Regulated Complex Assembly in Membrane-Associated Guanylate Kinases

膜相关鸟苷酸激酶（MAGUKs）是普遍存在的蛋白质，其由一个 PDZ结构域、SH 3结构域和GK结构域的共同核心。MAGUK超家族成员 参与组织分子组装，因此对于细胞连接的形成至关重要。中 假设模型，蛋白质结构域的分子内缔合的变构调节调节 MAGUK函数。多种MAGUK蛋白的体外结合试验先前证明了非- SH 3结构域与GK结构域的典型关联，其缺乏在细胞中常见的聚脯氨酸基序。 SH 3配体。此外，研究表明，连续连接的SH 3和GK的分子内缔合 结构域比分子间缔合更优选。这种相互作用被证明是重要的， MAGUK的功能，因为SH 3和GK结构域中的所有遗传鉴定的突变已被证明是 破坏SH 3-GK分子内相互作用。一种这样的突变等位基因dlgsw编码一种突变形式的 盘状大（Dig）肿瘤抑制蛋白，并破坏分子内缔合，同时留下 SH 3和GK结构域完整。只含有sw形式Dig的果蝇被证明是胚胎 致命的另外的研究提供了证据，表明蛋白质配体与HOOK结构域的相互作用， 连接SH 3和GK结构域调节这种分子内相互作用并最终调节复杂的组装。 我们所提出的研究的目的是检验SH 3-GK的变构修饰 分子内相互作用调节复杂装配。因此，提出了以下具体目标：（1） 确定SH 3-GK分子内相互作用对Dig在 通过野生型和sw Dig复合物的免疫共沉淀，随后通过鉴定蛋白质 使用Western印迹分析和质谱分析，（2）确定分子内的 免疫荧光实验研究上皮组织与细胞不对称分裂相互作用 评估WT与sw Dig中连接和细胞命运决定性标志物的细胞定位， 含有胚胎，和（3）确定HOOK配体的结合对Dig分子内 相互作用以评估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Dig组织的分子机制 信号传导复合物，以允许调节的不对称细胞分裂。这些研究还将提供 进一步了解Dig失活导致肿瘤形成和转移的原因。

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.