [URL], GABAA receptors are a family of ligand-gated ion channels that are pentamer composed pre-dominantly of α, β and γ subunits. They are the major target of the endogenous inhibitory neurotransmitter (GABA, γ-aminobutyric acid) and maintain the majority of fast inhibitory ion currents in the central nervous system, in addition to being drug targets for benzodiazepines, barbiturates, alcohols, neurosteroids, and some anesthetics. Moreover, modifications in GABAA receptor function are crucial in central nervous system diseases such as anxiety disorders, sleep disturbances, and seizure disorders. Therefore it is very important to understand the molecular mechanisms underlying the maintenance of functional inhibitory synapses including GABAA receptors. We have first isolated PRIP (phospholipase C-related, but catalytically inactive protein) as a novel inositol 1,4,5-trisphosphate binding protein, and subsequently found GABARAP (GABAA receptor associated protein) as one of binding partners that binds to γ2 subunit of the receptor and thus is implicated in the clustering and trafficking of the receptor to synaptic membrane. Further studies revealed that PRIP binds β subunit of the receptors and PP1c (catalytic subunit of protein phosphatase 1). These findings have prompted us to explore the possible involvement of PRIP in modulation of GABAA receptor signaling. Here we summarize our current understanding regarding how PRIP is involved in the modification of GABAA receptor signaling on the basis of the characteristics of these interacting molecules.. |
| 58. | Takashi Kanematsu, Atsushi Yasunaga, Yoshito Mizoguchi, Akiko Kuratani, Josef T. Kittler, Jasmina N. Jovanovic, Kei Takenaka, Keiichi I. Nakayama, Kiyoko Fukami, Tadaomi Takenawa, Stephen J. Moss, Junichi Nabekura, Masato Hirata, Modulation of GABAA receptor phosphorylation and membrane trafficking by phospholipase C-related inactive protein/protein phosphatase 1 and 2A signaling complex underlying brain-derived neurotrophic factor-dependent regulation of GABAergic inhibition, Journal of Biological Chemistry, 10.1074/jbc.M603118200, 281, 31, 22180-22189, 2006.08, [URL], Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABAA receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABAA receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABAA receptor β3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABAA receptors. We demonstrate that PRIP associates with phosphatases as well as with β subunits. PRIP was found to colocalize with GABAA receptor clusters in cultured neurons and with recombinant GABAA receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to β subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABAA receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABAA receptors by mediating the specific association between β subunits of these receptors with protein phosphatases.. |
| 59. | Isei Tanida, Mika Wakabayashi, Takashi Kanematsu, Naoko Minematsu-Ikeguchi, Yu Shin Sou, Masato Hirata, Takashi Ueno, Eiki Kominami, Lysosomal turnover of GABARAP-phospholipid conjugate is activated during differentiation of C2C12 cells to myotubes without inactivation of the mTor kinase-signaling pathway, Autophagy, 10.4161/auto.2871, 2, 4, 264-271, 2006.01, [URL], Although conjugation of overexpressed GABARP to phospholipid has been reported during starvation-induced autophagy, it is unclear whether endogenous GABARAP-phospholipid conjugation is also activated under starvation conditions. We observed little accumulation of GABARAP-phospholipid conjugate (GABARAP-PL) in mouse liver and kidney under starvation conditions, whereas endogenous LC3-phospholipid conjugate (LC3-II) accumulated. A small amount of endogenous GABARAP-PL was observed in the heart, independent of starvation. In rapamycin-treated HEK293 cells, there was little accumulation of endogenous GABARAP-PL, even in the presence of lysosomal protease-inhibitors, whereas there was significant accumulation of endogenous LC3-II, together with inactivation of the mTor kinase-signaling pathway. In HeLa and C2C12 cells, GABARAP-PL accumulation in the presence of lysosomal protease inhibitors was independent of starvation-induced autophagy, whereas LC3-II accumulation was significant during starvation-induced autophagy. Interestingly, we observed activation of lysosomal turnover of GABARAP-PL during the differentiation of C2C12 cells to myotubes, along with increased lysosomal turnover of LC3-II. Under these conditions, S6 ribosomal protein was still phosphorylated, suggesting that the mTor kinase-signaling pathway is active during the differentiation of C2C12 cells to myotubes, in contrast to starvation-induced autophagy. These results indicated that lysosomal turnover of GABARAP-PL was activated during the differentiation of C2C12 cells to myotubes without inactivation of the mTor kinase-signaling pathway, whereas little lysosomal turnover of GABARAP-PL was activated during starvation-induced autophagy.. |
| 60. | Satoko Yanagihori, Miho Terunuma, Kiyoshi Koyano, Takashi Kanematsu, Sung Ho Ryu, Masato Hirata, Protein phosphatase regulation by PRIP, a PLC-related catalytically inactive protein-Implications in the phospho-modulation of the GABAA receptor, Advances in Enzyme Regulation, 10.1016/j.advenzreg.2006.01.006, 46, 1, 203-222, 2006, [URL], PRIP, phospholipase C related, but catalytically inactive protein was first identified as a novel inositol 1,4,5-trisphosphate binding protein. It has a number of binding partners including protein phosphatase (PP1 and 2A), GABAA receptor associated protein, and the β subunits of GABAA receptors, in addition to inositol 1,4,5-trisphosphate. The identification of these molecules led us to examine the possible involvement of PRIP in the phospho-regulation of the β subunits of GABAA receptors using hippocampal neurons prepared from PRIP-1 and 2 double knock-out (DKO) mice. Experiments were performed with special reference to the dephosphorylation processes of the β subunits. The phosphorylation of β3 subunits by the activation of protein kinase A in cortical neurons of the control mice continued for up to 5 min, even after washing out of the stimulus, followed by a gradual dephosphorylation. That of DKO mice gradually increased in spite of the lower phosphorylation levels induced by the stimulation. There was little difference in the amount of cellular cyclic AMP and protein kinase A activity between the control and mutant mice, indicating that phosphatases such as PP1 and PP2A are primarily involved in the difference. The time course of PP1 activity changes in the vicinity of the receptors in control mice corresponded to the phosphorylation of PRIP, while that of the mutant mice decreased with the period of the incubation. This is a good agreement with the suggestion that PRIP binds to and inactivates PP1, which is regulated by the phosphorylation of PRIP at threonine 94. These results suggest that PRIP plays an important role in controlling the dynamics of GABAA receptor phosphorylation by through PP1 binding and, therefore, the efficacy of synaptic inhibition mediated by these receptors.. |
| 61. | Takashi Kanematsu, Hiroshi Takeuchi, Miho Terunuma, Masato Hirata, PRIP, a novel Ins(1,4,5)P3 binding protein, functional significance in Ca2+ signaling and extension to neuroscience and beyond, Molecules and cells, 20, 3, 305-314, 2005.12, Investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogs has led to the isolation of a novel binding protein with a molecular size of 130 kDa, characterized as a molecule with similar domain organization to phospholipase C-δ1 (PLC-δ1) but lacking the enzymatic activity. An isoform of the molecule was subsequently identified, and these molecules have been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and -2. Regarding its ability to bind Ins(1,4,5)P3 via the pleckstrin homology domain, the involvement of PRIP-1 in Ins(1,4,5)P3-mediated Ca2+ signaling was examined using COS-1 cells overexpressing PRIP-1 and cultured neurons prepared from PRIP-1 knockout mice. Yeast two hybrid screening of a brain cDNA library using a unique N-terminus as bait identified GABARAP (GABAA receptor associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible involvement of PRIP in GABAA receptor signaling. For this purpose PRIP knock-out mice were analyzed for GABAA receptor function in relation to the action of benzodiazepines from the electrophysiological and behavioral aspects. During the course of these experiments we found that PRIP also binds to the β-subunit of GABAA receptors and PP2A (protein phosphtase 2A). Here, we summarize how PRIP is involved in Ins(1,4,5)P3-mediated Ca2+ signaling and GABAA receptor signaling based on the characteristics of binding molecules.. |
| 62. | Ryosuke Inoue, Nori Aki Matsuki, Jing Gao, Takashi Kanematsu, Kihachiro Abe, Masato Hirata, The inhibitory effect of alendronate, a nitrogen-containing bisphosphonate on the PI3K-Akt-NFκB pathway in osteosarcoma cells, British Journal of Pharmacology, 10.1038/sj.bjp.0706373, 146, 5, 633-641, 2005.11, [URL], 1. Bisphosphonates are inhibitors of tumor cell growth as well as of bone resorption by inducing cell apoptosis. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. The aim of the present study was to determine the effect of alendronate, one of the nitrogen-containing bisphosphonates on the phoshoinositide 3-kinase (PI3K)-Akt-NFκB pathway, the major cell survival pathway. 2. The PI3K-Akt-NFκB pathway was activated in the osteosarcoma cell line MG-63 treated with tumor necrosis factor-α or insulin. Saos-2 was also used in some experiments. This was assessed by the production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3), increased PI3K activity, phosphorylation of Akt at serine 473 and threonine 308, increase in activity of the inhibitor of nuclear factor κB (IκB) kinase (IKK) and finally phosphorylation of IκB and its subsequent degradation. 3. Pretreatment with alendronate at 100 μM for 24 h prior to the stimulation with tumor necrosis factor-α or insulin partially inhibited the IκB phosphorylation and degradation. These events were more clearly observed in the presence of inhibitors of proteasomes, which are responsible for the degradation of IκB. The drug also partially inhibited the activity of IKK, but almost fully inhibited the phosphorylation of Akt and the production of PtdIns(3,4,5)P 3. 4. The inhibitory effect of alendronate on IκB phosphorylation and degradation was not attenuated by the exogenous addition of geranylgeraniol to replenish the cytosolic isoprenyl lipid substrate. 5. The present findings demonstrate that alendronate inhibited the PI3K-Akt-NFκB cell survival pathway at the point of PI3K activation, thus indicating the presence of new targets of alendronate.. |
| 63. | Hidefumi Goto, Miho Terunuma, Takashi Kanematsu, Yoshio Misumi, Stephen J. Moss, Masato Hirata, Direct interaction of N-ethylmaleimide-sensitive factor with GABA A receptor β subunits, Molecular and Cellular Neuroscience, 10.1016/j.mcn.2005.07.006, 30, 2, 197-206, 2005.10, [URL], GABAA receptors mediate most of the fast inhibitory neurotransmission in the brain, and are believed to be composed mainly of α, β, and γ subunits. It has been shown that GABAA receptors interact with a number of binding partners that act to regulate both receptor function and cell surface stability. Here, we reveal that GABA A receptors interact directly with N-ethylmaleimide-sensitive factor (NSF), a critical regulator of vesicular dependent protein trafficking, as measured by in vitro protein binding and co-immunoprecipitation assays. In addition, we established that NSF interacts with residues 395-415 of the receptor β subunits and co-localizes with GABAA receptors in hippocampal neurons. We also established that NSF can regulate GABAA receptor cell surface expression depending upon residues 395-415 in the β3 subunit. Together, our results suggest an important role for NSF activity in regulating the cell surface stability of GABAA receptors.. |
| 64. | Takahide Kouno, Mineyuki Mizuguchi, Isei Tanidal, Takashi Uenol, Takashi Kanematsu, Yoshihiro Mori, Hiroyuki Shinoda, Masato Hirata, Eiki Kominami, Keiichi Kawano, Solution structure of microtubule-associated protein light chain 3 and identification of its functional subdomains, Journal of Biological Chemistry, 10.1074/jbc.M413565200, 280, 26, 24610-24617, 2005.07, [URL], Microtubule-associated protein (MAP) light chain 3 (LC3) is a human homologue of yeast Apg8/Aut7/Cvt5 (Atg8), which is essential for autophagy. MAP-LC3 is cleaved by a cysteine protease to produce LC3-I, which is located in cytosolic fraction. LC3-I, in turn, is converted to LC3-II through the actions of E1- and E2-like enzymes. LC3-II is covalently attached to phosphatidylethanolamine on its C terminus, and it binds tightly to autophagosome membranes. We determined the solution structure of LC3-I and found that it is divided into N- and C-terminal subdomains. Additional analysis using a photochemically induced dynamic nuclear polarization technique also showed that the N-terminal subdomain of LC3-I makes contact with the surface of the C-terminal subdomain and that LC3-I adopts a single compact conformation in solution. Moreover, the addition of dodecylphosphocholine into the LC3-I solution induced chemical shift perturbations primarily in the C-terminal subdomain, which implies that the two subdomains have different sensitivities to dodecylphosphocholine micelles. On the other hand, deletion of the N-terminal subdomain abolished binding of tubulin and microtubules. Thus, we showed that two subdomains of the LC3-I structure have distinct functions, suggesting that MAP-LC3 can act as an adaptor protein between microtubules and autophagosomes.. |
| 65. | Kae Harada, Hiroshi Takeuchi, Masahiro Oike, Miho Matsuda, Takashi Kanematsu, Hitoshi Yagisawa, Kei Ichi I. Nakayama, Katsumasa Maeda, Christophe Erneux, Masato Hirata, Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated Ca2+ signaling, Journal of cellular physiology, 10.1002/jcp.20136, 202, 2, 422-433, 2005.02, [URL], PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-δ1 (PLC-δ1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1 -/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1 -/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,S)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1 PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P 3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.. |
| 66. | Taku Yamaguchi, Takashi Kubota, Takashi Kanematsu, Keiichi Nakayama, Masato Hirata, Tsuneyuki Yamamoto, Hypersensitivity to pentylenetetrazol-induced convulsion in mice lacking the PLC-related inactive protein-1, Brain Research, 10.1016/j.brainres.2004.08.009, 1025, 1-2, 237-240, 2004.10, [URL], In the present study, we investigated the effects of pentylenetetrazol (PTZ), a chemical convulsant that interacts with the GABA A receptor, in mice lacking the phospholipase C (PLC)-related inactive protein-1 (PRIP-1). PRIP-1 knockout mice did not develop spontaneous behavioral seizure. PRIP-1 knockout mice had markedly shorter latencies until the first clonic convulsion (CL) and tonic extensor (TE) following PTZ administration and increased incidence of convulsion compared to those in wild-type mice. Furthermore, the mortality rate by PTZ in mice lacking the PRIP-1 was also significantly increased in comparison with that in wild-type mice. These findings suggested that mice lacking the PRIP-1 were hypersensitive to PTZ-induced convulsion, and PRIP-1 might play roles in suppressing excessive excitability via interactions with the GABA A receptor.. |
| 67. | Miho Terunuma, Il Sung Jang, Sang Hoon Ha, Josef T. Kittler, Takashi Kanematsu, Jasmina N. Jovanovic, Keiichi I. Nakayama, Norio Akaike, Sung Ho Ryu, Stephen J. Moss, Masato Hirata, GABAA receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase 1 anchoring protein, Journal of Neuroscience, 10.1523/JNEUROSCI.1323-04.2004, 24, 32, 7074-7084, 2004.08, [URL], GABAA receptors are critical in controlling neuronal activity. Here, we examined the role for phospholipase C-related inactive protein type 1 (PRIP-1), which binds and inactivates protein phosphatase 1α (PP1α) in facilitating GABAA receptor phospho-dependent regulation using PRIP-1-/- mice. In wild-type animals, robust phosphorylation and functional modulation of GABAA receptors containing β3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP-1-/- mice. PRIP-1-/- mice exhibited enhanced PP1α activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABAA receptor β subunits, and moreover, these proteins were found to be PP1α substrates. Finally, phosphorylation of PRIP-1 on threonine 94 facilitated the dissociation of PP1α-PRIP-1 complexes, providing a local mechanism for the activation of PP1α. Together, these results suggest an essential role for PRIP-1 in controlling GABAA receptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors.. |
| 68. | Takashi Kanematsu, Miho Terunuma, Hidefumi Goto, Akiko Kuratani, Masato Hirata, The life cycle of the GABAA receptor and its regulating molecules, Folia Pharmacologica Japonica, 10.1254/fpj.123.105, 123, 2, 105-112, 2004.02, [URL], γ-Aminobutyric acidA (GABAA) receptors mediate most of the fast inhibitory neurotransmission in the central nervous system. These ligand-gated ion channels are crucial in the control of cell and network activity. Therefore, modulating their function or cell surface stability will have major consequences for neuronal excitation. This review highlights recent findings on the regulation of GABAA-receptor expression and function, focusing on the mechanisms of sorting, targeting, synaptic clustering, and endocytic events of GABAA receptors, all which are regulated by their associated proteins. Now these topics are an area of active interest in studies on inhibitory neurotransmission.. |
| 69. | Yoshito Mizoguchi, Takashi Kanematsu, Masato Hirata, Junichi Nabekura, A Rapid Increase in the Total Number of Cell Surface Functional GABA A Receptors Induced by Brain-derived Neurotrophic Factor in Rat Visual Cortex, Journal of Biological Chemistry, 10.1074/jbc.M305872200, 278, 45, 44097-44102, 2003.11, [URL], The number of postsynaptic γ-aminobutyric acid type A (GABA A) receptors is a fundamental determinant of the variability of inhibitory synaptic responses in the central nervous system. In rat visual cortex, [3H] SR-95531 binding assays revealed that brain-derived neurotrophic factor (BDNF), one of the neurotrophins, induced a rapid increase in the total number of cell surface GABAA receptors, through the activation of Trk B receptor tyrosine kinases. We also demonstrated that BDNF rapidly induced a sustained potentiation of GABAA receptor-mediated currents, using nystatin-perforated patch clamp recordings, in visual cortical layer 5 pyramidal neurons freshly isolated from P14 rats. The potentiation was caused by the activation of Trk B receptor tyrosine kinase and phospholipase C-γ. In addition, intracellular Ca2+ was important for the potentiation of GABAA responses induced by BDNF. The selective increase in mean miniature inhibitory postsynaptic (mIPSC) current amplitude without effects on mIPSC time courses supports the idea that BDNF rapidly induces an increase in the total number of cell surface functional GABA A receptors in visual cortical pyramidal neurons. These results suggest that BDNF could alter the number of cell surface GABAA receptors in a region-specific manner.. |
| 70. | Kiyoshi Hidaka, Takashi Kanematsu, James J. Caffrey, Hiroshi Takeuchi, Stephen B. Shears, Masato Hirata, The importance to chondrocyte differentiation of changes in expression of the multiple inositol polyphosphate phosphatase, Experimental Cell Research, 10.1016/S0014-4827(03)00337-9, 290, 2, 254-264, 2003.11, [URL], It is important to both physiological and pathological osteogenesis to understand the significance of changes in gene expression in growth-plate chondrocytes that transit between the proliferative and hypertrophic states. MINPP is one such gene of interest. The Minpp protein dephosphorylates highly phosphorylated inositol signaling molecules InsP5 and InsP 6. We show here that the ATDC5 chondrocyte progenitor cell line can recapitulate developmentally specific changes in MINPP expression previously only seen in longitudinal bone growth plates - both an initial 2-3-fold increase and a subsequent decrease back to initial levels during transition to hypertrophy. The increase in MINPP expression was accompanied by a 40% decrease in InsP6 levels in ATDC5 cells. However, InsP5 levels were not modified. Furthermore, throughout the hypertrophic phase, during which MINPP expression decreased, there were no alterations in InsP5 and InsP6 levels. We also created an ATDC5 line that stably overexpressed Minpp at 2-fold higher levels than in wild-type cells. This had no significant effect upon cellular levels of InsP5 and InsP 6. Thus, substantial changes in MINPP expression can occur without a net effect upon InsP5 and InsP6 turnover in vivo. On the other hand, Minpp-overexpressing cells showed impaired chondrogenesis. We noted that the expression of alkaline phosphatase activity was inversely correlated with the expression of MINPP. The ATDC5 cells that overexpress Minpp failed to show an insulin-dependent increase in alkaline phosphatase levels, which presumably affects phosphate balance [J. Biol. Chem. 276 (2001) 33995], and may be the reason cellular differentiation was impaired. In any case, we conclude that Minpp is important to chondrocyte differentiation, but in a manner that is, surprisingly, independent of inositol polyphosphate turnover.. |
| 71. | Takashi Kanematsu, Masato Hirata, PRIP-1 involved in GABAA receptor trafficking, Seikagaku. The Journal of Japanese Biochemical Society, 75, 5, 378-382, 2003.01. |
| 72. | Ayako Uji, Miho Matsuda, Toshio Kukita, Katsumasa Maeda, Takashi Kanematsu, Masato Hirata, Molecules interacting with PRIP-2, a novel Ins(1,4,5)P3 binding protein type 2 Comparison with PRIP-1, Life Sciences, 10.1016/S0024-3205(02)02275-0, 72, 4-5, 443-453, 2002.12, [URL], A family of phospholipase C-related, catalytically inactive proteins (designated PRIP) have been identified as a group of novel inositol 1,4,5-trisphosphate binding proteins with a domain organization similar to phospholipase C-δ but lacking the enzymatic activity. The PRIP family consists of at least two types of proteins (PRIP-1 and PRIP-2 subfamilies). In the present study, we examined the tissue distribution of PRIP-2, its expression in rat brain at the mRNA level, and the characteristics of its binding to inositol compounds, protein phosphatase 1, and γ-amino butyric acid receptor associated protein. We also compared these characteristics with those of PRIP-1. Northern blot analysis and reverse-transcription polymerase chain reaction showed that PRIP-1 was present mainly in the brain, whereas PRIP-2 was expressed ubiquitously. In situ hybridization studies using rat brain revealed that the mRNA for both PRIP-1 and PRIP-2 was similarly expressed; it was detected in the granular cell and Purkinje cell layers in the cerebellum, and in the hippocampal pyramidal cells, dentate granule cells, and pyramidal and/or granule cells of the cerebral cortex in the cerebrum. PRIP-2 bound inositol 1,4,5-trisphosphate and its parent lipid, phosphatidylinositol 4,5-bisphosphate, with a similar affinity, while PRIP-1 preferentially bound the former ligand by about 10-fold. PRIP-1 and PRIP-2 interacted with protein phosphatase 1 and γ-amino butyric acid receptor associated protein in a similar manner. These results indicate that, similar to PRIP-1, PRIP-2 may be involved in both inositol 1,4,5-trisphosphate-mediated and γ-amino butyric acid-related signaling.. |
| 73. | Ferry Sandra, Nori Aki Matsuki, Hiroshi Takeuchi, Tetsuro Ikebe, Takashi Kanematsu, Masamichi Ohishi, Masato Hirata, TNF inhibited the apoptosis by activation of Akt serine/threonine kinase in the human head and neck squamous cell carcinoma, Cellular Signalling, 10.1016/S0898-6568(02)00025-6, 14, 9, 771-778, 2002.06, [URL], Tumour necrosis factor (TNF) is known to induce apoptosis, but recently, TNF was shown to promote cell survival, a process regulated by phosphatidylinositol-3-OH kinase (PI3K) and the NFκB pathway. In this study, we investigated the relationship between the molecules implicated in regulating TNF-induced cell survival and apoptosis induced by TNF in a human head and neck squamous cell carcinoma cell line (SAS), with special reference to the Akt pathway, one of the pathways related to cell survival. In SAS cells, TNF induced the phosphorylation of Akt at both Ser473 and Thr308, causing the activation of Akt, and also induced the phosphorylation and degradation of IκB (inhibitor of NFκB). This phosphorylation and degradation was inhibited by pretreating the cells with the PI3K inhibitors, wortmannin or LY294002. The apoptosis of SAS cells induced by TNF was dependent on the concentration: a high concentration of TNF, but not a low concentration, induced apoptosis within 30 h. However, a low concentration of TNF in the presence of wortmannin or LY294002 induced apoptosis. Furthermore, expression of the kinase-negative form of Akt, IKKα or IKKβ, and the undegradable mutant of IκB, also induced apoptosis at low concentrations of TNF. When the SAS cells expressed constitutively activated Akt, apoptosis was not induced, even by high concentrations of TNF. These observations suggest that, in the SAS cell line, the PI3K-NFκB pathway contributes to TNF-induced cell survival and that inhibition of this pathway accelerates apoptosis.. |
| 74. | Takashi Kanematsu, Masato Hirata, The analysis of protein-protein interaction with special reference to PRIP-1, Folia Pharmacologica Japonica, 10.1254/fpj.119.241, 119, 4, 241-246, 2002.05, [URL], Analysis of protein-protein interaction is one of the powerful methods for elucidating the new functions of functionally unknown proteins. Using this approach, we isolated two proteins interacting with PRIP-1, which was isolated as a new Ins (1,4,5) P3 binding protein from brain. One was protein phosphatase 1 catalytic subunit (PP1c) and the other was GABARAP (GABAA-receptor-associated protein). The region of PRIP-1 responsible for their interaction was the site preceding to the pleckstrin homology domain of PRIP-1 for PP1c and the EF-hand motifs of PRIP-1 for GABARAP, which were determined by β-galactosidase assay of yeast two-hybrid system. The association between PRIP-1 and PP1c was confirmed in vitro by a pull-down assay, a far-western assay, an immunoprecipitation analysis and a surface plasmon resonance analysis. The interaction of PRIP-1 with PP1c resulted in inhibition of the catalytic activity of PP1c in a PRIP-1 concentration-dependent manner. The association between PRIP-1 and GABARAP was also confirmed by a pull-down assay, and we found that PRIP-1 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Our electrophysiological and behavioral analysis of PRIP-1 knockout mice revealed that PRIP-1 is essential for the function of GABAA receptors, especially in response to the agents acting on the γ2 subunit.. |
| 75. | Takashi Kanematsu, Il Sung Jang, Taku Yamaguchi, Hiroyasu Nagahama, Kenji Yoshimura, Kiyoshi Hidaka, Miho Matsuda, Hiroshi Takeuchi, Yoshio Misumi, Keiko Nakayama, Tsuneyuki Yamamoto, Norio Akaike, Masato Hirata, Keiichi Nakayama, Role of the PLC-related, catalytically inactive protein p130 in GABAA receptor function, EMBO Journal, 10.1093/emboj/21.5.1004, 21, 5, 1004-1011, 2002.03, [URL], The protein p130 was isolated from rat brain as an inositol 1, 4, 5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for γ-aminobutyric acid (GABA). Yeast two-hybrid screening identified GABARAP (GABAA receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABAA receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl-current by Zn2+ or diazepam, both of which act at GABAA receptors containing γ subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABAA receptors, especially in response to the agents acting on a γ2 subunit.. |
| 76. | F. Sandra, N. Nakamura, Takashi Kanematsu, Masato Hirata, M. Ohishi, The role of MDM2 in the proliferative activity of ameloblastoma, Oral Oncology, 10.1016/S1368-8375(01)00036-7, 38, 2, 153-157, 2002.03, [URL], Ameloblastoma is a unique tumor in the oral and maxillofacial region with various levels of proliferative activity in each type. p53 is most commonly found to be mutated in human cancer and sometimes is overexpressed also in other lesions, such as ameloblastoma. Murine Double Minute 2 (MDM2) is able to physically associate with the p53 tumor suppressor and therefore block the growth suppressive functions of p53. In the present study, immunohistochemistry, western blotting and enzyme-linked immunosorbent assay for p53 mutant selective test were done. MDM2 was overexpressed in ameloblastoma and the results showed different numbers of MDM2 labeling index based on both WHO classification and cytological pattern of outer layer cells. Basal ameloblastoma, which has a high proliferative activity, had the highest MDM2 labeling index. We suggest MDM2 protein caused the high proliferative activity of ameloblastoma, especially in basal cell ameloblastoma.. |
| 77. | Takahide Kouno, Kazunori Miura, Takashi Kanematsu, Masahiro Shirakawa, Masato Hirata, Keiichi Kawano, Letter to the editor 1H, 13C and 15N resonance assignments of GABARAP, GABAA receptor associated protein [4], Journal of Biomolecular NMR, 10.1023/A:1013884402033, 22, 1, 97-98, 2002.02, [URL]. |
| 78. | Kiyoshi Hidaka, Takashi Kanematsu, Hiroshi Takeuchi, Minoru Nakata, Ushio Kikkawa, Masato Hirata, Involvement of the phosphoinositide 3-kinase/protein kinase B signaling pathway in insulin/IGF-I-induced chondrogenesis of the mouse embryonal carcinoma-derived cell line ATDC5, International Journal of Biochemistry and Cell Biology, 10.1016/S1357-2725(01)00067-X, 33, 11, 1094-1103, 2001.09, [URL], The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin/insulin-like growth factor-I (IGF-I) stimulation. In the present study, we examined whether insulin/IGF-I stimulation caused activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway in ATDC5 cells. We also determined whether the insulin-stimulated differentiation of ATDC5 cells into chondrocytes could be mimicked by activation of the PKB pathway alone. ATDC5 cells produced phosphatidylinositol 3,4,5-trisphosphate and the pleckstrin homology domain of PKB was recruited to the plasma membrane in response to insulin stimulation. This was probably a result of activation of PI3K because the PI3K inhibitors, wortmannin and LY294002, inhibited both responses, although the effective concentrations were as high as 10 μM. Insulin stimulation caused the chondrogenic differentiation of ATDC5 cells as assessed by chondrogenic nodule staining with alcian blue. The addition of wortmannin or LY294002, PI3K inhibitors, suppressed the staining, and the suppression was reversible, indicating the effect of the inhibitors is not toxic. Finally, we exogenously expressed a constitutively-activated from of PKB (myristoylated PKB, myr-PKB) in ATDC5 cells, and found the chondrogenic differentiation of ATDC5 cells to form nodules occurred in the absence of insulin stimulation. The kinase-negative mutant of myr-PKB did not caused differentiation, indicating that kinase activity is required. These results support the hypothesis that the PI3K/PKB signaling pathway is involved in the chondrogenic differentiation of ATDC5 cells in response to insulin/IGF-I stimulation. This is the first report that demonstrates the involvement of phosphoinositide signaling in the induction of chondrogenesis from undifferentiated cells.. |
| 79. | Matsuki N., Takeuchi H., Oike M., Lia J., Sandra F., Ohishi M., Kanematsu T. & Hirata M., Role of the pleckstrin homology domain of PLC-related catalytically inactive protein, p130 in Ca2+ signaling., Curr. Top. Biochem., 4, 117~127, 2001.01. |
| 80. | Naoko Doira, Takashi Kanematsu, Miho Matsuda, Hiroshi Takeuchi, Hito O. Nakano, Yushi Ito, Keiko Nakayama, Kei Ichi Nakayama, Masato Hirata, Hyperinsulinemia in PRIP-1 gene deleted mice, Biomedical Research, 10.2220/biomedres.22.157, 22, 3, 157-165, 2001.01, [URL], The protein p130, named from its molecular size, was originally identified as an inositol 1, 4,5-trisphosphate binding protein similar to phospholipase C (PLC)-δ1, but lacking any PLC activity. It was recently renamed PLC-related catalytically inactive protein-1 (PRIP-1). In the present study, PRIP-1 gene-targeted mice were analyzed for glycogen metabolism based on the previous finding that PRIP-1 interacts with protein phosphatase-1 (26). Compared to the control mice, mutant mice exhibited lower phosphorylase activity and higher levels of glycogen in the liver, which appeared to be consistent with the inhibition of protein phosphatase-1 by PRIP-1. These observation could also be attributed to the increased levels of plasma insulin. Hyperinsulinemia, observed even in the young mice, was progressive with aging, and was accompanied by the accumulation of fat tissues, increased body weight and decreased sensitivity to insulin in the mutant mice at the age of 12 months. These results suggest that PRIP-1 is a molecule involved in the control of plasma insulin.. |
| 81. | Kenji Yoshimura, Hiroshi Takeuchi, Osamu Sato, Kiyoshi Hidaka, Naoko Doira, Miho Terunuma, Kae Harada, Yasuo Ogawa, Yushi Ito, Takashi Kanematsu, Masato Hirata, Interaction of p130 with, and Consequent Inhibition of, the Catalytic Subunit of Protein Phosphatase 1α, Journal of Biological Chemistry, 10.1074/jbc.M009677200, 276, 21, 17908-17913, 2001.01, [URL], The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1α (PP1cα) as a p130-binding protein. The association between p130 and PP1cα was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1cα resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1cα also detected the presence of a complex of p130 and PP1cα. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1cα, was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca2+ signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1cα.. |
| 82. | Hiroshi Takeuchi, Masahiro Oike, Hugh F. Paterson, Victoria Allen, Takashi Kanematsu, Yushi Ito, Christophe Erneux, Matilda Katan, Masato Hirata, Inhibition of Ca2+ signalling by p130, a phospholipase-C-related catalytically inactive protein Critical role of the p130 pleckstrin homology domain, Biochemical Journal, 10.1042/0264-6021:3490357, 349, 1, 357-368, 2000.07, [URL], p130 was originally identified as an Ins(1,4,5)P3-binding protein similar to phospholipase C-δ but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P3-mediated Ca2+ signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P3 with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P3. Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca2+ signalling. When fura-2-loaded COS-1(p130) cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca2+ concentration, observed in control cells, was inhibited in COS-1(p130). This inhibition was not accompanied by the decreased production of Ins(1,4,5)P3; the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P3 could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P3-mediated Ca2+ signalling.. |
| 83. | Takashi Kanematsu, Kenji Yoshimura, Kiyoshi Hidaka, Hiroshi Takeuchi, Matilda Katan, Masato Hirata, Domain organization of p130, PLC-related catalytically inactive protein, and structural basis for the lack of enzyme activity, European Journal of Biochemistry, 10.1046/j.1432-1327.2000.01291.x, 267, 9, 2731-2737, 2000.05, [URL], The 130-kDa protein (p130) was isolated as a novel inositol 1,4,5- trisphosphate [Ins(1,4,5)P3)-binding protein similar to phospholipase C-δ1 (PLC-δ1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. and Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525, Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. and Hirata, M. (1996) Biochem, J. 313, 319-3251. To test experimentally the domain organization of p130 and structural basis for lack of PLC activity, we subjected p130 to limited proteolysis and also constructed a number of chimeras with PLC-δ1. Trypsin treatment of p130 produced four major polypeptides with molecular misses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were CalCulated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC- δ1 and demonstrating that all domains of p130, including the unique region at the C-terminus, are stable, tightly folded structures. p130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that p130 has intrinsically no PLC activity. A number of chimeric molecules between p130 and PLC-δ1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two proteins, as only some domains of p130 could replace the corresponding structures in PLC-δ1 to form a functional enzyme. These results suggest that p130 and the related proteins could represent a new protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.. |
| 84. | Takashi Kanematsu, Masato Hirata, Novel Ins(1,4,5)P3 binding protein and GABA-A receptor signaling, Fukuoka igaku zasshi = Hukuoka acta medica, 91, 7, 159-164, 2000.01. |
| 85. | Takashi Kanematsu, Hiroshi Takeuchi, Masato Hirata, Studies on new ins(1,4,5)P3 binding proteins with reference to the pH domains, ACS Symposium Series, 718, 55-78, 1999.12, Two inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding proteins, having molecular masses of 85 kDa and 130 kDa, were purified from rat brain using an Ins(1,4,5)P3 affinity column; the former protein was found to be the δ1-isozyme of phospholipase C (PLC-δ1) and the latter protein was an unidentified novel protein. Subsequent molecular biology studies isolated the full-length cDNA for the 130 kDa-Ins(1,4,5)P3 binding protein (p130) which encoded 1,096 amino acids. The predicted amino acid sequence of p 130 had 38.2% homology to that of PLC-δ1. The region of p130 responsible for InS(1,4,5)P3 binding was mapped in a pleckstrin homology (PH) domain of the molecule. The PH domain of p130 could also bind to Ins(1,4,5,6)P4 with a similar affinity to Ins(1,4,5)P3. The 85 kDa protein (PLC-δ1) was also analyzed for the binding site by molecular biological, peptide synthetic chemical and immunological studies: the sequence 30-43 of PLC-δ1 was primarily involved in binding, which was mapped in the N-terminal of the PH domain of the molecule. Experiments as to the effect of Ins(1,4,5)P3 on PLC-δ1 activity showed that Ins(1,4,5)P3 at concentrations over 1 μM strongly inhibited PLC activity of PLC-δ1. The PH domains derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and p130, were analyzed for the capability and specificity of binding of inositol phosphates and derivatives of inositol lipids. We concluded from these studies that inositol phosphates and/or inositol lipids might be common ligands for the PH domains, and therefore inositol phosphates/inositol lipids might be involved in more aspects of cellular functions than originally thought, because more than 90 proteins to date are known to include PH domain. Which ligands are physiologically relevant for the PH domain, would depend on binding affinities and their cellular abundance.. |
| 86. | Tada Aki Yamamoto, Hiroshi Takeuchi, Takashi Kanematsu, Victoria Allen, Hitoshi Yagisawa, Ushio Kikkawa, Yutaka Watanabe, Akihiko Nakasima, Matilda Katan, Masato Hirata, Involvement of EF hand motifs in the Ca2+-dependent binding of the pleckstrin homology domain to phosphoinositides, European Journal of Biochemistry, 10.1046/j.1432-1327.1999.00786.x, 265, 1, 481-490, 1999.10, [URL], The pleckstrin homology (PH) domains of phospholipase C (PLC)-δ1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by PLC-δ1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca2+-dependent activation of PLC-δ1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-δ1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to Ptdlns(4,5)P2 is regulated by changes in free Ca2+ concentration within the physiological range. A Ca2+ dependent increase in the binding to PtdIns(4,5)P2 was observed with a full-length PLC-δ1, while the isolated PH domain did not show any Ca2+ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-δ1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca2+ dependent increase in the binding to PtdIns(4,5)P2. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P2 showed no Ca2+ dependent enhancement of binding. However, when present within a chimera also containing PLC-δ1 EF-hand motifs, the Ca2+ dependent binding was again observed. These results suggest that the binding of Ca2+ to the EF-hand motifs can modulate binding to PtdIns(4,5)P2 mediated by the PH domain.. |
| 87. | Nori Aki Matsuki, Kayoko Tateishi, Hiroshi Takeuchi, Hitoshi Yagisawa, Takashi Kanematsu, Masamichi Oishi, Masato Hirata, Antibodies against the PH domain of phospholipase C-δ1 inhibit Ins(1,4,5)P3-Mediated Ca2+ release from the endoplasmic reticulum, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1999.0869, 260, 1, 42-47, 1999.06, [URL], The pleckstrin homology domain (PH domain) is now well known as a structural module for the binding of inositol compounds. In the present study, polyclonal antibodies against the peptide KVKSSSWRRERFYK, derived from the N-terminal of the PH domain of phospholipase C-δ1 (PLC-δ1), were raised in rabbits. These were then tested for their ability to inhibit the binding of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to the binding proteins including the receptor molecule. The Fab fragment of the antibodies but not the whole molecule inhibited the binding of Ins(1,4,5)P3 not only to PLC-δ1 but also to the Ins(1,4,5)P3 receptor, indicating that the antibodies raised recognized the binding site for Ins(1,4,5)P3 in the receptor. Rat basophilic leukemic cells were permeabilized with saponin and assayed for Ins(1,4,5)P3mediated Ca2+ release. Pretreatment of permeabilized RBL cells with the Fab fragment of the antibodies diminished the release of Ca2+ caused by Ins(1,4,5)P3, and further absorption experiments using a variety of synthetic peptides suggested that the tripeptide KVK is the epitope of the antibodies. Structural information about KVK will help in screening for Ins(1,4,5)P3 antagonists.. |
| 88. | Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Masato Hirata, Membrane association of a new inositol 1,4,5-trisphosphate binding protein, p130 is not dependent on the pleckstrin homology domain, Chemistry and Physics of Lipids, 10.1016/S0009-3084(99)00016-X, 98, 1-2, 35-47, 1999.04, [URL], The 130-kDa protein was isolated as a novel inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding protein from rat brain and was molecularly cloned to be found similar to phospholipase C-δ1 (Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. and Hirata, M., 1992. Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol, J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. and Hirata, M., 1996. A new inositol 1,4,5-trisphosphate binding protein similar to phospholipase C-δ1, Biochem. J. 313, 319-325). The 130-kDa protein and its deleted protein expressed in COS-1 cells were seen in both the membrane and the cytosol fractions. Truncation of 232 residues from the N-terminus, the protein molecule lacking the pleckstrin homology (PH) domain was also localized in the membrane fraction as much as seen with a full-length protein and other deleted proteins, thereby indicating that the PH domain is not primarily involved in the membrane localization. The addition of Mg2+ to homogenates of COS-1 cells caused the translocation of expressed proteins from the cytosol to the membrane fraction, yet further addition of AlF4- which induced the activation of GTP binding proteins did not cause a further translocation. The protein translocated to the membrane by the addition of Mg2+ was hardly extracted with Triton X-100. The inclusion of Ins(1,4,5)P3 or phosphatidylinositol 4,5-bisphosphate in cell homogenates caused the very small reduction in the amounts of membrane-associated proteins expressed by some constructs. These results indicate that (i) the PH domain is not primarily involved in the membrane localization of the 130-kDa protein, (ii) the activation of GTP binding protein does not appear to cause the translocation of the 130-kDa protein, and (iii) intrinsic phosphatidylinositol 4,5-bisphosphate present in the membrane appears to be involved in the membrane association of the 130-kDa protein to a very small extent, probably through the binding site in the PH domain. Copyright (C) 1999 Elsevier Science Ireland Ltd.. |
| 89. | Xin H.B., Rogers K., Qi Y., Kanematsu T. & Fleischer S., Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine., Journal of Biological Chemistry , 274, 15315~15319, 1999.01. |
| 90. | Masato Hirata, Masako Yoshida, Takashi Kanematsu, Hiroshi Takeuchi, Intrinsic inhibitor of inositol 1,4,5-trisphosphate binding, Molecular and Cellular Biochemistry, 190, 1-2, 179-184, 1999, Rat brain cytosol was applied to a heparin column and eluted with 0.9 M-NaCl. The total binding activity of [3H] inositol 1,4,5-trisphosphate to the eluate was increased about 6-fold compared with the original cytosol. When the eluate was mixed with a flow-through fraction from the heparin column, however, the activity returned to the original lever, suggesting that the flow-through fraction contained an inhibitory factor(s) which prevented the binding. The factor(s) was purified by sequential column chromatography using gel permeation, a hydrophobic gel, and finally, a hydroxylapatite gel. Silver staining of sodium dedecyl sulfate gel electrphoresis of the sample thus purified showed a broad band located between the authentic molecular weight markers of 580 and 390 k. A carbohydrate staining method showed that the factor is a glycoprotein.. |
| 91. | Miho Matsuda, Takashi Kanematsu, Hiroshi Takeuchi, Toshio Kukita, Masato Hirata, Localization of a novel inositol 1,4,5-trisphosphate binding protein, p130 in rat brain, Neuroscience Letters, 10.1016/S0304-3940(98)00810-6, 257, 2, 97-100, 1998.11, [URL], We have isolated a novel inositol 1,4,5-trisphosphate binding protein with molecular mass of 130 kDa (p130), homologous to phospholipase C-δ1 in amino acid sequence but with no catalytic activity. Here we report the expression and localization of p130 at the mRNA level in rat brain. Northern blotting showed that gene expression encoding p130 was most abundant in brain. Brain localization of p130-mRNA using an in situ hybridization technique revealed that in the cerebellum, the mRNA was detected in the granular cell and Purkinje cell layers, and cerebellar nuclei. In the cerebrum, the mRNA was localized in hippocampal pyramidal cells, dentate granule cells and pyramidal and/or granule cells of the cerebral cortex. The brain localization of p130-mRNA was similar to that of the β-subtype of phospholipase C, indicating that p130 may be mainly involved in phospholipase Cβ-mediated signaling.. |
| 92. | Hiroshi Takeuchi, Miho Matsuda, Tada Aki Yamamoto, Takashi Kanematsu, Ushio Kikkawa, Hitoshi Yagisawa, Yutaka Watanabe, Masato Hirata, PTB domain of insulin receptor substrate-1 binds inositol compounds, Biochemical Journal, 10.1042/bj3340211, 334, 1, 211-218, 1998.08, [URL], We examined whether a phosphotyrosine binding (PTB) domain from the human insulin receptor substrate-1 (hIRS-1) is capable of binding inositol phosphates/phosphoinositides. The binding specificity was compared with that of the pleckstrin homology (PH) domain derived from the same protein because the three dimensional structure was found to be very similar to that of the PH domain, despite the lack of sequence similarity. We also attempted to locate the site of binding of the inositol compounds. The PTB domain bound [3H]Ins(1,4,5)P3, which was displaced most strongly by Ins(1,3,4,5,6)P5 and InsP6, indicating that these inositol polyphosphates show the highest affinity. The PTB domain bound to liposomes containing PtdIns(4,5)P2, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, but not phosphatidylinositol. In contrast, the PH domain showed a preference for Ins(1,4,5)P3, the polar head of PtdIns(4,5)P2. Site-directed mutagenesis studies were performed to map the binding site for inositol phosphates in the PTB domain. Mutation of K169Q, K171Q or K177Q, located in the loop connecting the β1 and β2 strands, which is partially responsible for binding inositol phosphates/phosphoinositides in the PH domains of several other proteins, reduced binding activity, probably because of a reduction in affinity. Mutation of R212Q or R227Q, shown to be involved in the binding of a phosphotyrosine, had little effect on the binding capacity. These results indicate that the PTB domain of hIRS-1 can bind inositol phosphates/phosphoinositides. Therefore signalling through the PTB domain could be regulated by the binding not only of proteins with phosphotyrosine but also of inositol phosphates/phosphoinositides, implying that PTB domains could be involved in a myriad of interconnections between intracellular signalling pathways.. |
| 93. | Masato Hirata, Takashi Kanematsu, Hiroshi Takeuchi, Hitoshi Yagisawa, Pleckstrin homology domain as an inositol compound binding module, Japanese Journal of Pharmacology, 10.1254/jjp.76.255, 76, 3, 255-263, 1998.03, [URL], Many of the proteins that participate in cell signalling contain structural modules involved in regulatory interactions between components of signal transduction cascades. One of such modules is the pleckstrin homology (PH) domain, a region of approximately 120 amino acids that can form an electrostatically polarized tertiary structure. Several molecules such as inositol 1,4,5-trisphosphate/phosphatidylinositol 4,5-bisphosphate, the βγ-subunits of heterotrimeric G proteins and protein kinase C have been proposed as common ligands for the PH domain. Through these potential interactions, the PH domain has been proposed to play a role in membrane recruitment of proteins containing the PH domain, thus targeting them to appropriate cellular compartment or enabling them to interact with other components of the signal transduction pathway. In this review, we mainly focus on membrane targeting through the binding to inositol phosphates/phosphoinositides.. |
| 94. | Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Fumio Sakane, Hiroaki Konishi, Ushio Kikkawa, Yutaka Watanabe, Matilda Katan, Masato Hirata, Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein, Biochimica et Biophysica Acta - Molecular Cell Research, 10.1016/S0167-4889(97)00109-2, 1359, 3, 275-285, 1997.12, [URL], The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domain from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6- tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6- tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphonoisitide derivative. However, all PH domains had similar affinity inositol 1,3,4,5- tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important lipids could be important ligands for the PH domain, and therefore inositol phosphate/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.. |
| 95. | H. Takeuchi, Miho Matsuda, Takashi Kanematsu, M. Hirata, Both PTB domain and PH domain of IRS-1 bind inositol polyphosphates with distinct specificity, FASEB Journal, 11, 9, 1997, Studies in the laboratory using the recombinant pleckstrin homology (PH) domains from several proteins have confirmed our proposal that inositol phosphates/inositol lipids would be physiologically relevant ligands for the PH domain. In this study, we examined whether a phospho-tyrosine binding (PTB) domain is capable of binding specific inositol phosphates, and compared the specificity with that seen for the PH domain originated from the same protein, human insulin receptor substrate (IRS)-l. The PTB domain, the 3D structure of which is similar to that of PH domain, bound D[3H]Ins(l,4,5)P3 and the binding was displaced by Hns(l,4,5)P3 and oIns(l,3,4)P3 to the lesser extent than that by D-[ns(l,4,5)P3, indicating the specific binding. D-Ins(l,3,4,5)P4 and D-Ins(l,3,4,5,6)P5 as well as Ins?6 had the same affinity as D-Ins(l,4,5)P3. In contrast, the PH domain showed the specific preference to u-Ins(l,4,5)P3, the polar head of phosphatidylinositol 4,5-bisphosphate (PIP2). Therefore, IRS-1 is possible to be recruited for the function to the plasma membrane, in which PlPa is present, by the binding via the PH domain. On the other hand, the binding of the PTB domain to PIP2 in the plasma membrane or phosphotyrosine on the receptor molecules or adaptor molecules may be inhibited by the binding to D-Ins(l,3,4,5,6)P5 as well as InsPe which are the most abundant inositol compounds in cells.. |
| 96. | Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hassan Bin Yaakob, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B. Shears, Masato Hirata, Localization of a high-affinity inositol 1,4,5-trisphosphate/inositol 1,4,5,6-tetrakisphosphate binding domain to the pleckstrin homology module of a new 130 KDa protein Characterization of the determinants of structural specificity, Biochemical Journal, 10.1042/bj3180561, 318, 2, 561-568, 1996.09, [URL], We have previously identified a novel 130 kDa protein (p130) which binds Ins(1,4,5)P3 and shares 38% sequence identity with phospholipase C-δ1 [Kanematsu, Misumi, Watanabe, Ozaki, Koga, Iwanaga, Ikehara and Hirata (1996) Biochem. J. 313, 319-325]. We have now transfected COS-1 cells with genes encoding the entire length of the molecule or one of several truncated mutants, in order to locate the region for binding of Ins(1,4,5)P3. Deletion of N-terminal residues 116-232, the region which corresponds to the pleckstrin homology (PH) domain of the molecule, completely abolished binding activity. This result was confirmed when the PH domain itself (residues 95-232), isolated from a bacterial expression system, was found to bind [3H]Ins(1,4,5)P3. We also found that Ins(1,4,5,6)P4 was as efficacious as Ins(1,4,5)P3 in displacing [3H]Ins(1,4,5)P3, suggesting that these two polyphosphates bind to p130 with similar affinity. This conclusion was confirmed by direct binding studies using [3H]Ins(1,4,5,6)P4 with high specific radioactivity which we prepared ourselves. Binding specificity was also examined with a variety of inositol phosphate derivatives. As is the case with other PH domains characterized to date, we found that the 4,5-vicinal phosphate pair was an essential determinant of ligand specificity. However, the PH domain of p130 exhibited some novel features. For example, the 3- and/or 6-phosphates could also contribute to overall binding; this contrasts with some other PH domains where these phosphate groups decrease ligand affinity by imposing a steric constraint. Secondly, a free monoester 1-phosphate substantially increased binding affinity, which is a situation so far unique to the PH domain of p130.. |
| 97. | Takashi Kanematsu, Yoshio Misumi, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, Sadaaki Iwanaga, Yukio Ikehara, Masato Hirata, A new inositol 1,4,5-trisphosphate binding protein similar to phospholipase C-δ1, Biochemical Journal, 10.1042/bj3130319, 313, 1, 319-325, 1996.01, [URL], We have reported that two inositol 1,4,5-trisphosphate binding proteins, with molecular masses of 85 and 130 kDa, were purified from rat brain; the former protein was found to be the δ1-isoenzyme of phospholipase C (PLC-δ1) and the latter was an unidentified novel protein [Kanematsu, Takeya, Watanabe, Ozaki, Yoshida, Koga, Iwanaga and Hirata (1992) J. Biol. Chem. 267, 6518-6525]. Here we describe the isolation of the full-length cDNA for the 130 kDa Ins(1,4,5)P3 binding protein, which encodes 1096 amino acids. The predicted sequence of the 130 kDa protein had 38.2% homology to that of PLC-δ1. Three known domains of PLC-δ1 (pleckstrin homology and putative catalytic X and Y domains) were located at residues 110-222, 377-544 and 585-804 with 35.2%, 48.2% and 45.8% homologies respectively. However, the protein showed no PLC activity to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol. The 130 kDa protein expressed by transfection in COS-1 cells bound Ins(1,4,5)P3 in the same way as the molecule purified from brain. Thus the 130 kDa protein is a novel Ins(1,4,5)P3 binding protein homologous to PLC-δ1, but with no catalytic activity. The functional significance of the 130 kDa protein is discussed.. |
| 98. | Masato Hirata, Yutaka Watanabe, Takashi Kanematsu, Shoichiro Ozaki, Toshitaka Koga, d-myo-Inositol 1,4,5-trisphosphate analogues substituted at the 3-hydroxyl group, BBA - General Subjects, 10.1016/0304-4165(95)00043-B, 1244, 2-3, 404-410, 1995.06, [URL], d-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogues derived at 3-OH with a bulky substituent were chemically synthesized and structural features of vicinity surrounding the 3-OH of Ins(1,4,5)P3, recognized by metabolic enzymes and by the receptor were explored. 3-Benzoyl-, 3-methylbenzoyl- and 3-para-aminobenzoyl-Ins(1,4,5)P3 inhibited the dephosphorylation of [3H]Ins(1,4,5)P3 by the 5-phosphatase present in erythrocyte ghosts, but the potency varied. The inhibitory potency for the former two compounds was slightly lower than that for Ins(1,4,5)P3, while that for the latter compound was higher. Transfer of the amino group to the meta-position of the benzoyl group led to a less potent analogue. In an assay of [3H]Ins(1,4,5)P3 3-kinase at a low Ca2+ concentration, catalyzed by rat brain cytosol, 3-meta-aminobenzoyl-Ins(1,4,5)P3 was the most potent among compounds examined, including Ins(1,4,5)P3 in inhibiting the phosphorylation, whereas both 3-benzoyl- and 3-methylbenzoyl-Ins(1,4,5)P3 at concentrations up to 30 μM, were without effect. All analogues examined were effective in inhibiting [3H]Ins(1,4,5)P3 binding to purified Ins(1,4,5)P3 receptor, but all 3-derived analogues were less potent and 3-benzoyl-Ins(1,4,5)P3 was the least potent. It would thus appear that the space in the vicinity surrounding the 3-hydroxyl group of Ins(1,4,5)P3 is sterically restrictive with regard to recognition by metabolic enzymes and the receptor, whereas the amino group providing arms for either the electrostatic interaction or the hydrogen bond, makes the analogues more potent.. |
| 99. | Masako Yoshida, Takashi Kanematsu, Yutaka Watanabe, Toshitaka Koga, Shoichiro Ozaki, Sadaaki Iwanaga, Masato Hirata, D-myo-inositol 1,4,5-trisphosphate-binding proteins in rat brain membranes, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a124447, 115, 5, 973-980, 1994.01, [URL], Rat brain membrane fractions obtained using Triton X-100 were applied to a D-myo-inositol 1,4,5-trisphosphate [D-Ins(1,4,5)P3] immobilized column, followed by gel filtration and anion-exchange chromatography. Two proteins with molecular masses of 130 and 85 kDa, as assessed by SDS-polyacrylamide gel electrophoresis, were purified to apparent homogeneity as D-[3H] Ins(1,4,5)P3-binding proteins with no D-Ins(1,4,5)P3-metabolizing activity. Partial amino acid sequence determinations of these proteins revealed that the 130 kDa protein appears to be a new D-Ins(1,4,5)P3-binding protein and the 85 kDa protein is δ1-isozyme of phospholipase C. We have previously purified 130 and 85 kDa proteins, as D-[3H]Ins(1, 4, 5)P3-binding proteins, from rat brain cytosol fraction. Antibodies against the 130 kDa protein from the cytosol cross-reacted with the membrane 130 kDa protein purified in this study, suggesting that the membrane 130 kDa protein is likely to be the same as the protein from the cytosol fraction. The inhibition of D-[3H]Ins(1,4,5)P3 binding by D-isomers of inositol phosphates available clarified that the 130 kDa protein has a similar affinity for D-Ins(1,4,5,6)P4 to that for D-Ins(1,4,5)P3, while the 85 kDa protein is specific to D-Ins(1,4,5)P3.. |
| 100. | Hitoshi Yagisawa, Masato Hirata, Takashi Kanematsu, Yutaka Watanabe, Shoichiro Ozaki, Kaori Sakuma, Hideko Tanaka, Norikazu Yabuta, Hideaki Kamata, Hajime Hirata, Hiroshi Nojima, Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-δ1, Journal of Biological Chemistry, 269, 31, 20179-20188, 1994.08, It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilized matrix was the δ1 isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-δ1 in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P3 binding. The lysate was applied to Ins(1,4,5)P3-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P3 binding. The lysate was also purified to near homogeneity using a glutathione- Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-δ1 consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P3 binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-δ1 which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P3 binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-δ1, which contains 6 basic amino acids, binds to an Ins(1,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P3 binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-δ1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.. |
| 101. | M. Hirata, N. Narumoto, Y. Watanabe, T. Kanematsu, T. Koga, S. Ozaki, DL-myo-inositol 1,2,4,5-tetrakisphosphate, a potent analog of D-myo- inositol 1,4,5-trisphosphate, Molecular Pharmacology, 45, 2, 271-276, 1994.03, Synthetic DL-myo-inositol 1,2,4,5-tetrakisphosphate (DL-Ins-(1,2,4,5)P4) functioned as a full agonist, with only 3-fold less potency than D- Ins(1,4,5)P3 in eliciting the release of Ca2+ from nonmitochondrial pools of permeabilized rat basophilic leukemic cells. DL-Ins(1,2,4,5)P4 inhibited the binding of D-[3H]Ins(1,4,5)P3 to the purified D-Ins(1,4,5)P3 receptor with almost the same potency as seen for the Ca2+ release. This compound inhibited the hydrolysis of D-[3H]Ins(1,4,5)P3 to D-[3H]Ins(1,4)P2 catalyzed by erythrocyte ghosts, with a K(i) value of as low as 1.4 μM, but it could not serve as a substrate for the same enzyme. D-Ins(1,4,5)P3 3- kinase in rat brain cytosol did not recognize the compound at concentrations up to 30 μM. Thus, it would appear that DL-Ins(1,2,4,5)P4 can serve as a potent and long lasting experimental and pharmacological tool for stimulating D-Ins(1,4,5)P3-mediating processes.. |
| 102. | Shoichiro Ozaki, Yutaka Watanabe, Tomio Ogasawara, Masato Hirata, Takashi Kanematsu, Synthesis and biological properties of 2-substituted myo-inositol 1,4,5-trisphosphate analogues directed toward affinity chromatography and photoaffinity labeling, Carbohydrate Research, 10.1016/0008-6215(92)85048-5, 234, C, 189-206, 1992.10, [URL], A series of myo-inositol 1,4,5-trisphosphate analogues with the 2-acyl substituents p-aminobenzoyl (7), p-azidobenzoyl (8), 4-{5-[2-(benzamido)ethyl]-2-hydroxyphenylazo}benzoyl (9), and cis,trans-4-aminocyclohexylcarbonyl (10) were synthesised and examined for their effects on the 5-phosphatase, the 3-kinase, the tritiated trisphosphate-binding activity, and the Ca2+-releasing activity. Each analogue inhibited the hydrolysis of d-[5-32P]Ins(1,4,5)P3 and the phosphorylation of d-[3H]Ins(1,4,5)P3, catalysed by erythrocyte ghosts and brain cytosol, respectively. The analogues acted as full agonists in releasing Ca2+ from permeabilised cells and also inhibited the binding of d-[3H]Ins(1,4,5)P3 to cerebellum microsomes. The analogues 7 and 10 were utilised for immobilisation of the trisphosphate on SepharoseTM and the subsequent affinity chromatography effected purification of the above proteins. A photoaffinity probe, the appendage of which acted as the photoaffinity probe as well as a non-radioactive molecular marker, was also derived from the analogue 7.. |
| 103. | T. Kanematsu, H. Takeya, Y. Watanabe, S. Ozaki, M. Yoshida, T. Koga, S. Iwanaga, M. Hirata, Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol, Journal of Biological Chemistry, 267, 10, 6518-6525, 1992.01, In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3- binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the δ-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 μM strongly inhibited 85-kDa phospholipase Cδ activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the δ isozyme of phospholipase C.. |
| 104. | Masato Hirata, Takashi Kanematsu, Kaori Sakuma, Toshitaka Koga, Yutaka Watanabe, Shoichiro Ozaki, Hitoshi Yagisawa, D-myo-inositol 1,4,5-trisphosphate binding domain of phospholipase C-δ1, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1994.2845, 205, 3, 1563-1571, 1994.01, [URL], D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding domain of phospholipase C-δ1 (PLC-δ1) was determined by examining binding activity of the synthetic peptide corresponding to residues 30-43 of PLC-δ1. The peptide coupled with carrier proteins such as keyhole limpet hemocyanin or bovine serum albumin bound Ins(1,4,5)P3, whereas a scrambled peptide with the same amino acids did not do so. Polyclonal antibody against the peptide was examined to determine whether it would cause inhibition of the Ins(1,4,5)P3 binding to PLC-δ1. Fab fragment of antibody to the peptide did inhibit binding to PLC-δ1, in a dose-dependent manner. Thus it seems likely that the region of residues 30-43 of PLC-δ1 is responsible for the binding of Ins(1,4,5)P3.. |
| 105. | Yuichi Kimura, Takashi Kanematsu, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, Masato Hirata, Synthetic inositol 1,3,4,5-tetrakisphosphate analogs and their effect on the binding to microsomal fraction of rat cerebellum, BBA - Biomembranes, 10.1016/0005-2736(91)90127-T, 1069, 2, 218-222, 1991.11, [URL], Binding activity of [3H]inositol 1,3,4,5-tetrakisphosphate (InsP4) was characterized with rat cerebellar membranes. Two types of InsP4 analog with either the aminobenzoyl or the aminocyclohexanecarbonyl group on the 2nd position of InsP4 have been synthesized and their effects on the binding activity were also examined. [3H]InsP4 binding was gradually displaced by increasing amounts of unlabeled InsP4, with an IC50 of 60-170 nM, depending on the pH values. The binding was sharply increased at acidic pH and millimolar concentrations of Ca2+, this being in clear contrast with [3H]InsP3 binding noted in the same species of tissue. Heparin inhibited the binding, with an IC50 of 1.7, 3 or 20 μg/ml at pH 8.3, 7.2 or 5.0, respectively. Adenine nucleotide inhibited the binding more potently than did [3H]InsP3 binding. InsP4 analogs were as effective as InsP4 in displacing [3H]InsP4 from rat cerebellar membranes, thereby indicating that the 2nd hydroxl group may not be involved in recognition of InsP4 by its binding sites.. |
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