Mechanisms of CaM Kinase II Signal Transduction

描述（由申请人提供）：Ca2+/钙调蛋白（CaM）依赖性蛋白激酶II （CaMKII）的活性和亚细胞定位受Ca2+/CaM结合、Thr286和Thr305/306的自磷酸化以及与CaMKII相关蛋白（CaMKAPs）的相互作用调节。CaMKII的抑制，CaMKII自磷酸化位点的突变，或阻断CaMKII与NMDA受体（NMDAR） GluN2B（以前的NR2B）亚基的结合，会破坏海马谷氨酸能突触的正常LTP。然而，在其他兴奋性突触中，CaMKII对LTD诱导或独立于激酶活性的突触变化很重要。CaMKII在不同生理情况下诱导突触功能变化的具体分子过程尚不清楚。该项目的总体目标是确定CaMKII对兴奋性突触传递的正常调节至关重要的特定下游靶点的作用机制。我们的统一假设是，通过改变CaMKII复合物与其底物和其他蛋白质的组成，CaMKII的作用在离散的亚细胞区室中受到“微调控”。因此，下游CaMKII在特定细胞/生理环境下的作用将由这些复合物的其他组分决定。在之前的资助期内，我们在表征含有NMDAR亚基、电压门控Ca2+通道21/22亚基、致密素、1-肌动蛋白和/或SAP97的CaMKII复合物方面取得了出色的进展，发表了20篇主要研究论文。有趣的是，这些CaMKAPs通过多种分子机制与CaMKII相互作用，通常也与其他突触蛋白相互作用。我们的发现提示了精确调节camkii对关键突触信号蛋白依赖作用的新机制，以及突触后密度（psd）中多蛋白复合物的调节组装。这一竞争性更新应用提出了在体外、异种细胞和神经元/脑片中使用生化、分子和电生理方法的组合来测试以下特定假设：目的1：测试2个亚基和密度不同地靶向CaMKII亚型以调节l型电压门控Ca2+通道的假设。目的2：验证1- actitinin激活CaMKII调节GluN2B- NMDARs以及磷脂调节这一途径的假设。目的3：验证CaMKII与特定SAP97剪接变体相互作用并磷酸化以调节AMPARs的假设。目的4：验证CaMKII自磷酸化被psd内的CaMKAPs修饰的假设，并反过来调节相关的蛋白-蛋白相互作用，使CaMKII发挥非催化的结构作用。这些研究将确定生理控制的基本机制，使CaMKII能够选择性地调节对不同形式的突触可塑性、学习和记忆至关重要的靶标。

DESCRIPTION (provided by applicant): The activity and subcellular localization of Ca2+/calmodulin(CaM)-dependent protein kinase II (CaMKII) are regulated by Ca2+/CaM binding, autophosphorylation at Thr286 and Thr305/306, and interactions with CaMKII- associated proteins (CaMKAPs). Inhibition of CaMKII, mutation of CaMKII autophosphorylation sites, or blocking CaMKII binding to NMDA receptor (NMDAR) GluN2B (formerly NR2B) subunits disrupts normal LTP at hippocampal glutamatergic synapses. However, at other excitatory synapses CaMKII is important for LTD induction or for synaptic changes independent of kinase activity. The specific molecular processes engaged by CaMKII to induce these disparate changes of synaptic function in different physiological situations remain unclear. The overarching goal of this project is to define mechanisms of CaMKII action toward specific downstream targets that are critical for normal regulation of excitatory synaptic transmission. Our unifying hypothesis is that CaMKII actions are "micro-regulated" in discrete subcellular compartments by changing the composition of CaMKII complexes with its substrates and other proteins. Thus, the repertoire of downstream CaMKII actions in a specific cellular/physiological context will be dictated by other components of these complexes. In previous funding periods, we made excellent progress characterizing CaMKII complexes containing NMDAR subunits, 21/22 subunits of voltage-gated Ca2+ channels, densin, 1-actinin and/or SAP97, generating 20 primary research publications. Interestingly, these CaMKAPs interact with CaMKII by multiple molecular mechanisms, and also typically interact with other synaptic proteins. Our findings suggest novel mechanisms for precise modulation of CaMKII-dependent actions on key synaptic signaling proteins, as well as for regulated assembly of multi-protein complexes in postsynaptic densities (PSDs). This competing renewal application proposes the use of a combination of biochemical, molecular and electrophysiological approaches in vitro, in heterologous cells, and in neurons/brain slices to test the following specific hypotheses: Aim 1: Test the hypothesis that 2 subunits and densin differentially target CaMKII isoforms to regulate L-type voltage-gated Ca2+ channels. Aim 2: Test the hypothesis that 1-actinin activates CaMKII to regulate GluN2B- NMDARs and that phospholipids modulate this pathway. Aim 3: Test the hypothesis that CaMKII interacts with and phosphorylates specific SAP97 splice variants to regulate AMPARs. Aim 4: Test the hypothesis that CaMKII autophosphorylation is modified by CaMKAPs within PSDs, and in turn regulates relevant protein- protein interactions, allowing CaMKII to play a non-catalytic, structural role. These studies will define fundamental mechanisms of physiological control that allow CaMKII to selectively regulate targets critical to different forms of synaptic plasticity, learning and memory. PUBLIC HEALTH RELEVANCE: Normal human behaviors such as learning and memory require precise control of connections (called synapses) between nerve cells in the brain, which are dependent on the activities of many receptor and ion channel proteins. CaMKII is one of the most abundant proteins at the synapse, where it can chemically modify and regulate several different target proteins, such as receptor and ion channel proteins, to elicit different responses depending on physiological demand. By defining mechanisms that allow CaMKII to selectively modify different target proteins, the proposed studies will guide the development of novel approaches to treat many neurodevelopmental, psychiatric and neurological disorders linked to abnormal synaptic functions.