Mechanisms of CaM Kinase II Signal Transduction

描述（来自申请人摘要）： 神经元信号转导和蛋白质的详细表征 磷酸化/去磷酸化对于理解许多脑 功能协调发展的例如，NMDA型谷氨酸受体的激活刺激了 蛋白激酶和蛋白磷酸酶，反馈调节AMPA- 和NMDA型谷氨酸受体。钙/钙调蛋白依赖性蛋白激酶II （CaMKII）是由NMDA受体刺激激活的主要树突激酶， 导致Thr 286-自身磷酸化和AMPA-和 NMDA受体和其他几种蛋白质，包括致密蛋白-180， 具有PDZ结构域的O-唾液酸糖蛋白。我们发现CaMKII 自身磷酸化促进其向突触后致密物的转位 （PSD），膜下细胞骨架特化，并确定了NR 2B NMDA受体亚基和密度蛋白-180作为两种蛋白质， 有助于易位。五个具体目标解决了我们的假设， 与NR 2B和densin-180的结合调节CaMKII，导致突触特异性 谷氨酸受体的调节。 1.将通过以下方法验证CaMKII和dens- 180的神经元相互作用： 共定位使用免疫荧光共聚焦显微镜和 免疫共沉淀测定。NR 2B和致密蛋白-180对 将测定PSD中的CaMK II结合活性。2.中的相互作用域 NR 2B、致密蛋白-180和CaMKII将通过截短/缺失在体外鉴定 和定点突变，它们的重要性将在HEK 293中得到证实 细胞和神经元。这些信息将用于开发试剂， 特异性操纵CaMK II在细胞中定位。3.动力学 CaMKII.densin-180和CaMKII.NR2B的相互作用以及CaMKII.NR2B的调节作用 致密蛋白-180、NR 2B和CaMKII的磷酸化/去磷酸化，以及 将在体外和完整细胞中检查NMDA受体活化。4. 与NR 2B或densin-180相互作用对CaMKJI自磷酸化的影响 将在体外和完整细胞中进行研究。AMPA受体 将比较HEK 293细胞和神经元中的磷酸化和增强 在NR 2B和致密蛋白-180用于差异靶向 CaMKJI。5. CaMKII结合和CaMKII介导的NR 2B磷酸化在 将测定NMDA受体的调节。更长远的目标是 建立NR 2B和致密蛋白-180靶向CaMK Ⅱ在调节 突触传递和突触特异性突触可塑性。这些研究 将为信号转导机制提供基本的见解 学习和记忆等正常大脑功能的基础。的试剂 蛋白质相互作用也有发展潜力， 用于治疗精神障碍如精神分裂症或 抑郁症可能还有脑损伤

DESCRIPTION(From applicant's abstract): Detailed characterizations of neuronal signal transduction and protein phosphorylation/dephosphorylation are critical for understanding many brain functions. For example, activation of NMDA-type glutamate receptors stimulates both protein kinases and protein phosphatases, which feedback to modulate AMPA- and NMDA-type glutamate receptors. Ca-+/calmodulin-dependent protein kinase II (CaMKII) is a major dendritic kinase activated by NMDA receptor stimulation, resulting in Thr286-autophosphorylation and phosphorylation of AMPA- and NMDA-receptors and several other proteins including densin-180, an O-sialoglycoprotein with a PDZ domain. We showed that CaMKII autophosphorylation promotes its translocation to postsynaptic densities (PSDs), submembranous cytoskeletal specializations, and identified the NR2B subunit of NMDA receptors and densin- 180 as two proteins that likely contribute to translocation. Five Specific Aims address our hypothesis that binding to NR2B and densin-180 modulates CaMKII, resulting in synapse-specific regulation of glutamate receptors. 1. Neuronal interaction of CaMKII and dens in- 180 will be verified by colocalization using immunofluorescent confocal microscopy and by coimmunoprecipitation assays. Relative contributions of NR2B and densin- 180 to CaMKII binding activities in PSDs will be determined. 2. Interaction domains in NR2B, densin-180 and CaMKII will be identified in vitro by truncation/deletion and site-directed mutagenesis, and their importance will be confirmed in HEK293 cells and neurons. This information will be used to develop reagents that specifically manipulate CaMKII localization in cells. 3. Dynamics of CaMKII.densin-180 and CaMKII.NR2B interactions, and regulatory roles of phosphorylation/dephosphorylation of densin-180, NR2B and CaMKII, as well as NMDA receptor activation, will be examined in vitro and in intact cells. 4. Effects of interaction with NR2B or densin-180 on CaMKJI autophosphorylation will be investigated in vitro and in intact cells. AMPA receptor phosphorylation and potentiation in HEK293 cells and neurons will be compared under conditions where NR2B and densin-180 are used to differentially target CaMKJI. 5. Roles of CaMKII-binding and CaMKII-mediated NR2B phosphorylation in regulation of NMDA receptors will be determined. More long-range goals are to establish the roles of CaMKII targeting by NR2B and densin-180 in regulation of synaptic transmission and synapse-specific synaptic plasticity. These studies will provide fundamental insights into signal transduction mechanisms underlying normal brain functions such as learning and memory. Reagents that block these protein.protein interactions also have potential for development as therapeutic compounds to treat mental disorders, such as schizophrenia or depression, and possibly brain injuries.