Autophosphorylation of CaMKII in Neural Signal Transduction

描述(由申请人提供)：该项目的目标是建立谷氨酸能脊椎突触传递过程中钙依赖信号事件的定量动力学模型。钙/钙调蛋白依赖的蛋白激酶II(CaMKII)是NMDA型谷氨酸受体钙离子流动的主要靶点，也是兴奋性突触突触后膜下几个重要的神经蛋白磷酸化途径的重要组成部分。CaMKII的激活涉及四个钙离子与单个钙调蛋白(CaM)分子的结合，以及CaM与每个CaMKII亚基上的结合位置的结合，从而导致催化结构域的激活。钙离子和钙调素激活CaMKII的动力学是特别有趣的。 我将进行生化分析，以确定钙离子与CaM的结合常数和钙离子与CaMKII的结合常数，并确定CaMKII的存在是否增强了协同作用。我将使用突触上可能发生的钙、钙调素、钙MKII的浓度来确定CaMKII的自磷酸化的固有速率。我将使用不能在特定位点结合钙的突变形式的CaM，以及含有氨基或羧基EF手的CaM的胰酶片段，以便直接测量这些独立位点以其钙结合形式与CaMKII的结合亲和力。使用我得到的动力学参数，我将模拟当钙离子水平变化时自动磷酸化的初始水平，并将预测与使用急流装置的实验进行比较。然后，我将与索尔克研究所的研究人员合作，使用Mcell程序模拟脊椎突触后密度中CaMKII的激活。CaMKII参与了复杂的信号通路，导致突触强度(LTP)的增强，或在不同情况下导致突触强度(LTD)的减弱。这项工作将帮助我们理解脊椎中的钙信号如何以如此高的特异性对这些变化进行编码。

DESCRIPTION (provided by applicant): The goal of the project is to build a quantitative, kinetic model for calcium-dependent signaling events during synaptic transmission in glutamatergic spines. Calcium/calmodulin-dependent protein kinase II (CaMKII) is a major target of the Ca 2+flux through NMDA-type glutamate receptors and is known to be a crucial component of several important neural protein phosphorylation pathways beneath the post-synaptic membrane of excitatory synapses. Activation of CaMKII involves the binding of four Ca 2+ ions to individual calmodulin (CaM) molecules and the association of CaM with a binding site on each CaMKII subunit that leads to activation of the catalytic domain. Of particular interest is the kinetics of activation of CaMKII by Ca 2+ and CaM. I will perform biochemical assays to determine binding constants for Ca 2+ to CaM and for Ca 2+ CaM to CaMKII, and determine whether cooperativity is enhanced in the presence of CaMKII. I will determine the intrinsic rate of autophosphorylation of CaMKII using concentrations of Ca, CaM, CaMKII that will be likely to occur at synapses. I will use mutant forms of CaM that cannot bind Ca at particular sites, and tryptic fragments of CaM containing either the amino or carboxyl EF hands, in order to directly measure the binding affinity for CaMKII of these separate sites in their Ca 2+ bound form. Using the kinetic parameters that I obtain, I will simulate the initial level of autophosphorylation when the Ca 2+ level changes, and compare predictions with experiments using a quench flow apparatus. I will then collaborate with investigators at the Salk Institute to simulate activation of CaMKII in the post-synaptic density in spines using the program MCell. CaMKII is involved in complex signaling pathways that lead to strengthening of synaptic strength (LTP), or, under different circumstances, weakening of synaptic strength (LTD). The proposed work will help us to understand how a Ca 2+ signal in a spine achieves the encoding of these changes with such high specificity.