ROLE OF SH3 DOMAINS IN NON RECEPTOR PTKS

非受体蛋白酪氨酸激酶（PTK）、Abl、Csk、Hck和其他Src家族成员是信号转导的中心调节剂，并且参与多种疾病状态，包括许多癌症和自身免疫综合征。 它们共享三个高度同源的结构域：SH 3（Src同源3）结构域、SH 2结构域和催化激酶结构域。 SH 3结构域参与体外和体内激酶活性的调节。 我们提出了结构表征的分子机制SH 3域控制的激酶活性，使用杂波NMR加上蛋白质片段同位素标记。 我们将使用化学位移映射和直接NMR结构测定来确定SH 3和Csk的催化结构域之间的分子内相互作用表面，这可能涉及SH 3/配体识别的新机制。 Abl SH（3，2，激酶）构建体中SH 3结构域的节段同位素标记将用于绘制SH 3与分子其他部分的接触图，并评估酶和SH相关激活剂和抑制剂存在下的结构修饰和变化。 我们将对Lek和Hck以及其他Src家族成员的溶解度和缔合特性进行广泛的调查，以确定未来NMR研究最有可能的实际候选者。 根据调查结果，将进行非受体酪氨酸激酶Src家族活性形式的SH 3自抑制机制的结构表征。 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，并且可能代表设计用于调节细胞PTK活性的高度特异性治疗剂的第一步。

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.