Regulated Complex Assembly in Membrane-Associated Guanylate Kinases

描述（由申请人提供）：膜相关鸟苷酸激酶（MAGUKs）是普遍存在的蛋白质，由PDZ结构域、SH3结构域和GK结构域的共同核心组成。MAGUK超家族的成员参与组织分子组装，因此对细胞连接的形成至关重要。在一个假设的模型中，蛋白质结构域的分子内缔合的变构调节调节MAGUK功能。多种MAGUK蛋白的体外结合测定先前证明了SH3结构域与GK结构域的非典型缔合，所述GK结构域缺乏通常在SH3配体中发现的聚脯氨酸基序。此外，它表明，连续连接的SH3和GK结构域的分子内缔合优于分子间缔合。这种相互作用对MAGUK功能很重要，因为SH3和GK结构域中的所有遗传鉴定的突变都显示出破坏SH3-GK分子内相互作用。一种这样的突变等位基因dlgsw编码椎间盘大（Dig）肿瘤抑制蛋白的突变形式，并破坏分子内缔合，同时使SH3和GK结构域保持完整。仅含有sw形式Dig的果蝇显示出胚胎致死性。其他研究提供的证据表明，蛋白质配体与连接SH3和GK结构域的HOOK结构域的相互作用调节这种分子内相互作用并最终调节复杂的组装。我们提出的研究的目的是测试的假设，即变构修饰的SH3-GK分子内相互作用调节复杂的组装。因此，提出了以下具体目标：（1）通过野生型和sw Dig复合物的共免疫沉淀，随后使用蛋白质印迹分析和质谱法鉴定蛋白质组分，确定SH3-GK分子内相互作用对Dig在体内形成的复合物组的影响，（二）通过免疫荧光实验确定上皮组织和不对称细胞分裂中的分子内相互作用，和细胞命运决定性标志物，以及（3）确定HOOK配体结合对Dig分子内相互作用的影响，以评估MAGUK组织细胞信号传导复合物的分子机制。这些研究将有助于我们理解的分子机制，其中迪格组织信号复合物，以允许调节不对称细胞分裂。这些研究还将进一步深入了解Dig失活导致肿瘤形成和转移的原因。

DESCRIPTION (provided by applicant): Membrane-associated guanylate kinases (MAGUKs) are ubiquitous proteins that are composed of a common core of a PDZ domain, an SH3 domain, and a GK domain. Members of the MAGUK superfamily are involved in organizing molecular assemblies and thus are vital for formation of cellular junctions. In a hypothesized model, allosteric regulation of the intramolecular association of protein domains regulates MAGUK function. In vitro binding assays of a variety of MAGUK proteins previously demonstrated non- canonical association of the SH3 domains with GK domains that lack polyproline motifs commonly found in SH3 ligands. Furthermore, it was shown that intramolecular association of contiguously linked SH3 and GK domains was preferred over intermolecular association. This interaction was shown to be important for MAGUK function as all genetically identified mutations in the SH3 and GK domains have been shown to disrupt the SH3-GK intramolecular interaction. One such mutant allele, dlgsw, encodes a mutant form of the Discs Large (Dig) tumor suppressor protein and disrupts the intramolecular association while leaving the SH3 and GK domains intact. Drosophila containing only the sw form of Dig are shown to be embryonic lethal. Additional studies provide evidence that interaction of protein ligands with the HOOK domain that joins the SH3 and GK domains regulate this intramolecular interaction and ultimately complex assembly. The objective of our proposed research is to test the hypothesis that allosteric modification of the SH3-GK intramolecular interaction regulates complex assembly. Thus, the following specific aims are proposed: (1) Determmine the effect of the SH3-GK intramolecular interaction on the set of complexes that Dig forms in vivo by co-immunoprecipitation of wild-type and sw Dig complexes followed by identification of protein components using Western blot analyses and mass spectrometry, (2) determine the intramolecular interaction in epithelial organization and asymmetric cell division by immunofluorescence experiments assessing the cellular localization of junctional and cell fate determinant markers in WT versus sw Dig- containing embryos, and (3) determine the effect of binding of HOOK ligands on the Dig intramolecular interaction to assess the molecular mechanism by which MAGUKs organize cell signaling complexes. These studies will contribute to our understanding of the molecular mechanism by which Dig organizes signaling complexes in order to allow for regulated asymmetric cell division. Such studies will also provide further insight into why inactivation of Dig results in tumor formation and metastasis.