The Pathway to Activation of the Vav Proto-Oncogene

Vav 原癌基因的激活途径

• 批准号: 6848307
• 负责人: Michael K Rosen
• 金额: $ 25.48万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6848307
• 关键词: chemical kinetics; gene induction /repression; guanine nucleotide exchange factors; nuclear magnetic resonance spectroscopy; phosphorylation; posttranslational modifications; protein kinase; protooncogene; surface plasmon resonance; thermodynamics

DESCRIPTION (provided by applicant): The protooncoprotein Vav, a key regulator of lymphocyte signal transduction pathways, is a guanine nucleotide exchange factor (GEF) for Rho GTPases. The catalytic DH domain of Vav is autoinhibited by an adjacent N-terminal peptide, which binds the GEF active site. Phosphorylation of a buried tyrosine in this arm by Lck dissociates the arm from the DH domain, activating Vav. This proposal integrates biochemical and NMR spectroscopic investigations to discover the pathway to Vav activation. We hypothesize that A) additional tyrosine motifs (Y-motifs) in the Vav N-terminus may bind the DH domain and contribute to autoinhibition, and B) initial phosphorylation of exposed Y-motifs recruits Lck through its SH2 domain, enabling the kinetically disfavored phosphorylation event to occur in intramolecular fashion. Affinities of phosphorylated Vav proteins for the Lck SH2 domain, and of Vav N-terminal peptides for the DH domain will be determined, revealing which Y-motifs in the Vav N-terminus can bind the DH domain, and, when phosphorylated, recruit Lck. Effects of phosphorylation and SH2 binding on DH-inhibitory peptide affinity and binding kinetics will be determined, revealing whether these initial steps could contribute to relief of autoinhibition. Kinetic parameters for Lck kinase domain-catalyzed phosphorylation of Vav peptides, both free and bound intramolecularly to the DH domain, will be determined. Parameters will also be determined for full-length Lck, revealing the extent to which SH2-mediated docking to phosphorylated sites can overcome the sequestering effect of the DH domain toward the buried tyrosine. Amide and methyl group [mu]s-ms timescale dynamics in autoinhibited Vav will be measured by NMR relaxation dispersion analysis at multiple fields. The data will quantitate the rates of these motions, as well as the populations of the ground and excited states and the chemical shift differences between them. The chemical shift and rate data, combined with biochemically-measured helix dissociation rates, will reveal if the NMR dynamics represent excursions to a helix-dissociated state. Comparison of NMR dynamics rates with kinetics of phosphorylation will reveal if NMR dynamics could represent motions that govern the rate of Vav activation by Lck. These results will test our ideas that autoinhibited proteins often possess exposed "access points" to overcome kinetic barriers to activation inherent in their design.

描述(由申请人提供)：原癌蛋白Vav是淋巴细胞信号转导途径的关键调节因子，是Rho GTP酶的鸟嘌呤核苷酸交换因子(Global)。Vav的催化水解区被相邻的N-末端多肽自动抑制，该多肽与全球环境基金的活性部位结合。Lck将该臂中埋藏的酪氨酸磷酸化，使该臂与水解区分离，激活Vav。这一建议结合了生化和核磁共振波谱研究，以发现Vav激活的途径。我们假设，A)Vav N-末端的额外酪氨酸基序(Y-基序)可能与DH域结合并有助于自身抑制，以及B)暴露的Y-基序的初始磷酸化通过其SH2结构域招募Lck，使动力学上不受欢迎的磷酸化事件以分子内方式发生。将确定磷酸化的Vav蛋白与Lck SH2结构域的亲和力，以及Vav N-末端多肽与DH域的亲和力，揭示Vav N-末端的哪些Y基序可以与DH域结合，并在磷酸化时招募Lck。将确定磷酸化和SH2结合对氢脱氢酶抑制肽亲和力和结合动力学的影响，揭示这些初始步骤是否有助于解除自身抑制。将确定LCK激酶结构域催化的Vav多肽的磷酸化动力学参数，无论是游离的还是分子内结合到DH域的。还将确定全长Lck的参数，揭示SH2介导的与磷酸化位点的对接在多大程度上可以克服水解域对埋藏的酪氨酸的隔离效应。用核磁共振弛豫色散分析方法测量自抑制VaV中酰胺和甲基[MU]S-MS的时间尺度动力学。这些数据将量化这些运动的速率，以及基态和激发态的数量以及它们之间的化学位移差异。化学位移和速率数据，结合生物化学测量的螺旋解离速率，将揭示核磁共振动力学是否代表向螺旋解离状态的漂移。将核磁共振动力学速率与磷酸化动力学进行比较，将揭示核磁共振动力学是否能代表支配Lck激活Vav速率的运动。这些结果将检验我们的想法，即自抑制蛋白通常具有暴露的“接入点”，以克服其设计中固有的激活动力学障碍。