Mechanisms of CaM Kinase II Signal Transduction

描述(摘自申请人的摘要)： 神经元信号转导和蛋白质的详细特征 磷酸化/去磷酸化是理解许多大脑的关键 功能。例如，NMDA型谷氨酸受体的激活刺激 蛋白激酶和蛋白磷酸酶都能反馈调节AMPA- 和NMDA型谷氨酸受体。钙/钙调蛋白依赖的蛋白激酶II (CaMKII)是一种由NMDA受体刺激激活的主要树突状细胞激酶， 导致Thr286-AMPA-和-的自动磷酸化和磷酸化 NMDA受体和其他几种蛋白质，包括致密蛋白-180和 带有PDZ结构域的O-唾液酸糖蛋白。我们展示了CaMKII 自身磷酸化促进其向突触后密度移位 (PSD)，膜下细胞骨架特化，并确定了NR2B NMDA受体亚单位和致密蛋白-180作为两种可能的蛋白质 会导致移位。五个具体目标解决了我们的假设 与NR2B和致密蛋白-180的结合调节CaMKII，导致突触特异性 谷氨酸受体的调节。 1.CaMKII和DIENS在-180中的神经元相互作用将被验证 使用免疫荧光共聚焦显微镜和 免疫共沉淀法。NR2B和Densin-180的相对贡献率 将测定PSD中的CaMKII结合活性。2.中的互动域 NR2B、Densin-180和CaMKII将在体外通过截断/缺失的方式进行鉴定 和定点突变，它们的重要性将在HEK293中得到确认 细胞和神经元。这些信息将被用来开发 特异性地操纵CaMKII在细胞中的定位。3.动态的 CaMKII.致密蛋白-180和CaMKII.NR2B的相互作用和调节作用 致密蛋白-180、NR2B和CaMKII的磷酸化/去磷酸化以及 NMDA受体的激活，将在体外和完整的细胞中进行检测。4. 与NR2B或Densin-180相互作用对CaMKJI自身磷酸化的影响 将在体外和完整的细胞中进行研究。AMPA受体 将比较HEK293细胞和神经元中的磷酸化和增强 在使用NR2B和Densin-180差异靶向的情况下 CaMKJI5.CaMKII结合和CaMKII介导的NR2B磷酸化在细胞周期中的作用 NMDA受体的调节将被确定。更长远的目标是 确定NR2B和Densin-180靶向CaMKII在调控中的作用 突触传递和突触特异性突触可塑性。这些研究 将提供对信号转导机制的基本见解 潜在的正常大脑功能，如学习和记忆。这些试剂 阻断这些蛋白质。蛋白质之间的相互作用也有可能发展为 治疗精神障碍的治疗化合物，如精神分裂症或 抑郁，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.