Autophosphorylation of CaMKII in Neural Signal Transduction

神经信号转导中 CaMKII 的自磷酸化

• 批准号: 6707916
• 负责人: Mee H Choi
• 金额: $ 8.13万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6707916
• 关键词: NMDA receptors; SDS polyacrylamide gel electrophoresis; biological signal transduction; calcium flux; calmodulin; calmodulin dependent protein kinase; neural transmission; phosphorylation; postdoctoral investigator; receptor binding; synapses

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.

描述（由申请人提供）：该项目的目标是建立一个定量的，动力学模型的钙依赖性信号事件在突触传递过程中的突触能脊髓。钙/钙调素依赖性蛋白激酶II（CaMKII）是NMDA型谷氨酸受体介导的Ca 2+流的主要靶点，也是兴奋性突触突触后膜下几条重要神经蛋白磷酸化途径的重要组成部分。CaMKII的激活涉及4个Ca 2+离子与单个钙调素（CaM）分子的结合以及CaM与每个CaMKII亚基上的结合位点的缔合，其导致催化结构域的激活。特别感兴趣的是Ca 2+和CaM激活CaMKII的动力学。 我将进行生化分析，以确定结合常数的钙2+钙调素和钙2+钙调素钙MKII，并确定是否协同性增强的存在下，钙MKII。我将使用可能发生在突触处的Ca、CaM、CaMKII的浓度来确定CaMKII自磷酸化的内在速率。我将使用不能在特定位点结合Ca的CaM突变体和含有氨基或羧基EF手的CaM胰蛋白酶片段，以直接测量这些单独位点在其Ca 2+结合形式下对CaMK II的结合亲和力。使用的动力学参数，我得到的，我将模拟初始水平的自磷酸化时的Ca 2+水平的变化，并比较预测与实验使用淬火流动装置。然后，我将与索尔克研究所的研究人员合作，使用MCell程序模拟脊髓突触后密度中CaMKII的激活。CaMKII参与导致突触强度（LTP）增强或在不同情况下导致突触强度（LTD）减弱的复杂信号传导途径。这项工作将有助于我们理解脊椎中的Ca 2+信号如何以如此高的特异性编码这些变化。