Kawaai Katsuhiro, Hisatsune Chihiro, Kuroda Yukiko, Mizutani Akihiro, Tashiro Tomoko, Mikoshiba Katsuhiko
Department of Chemistry and Biological Science, Aoyama-Gakuin University, 5-10-1 Fuchinobe, Sagamihara, Kanagawa 229-8558, Japan, the Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, and the Calcium Oscillation Project, International Cooperative Research Project-Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Department of Chemistry and Biological Science, Aoyama-Gakuin University, 5-10-1 Fuchinobe, Sagamihara, Kanagawa 229-8558, Japan, the Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, and the Calcium Oscillation Project, International Cooperative Research Project-Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Department of Chemistry and Biological Science, Aoyama-Gakuin University, 5-10-1 Fuchinobe, Sagamihara, Kanagawa 229-8558, Japan, the Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, and the Calcium Oscillation Project, International Cooperative Research Project-Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
J Biol Chem. 2009 Jan 2;284(1):372-380. doi: 10.1074/jbc.M805828200. Epub 2008 Nov 6.
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular channel proteins that mediate calcium (Ca2+) release from the endoplasmic reticulum, and they are involved in many biological processes (e.g. fertilization, secretion, and synaptic plasticity). Recent reports show that IP3R activity is strictly regulated by several interacting molecules (e.g. IP3R binding protein released with inositol 1,4,5-trisphosphate, huntingtin, presenilin, DANGER, and cytochrome c), and perturbation of this regulation causes intracellular Ca2+ elevation leading to several diseases (e.g. Huntington disease and Alzheimer disease). In this study, we identified protein kinase C substrate 80K-H (80K-H) to be a novel molecule interacting with the COOH-terminal tail of IP3Rs by yeast two-hybrid screening. 80K-H directly interacted with IP3R type 1 (IP3R1) in vitro and co-immunoprecipitated with IP3R1 in cell lysates. Immunocytochemical and immunohistochemical staining revealed that 80K-H colocalized with IP3R1 in COS-7 cells and in hippocampal neurons. We also showed that the purified recombinant 80K-H protein directly enhanced IP3-induced Ca2+ release activity by a Ca2+ release assay using mouse cerebellar microsomes. Furthermore 80K-H was found to regulate ATP-induced Ca2+ release in living cells. Thus, our findings suggest that 80K-H is a novel regulator of IP3R activity, and it may contribute to neuronal functions.
肌醇1,4,5-三磷酸受体(IP3Rs)是介导内质网释放钙离子(Ca2+)的细胞内通道蛋白,参与许多生物学过程(如受精、分泌和突触可塑性)。最近的报道表明,IP3R活性受到几种相互作用分子(如与肌醇1,4,5-三磷酸一起释放的IP3R结合蛋白、亨廷顿蛋白、早老素、DANGER和细胞色素c)的严格调控,这种调控的紊乱会导致细胞内Ca2+升高,进而引发多种疾病(如亨廷顿病和阿尔茨海默病)。在本研究中,我们通过酵母双杂交筛选鉴定出蛋白激酶C底物80K-H(80K-H)是一种与IP3Rs羧基末端尾巴相互作用的新分子。80K-H在体外直接与1型IP3R(IP3R1)相互作用,并在细胞裂解物中与IP3R1进行共免疫沉淀。免疫细胞化学和免疫组织化学染色显示,80K-H在COS-7细胞和海马神经元中与IP3R1共定位。我们还通过使用小鼠小脑微粒体的Ca2+释放试验表明,纯化的重组80K-H蛋白直接增强了IP3诱导的Ca2+释放活性。此外,发现80K-H可调节活细胞中ATP诱导的Ca2+释放。因此,我们的研究结果表明,80K-H是IP3R活性的新型调节因子,可能对神经元功能有贡献。