ELUCIDATING THE KINETIC MECHANISM OF SCAFFOLD-MEDIATED MAP KINASE SIGNALING

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 活细胞通过蛋白质网络与环境相互作用并对其做出反应，这些蛋白质网络将细胞外信号转化为细胞内的特定生化反应。 每个通路内的特异性对于适当调节不同的细胞行为是必不可少的。 支架蛋白已经被认为通过将单个信号传导蛋白拴在一起形成多蛋白复合物而有助于特异性。 新出现的证据表明，支架蛋白也可以发挥更直接的作用，通过变构促进特定的激酶-激酶反应，并通过响应上游激活剂的构象变化的信号传递。 为了构建支架蛋白如何促进特定激酶-激酶反应的物理模型，我们将使用动力学和生物物理方法相结合的方法。 首先，我们将测量在支架存在和不存在的情况下单个激酶-激酶反应的速率常数。 其次，我们将确定关键的表面和相互作用所需的支架行动使用结晶学和生物化学方法。 该项目的一个必要组成部分是能够确定不同的蛋白质样品是否已被磷酸化，并确定靶蛋白中磷酸化的特定位点。 UCSF质谱设备将有助于获得这些信息。 这些实验将为支架介导的信号转导提供动力学和热力学框架，这将为理解细胞信号传导中如何保持特异性提供基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Living cells interact with and respond to their environment through networks of proteins that transduce extracellular signals into specific biochemical responses inside the cell. Specificity within each pathway is essential for proper regulation of diverse cellular behaviors. Scaffold proteins have been suggested to contribute to specificity by tethering individual signaling proteins together into multi-protein complexes. Emerging evidence suggests that scaffold proteins can also play a more direct role in signal transmission by allosterically promoting specific kinase-kinase reactions and by undergoing conformational changes in response to upstream activators. To construct a physical model for how scaffold proteins promote specific kinase-kinase reactions, we will use a combination of kinetic and biophysical approaches. First, we will measure rate constants for individual kinase-kinase reactions in the presence and absence of scaffolds. Second, we will identify key surfaces and interactions required for scaffold action using crystallographic and biochemical methods. A necessary component of this project is the ability to determine if different protein samples have been phosphorylated, and to identify specific sites of phosphorylation in target proteins. The UCSF mass spectrometry facility will be instrumental in obtaining this information. These experiments will provide kinetic and thermodynamic framework for scaffold-mediated signal transduction, which will provide the basis for understanding how specificity is maintained in cell signaling.