ROLE OF SH3 DOMAINS IN NON RECEPTOR PTKS

非受体蛋白酪氨酸激酶(PTK‘s)、Abl、CSK、HCK等Src家族成员是信号转导的中枢调节因子，参与多种疾病状态，包括多种癌症和自身免疫综合征。它们有三个高度同源的结构域：一个SH3(Src Homology 3)结构域、一个SH2结构域和一个催化激活区。SH3结构域在体外和体内都参与了对激酶活性的调节。我们提出了用异核核磁共振结合蛋白质片段同位素标记的方法来表征SH3结构域控制激酶活性的分子机制。我们将使用化学位移图谱和直接核磁共振结构测定来确定SH3与CSK催化结构域之间的分子内相互作用表面，这可能涉及一种新的SH3/配体识别机制。在Abl SH(3，2，Kinase)结构中SH3结构域的分段同位素标记将被用来定位SH3与分子其他部分的接触，并评估在存在酶和SH相关的激活剂和抑制剂的情况下的结构修改和变化。我们将对Lek和HCK以及其他Src家族成员的溶解性和缔合性质进行广泛的调查，以确定最有可能用于未来核磁共振研究的候选化合物。在调查结果的基础上，将对非受体酪氨酸激酶的Src家族活性形式的SH3自抑制机制进行结构表征。从这些研究中获得的结构信息对于理解激酶调节的分子内机制将是重要的，并可能是设计高度特异的治疗药物来调节细胞PTK活性的第一步。

Non-receptor protein tyrosine kinases (PTK's), Abl, Csk, Hck, and other Src family members, are central regulators of signal transduction and involved in multiple disease states, including many cancers and autoimmune syndromes. They share three highly homologous domains: an SH3 (Src homology 3) domain, an SH2 domain and a catalytic kinase domain. SH3 domains are involved in regulation of kinase activity in vitro and vivo. We propose structural characterization of the molecular mechanisms of SH3 domain control of the kinase activity using heteronuclear NMR coupled with protein segmental isotopic labeling. We will use chemical shift mapping and direct NMR structure determination to identify the intramolecular interaction surface between SH3 and catalytic domains of Csk which is likely to involve a novel mechanism of SH3/ligand recognition. Segmental isotopic labeling of SH3 domain within Abl SH(3,2,kinase) construct will be used to map the contacts of the SH3 with other parts of the molecule and assess structural modifications and changes in the presence of enzymatic and SH-related activators and inhibitors. We will conduct a broad survey of the solubility and association properties of Lek and Hck and other Src family members to establish the most likely practical candidate for future NMR studies. Based on the results of the survey, structural characterization of the SH3 autoinhibitory mechanisms in the active form of the Src-family of non-receptor tyrosine kinases will be conducted. The structural information derived from these studies will be important for understanding intramolecular mechanisms of kinase regulation and may represent a first step in designing highly specific therapeutic agents for regulating cellular PTK's activity.