Autophosphorylation of CaMKII in Neural Signal Transduction

神经信号转导中 CaMKII 的自磷酸化

• 批准号: 7176109
• 负责人: Mee H Choi
• 金额: $ 11.29万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 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/7176109
• 关键词: Affinity; Binding; Binding Sites; Biochemical; Biological Assay; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcium; Calcium/calmodulin-dependent protein kinase; Calmodulin; Catalytic Domain; Chromosome Pairing; Collaborations; Complex; Condition; Data; Dissociation; Docking; EF Hand Motifs; Event; Excitatory Synapse; Face; Fluorescence; Glutamate Receptor; Glutamates; Goals; Holoenzymes; In Vitro; Individual; Institutes; Ions; Kinetics; Laboratories; Lead; Measures; Modeling; Oral cavity; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Physiologic pulse; Protein phosphatase; Pulse taking; Rate; Reaction; Research Personnel; Resolution; Role; Signal Pathway; Signal Transduction; Simulate; Site; Specificity; Synapses; Synaptic Membranes; Synaptic Transmission; Techniques; Testing; Threonine; Time; Vertebral column; Work; calmodulin-dependent protein kinase II; density; interest; member; mutant; postsynaptic; programs; receptor; relating to nervous system; research study; simulation

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）：该项目的目的是在谷氨酸能刺突触传播期间建立一个定量的，动力学模型，以构建依赖钙的信号事件。钙/钙调蛋白依赖性蛋白激酶II（CAMKII）是通过NMDA型谷氨酸受体的Ca 2+通量的主要靶标，已知是几种重要的神经蛋白磷酸化途径的至关重要组成部分。 CAMKII的激活涉及四个Ca 2+离子与单个钙调蛋白（CAM）分子的结合，而CAM与每个CAMKII亚基上的结合位点的结合，从而导致催化结构域的激活。特别有趣的是Ca 2+和CAM激活CAMKII的动力学。 我将执行生化测定，以确定Ca 2+与CAM的结合常数以及Ca 2+ CAM与Camkii的结合常数，并确定在CAMKII存在下是否会增强合作性。我将使用CA，CAM，CAMKII的浓度来确定CAMKII的自磷酸化速率，这些浓度可能会在突触时发生。我将使用无法在特定位点结合Ca的凸轮的突变形式，以及包含氨基或羧基EF手的CAM的胰蛋白酶片段，以直接测量以其Ca 2+结合形式的CAMKII对CAMKII的结合亲和力。使用我获得的动力学参数，当Ca 2+水平变化时，我将模拟自磷酸化的初始水平，并将预测与使用淬灭流动器的实验进行比较。然后，我将与Salk Institute的调查人员合作，使用程序McEll在刺中模拟CAMKII的激活后密度。 CAMKII参与了导致突触强度（LTP）加强的复杂信号通路，或者在不同情况下，突触强度（LTD）弱化。拟议的工作将帮助我们了解脊柱中的Ca 2+信号如何以如此高的特异性来实现这些变化的编码。