Intramolecular Interactions Between the Src Homology 3 Guanylate Kinase Domains of Discs Large Regulate Its Function in Asymmetric Cell Division

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.The membrane-associated guanylate kinase (MAGUK) 2 superfamily consists of ubiquitous scaffolding proteins that are 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). MAGUKs are concentrated at sites of cellcell contact (1) and organize a variety of cell adhesion molecules, cytoskeletal proteins, receptors, ion channels, and associated signaling molecules at specialized regions of the membrane (2). Protein complex organization by MAGUKs has been thought to occur, at least in part, through allosteric regulation that arises from an intramolecular interaction between the SH3 and GK domains. This interaction has been shown to regulate binding of numerous MAGUK ligands in vitro (3-5). However, whereas the SH3-GK interaction has been extensively characterized biochemically (6 -8), very little data exists regarding its physiological relevance. Here we investigate the role of the SH3-GK intramolecular interaction of Discs large (Dlg), a prototype of the MAGUK superfamily.Loss of dlg activity results in overgrowth of imaginal discs and tumor formation (9). Dlg localizes to septate and neuromuscular junctions and is essential for establishing and maintaining apicobasal polarity of Drosophila epithelia (10). Dlg also plays an important role in regulating the process of asymmetric cell division (ACD) (11-13). ACD is a mechanism for generating cellular diversity via unequal mitotic divisions of progenitor cells. For instance, in wild-type Drosophila...

The Structure of the PDZ3-SH3-GuK Tandem of ZO-1 Protein Suggests a Supramodular Organization of the Membrane-associated Guanylate Kinase (MAGUK) Family Scaffold Protein Core

“…Deletion of or replacement of the PDZ3/SH3-linker with flexible residues diminishes the GuK-mediated binding to GuK-Holder (5). Additionally, binding of a target peptide to PDZ3 weakens the GuK-Holder binding ability of the SH3-GuK module, implying a conformational coupled allosteric regulation between PDZ3 and SH3-GuK (6). In another example, the interaction of the GuK domain of mammalian PSD-93/ DLG2 to MAP1a is also allosterically regulated by its PDZ3, albeit in this case, binding of the PDZ ligands releases its inhibition of the GuK/MAP1a interaction (22).…”

“…1A). Recent studies have demonstrated that functional interaction between Dlg SH3-GuK tandem and its partner GuKHolder is dependent on the presence of the upstream PDZ domain of Dlg and that this interaction is further regulated by the PDZ domain binding peptide (5,6). Considering the highly conserved domain organization feature of MAGUKs throughout evolution, we hypothesized that the signature PDZ-SH3-GuK tandem of MAGUKs may form a structural supramodule with three domains interacting with each other to assemble into an integral structural unit, which has functions distinct from those of the isolated domains or the simple sum of the individual domains.…”

Protein Kinase Cα Promotes Cell Migration through a PDZ-Dependent Interaction with its Novel Substrate Discs Large Homolog 1 (DLG1)

“…In the fly, SH3-Hook mutants or mutations that otherwise disrupt SH3-GUK binding phenocopy DLG null mutants (50,51); thus, this region is critical for the function of this scaffolding protein. The Hook domain of DLG1 cooperates with the SH3 domain to bind the GUK intramolecularly and regulate the accessibility of this domain to binding partners (52).…”

Clustering of CARMA1 through SH3–GUK domain interactions is required for its activation of NF-κB signalling

“…1a,b). A mutant of Dlg, dlg sw , bearing a COOHterminal truncation following the GUK domain, comparable to a 13-aa truncation of PSD-95 containing the bF strand, disrupted the intramolecular SH3-GUK interaction both in Dlg and PSD-95 while leaving the SH3 and GUK domains intact 12 . The bF truncation of CARMA1 corresponding to the dlg sw mutation disrupted the intermolecular SH3-GUK interaction of CARMA1 (Fig.…”

“…All MAGUK proteins comprise one to three PDZ domains, an SH3 domain and a GUK domain that lacks catalytic activities but which acts as a protein interaction domain. Previous studies have suggested that organization of the protein complex by MAGUKs occurs through allosteric regulation of intra-and intermolecular interactions via the SH3 and GUK domains [6][7][8][9][10][11][12] . The importance of the SH3-GUK domain interaction for MAGUK function was demonstrated by genetic studies in invertebrates that identified numerous loss-of-function mutations in the SH3 and GUK domains of disc large (dlg) in Drosophila 13 and lin-2 in Caenorhabditis elegans 14 , all of which could disrupt SH3-GUK interactions 6 .…”