Abstract
Membrane-associated guanylate kinases (MAGUKs) regulate the formation and function of molecular assemblies at specialized regions of the membrane. Allosteric regulation of an intramolecular interaction between the Src homology 3 (SH3) and guanylate kinase (GK) domains of MAGUKs is thought to play a central role in regulating MAGUK function. Here we show that a mutant of the Drosophila MAGUK Discs large (Dlg), dlg(sw), encodes a form of Dlg that disrupts the intramolecular association while leaving the SH3 and GK domains intact, providing an excellent model system to assess the role of the SH3-GK intramolecular interaction in MAGUK function. Analysis of asymmetric cell division of maternal-zygotic dlg(sw) embryonic neuroblasts demonstrates that the intramolecular interaction is not required for Dlg localization but is necessary for cell fate determinant segregation to the basal cortex and mitotic spindle alignment with the cortical polarity axis. These defects ultimately result in improper patterning of the embryonic central nervous system. Furthermore, we demonstrate that the sw mutation of Dlg results in unregulated complex assembly as assessed by GukHolder association with the SH3-GK versus PDZ-SH3-GK modules of Dlg(sw). From these studies, we conclude that allosteric regulation of the SH3-GK intramolecular interaction is required for regulation of MAGUK function in asymmetric cell division, possibly through regulation of complex assembly.
Highlights
12924 JOURNAL OF BIOLOGICAL CHEMISTRY composed of a common core of contiguously linked modular domains
The Dlgsw Mutant Selectively Disrupts the Src homology 3 (SH3)-guanylate kinase (GK) Intramolecular Interaction While Leaving the Domains Intact—As alternative splicing and multiple promoters lead to numerous Discs large (Dlg) isoforms [17, 21], we utilized the sw allelle that includes a frameshift mutation near the end of the coding region of the dlg locus to test for the role of the SH3-GK intramolecular interaction
Qualitative binding assays of an SH3 domain construct by WT or mutant GK domains to test for the ability of wild-type and mutant proteins to form an intramolecular interaction
Summary
12924 JOURNAL OF BIOLOGICAL CHEMISTRY composed of a common core of contiguously linked modular domains (protein-protein interaction domains, PDZ and SH3 domains, and a domain with homology to the yeast guanylate kinase, GK domain). In the studies presented here, we utilized the dlgsw mutant allele to explore the role of the intramolecular interaction in regulating complex assembly and to assess the in vivo, physiological significance of this interaction for Dlg function in neuroblast asymmetric cell division.
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