Regulated Complex Assembly in Membrane-Associated Guanylate Kinases

膜相关鸟苷晚期蛋白(MAGUK)是一种普遍存在的蛋白质，由一种 PDZ域、SH3域和GK域的共同核心。Maguk超级家族的成员 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 假想模型，蛋白质结构域分子内结合的变构调节 Maguk函数。多种Maguk蛋白的体外结合分析表明， SH3结构域与GK结构域的典型关联，这些结构域缺乏通常在 SH3配体。此外，研究还表明，SH3和GK的分子内缔合 结构域优先于分子间缔合。这种相互作用被证明是重要的 MAGUK的功能是因为SH3和GK结构域的所有基因突变都已被证明是 破坏SH3-GK的分子内相互作用。一个这样的突变等位基因dlgsw编码了一种突变形式的 Diss Large(Dig)肿瘤抑制蛋白，并破坏分子内结合，同时离开 SH3和GK结构域完好无损。只含有Sw形式的Dig的果蝇被证明是胚胎 致命的。其他研究提供的证据表明，蛋白质配体与钩状域的相互作用 加入SH3和GK结构域调节这种分子内相互作用，最终形成复杂的组装。 我们提议的研究的目的是检验SH3-GK的变构修饰的假设 分子内相互作用调节复杂的组装。为此，提出了以下具体目标：(1) SH3-GK分子内相互作用对Dig形成的一系列络合物的影响 免疫共沉淀法鉴定野生型和SW地高辛复合体的蛋白质 采用Western印迹分析和质谱分析，(2)确定分子内 免疫荧光实验研究上皮组织与细胞不对称分裂的相互作用 评估连接和细胞命运决定标记在WT和SWDIG中的细胞定位 包含胚胎，以及(3)确定挂钩配体结合对分子内DIG的影响 相互作用，以评估MAGUK组织细胞信号复合体的分子机制。 这些研究将有助于我们理解Dig组织的分子机制。 信号复合体，以允许调节的不对称细胞分裂。这类研究还将提供 进一步了解为什么DIG失活会导致肿瘤形成和转移。

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.