Regulated Complex Assembly in Membrane-Associated Guanylate Kinases

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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.