Intramolecular Interactions Between the Src Homology 3 Guanylate Kinase Domains of Discs Large Regulate Its Function in Asymmetric Cell Division
2009; Elsevier BV; Volume: 284; Issue: 19 Linguagem: Inglês
10.1074/jbc.m809304200
ISSN1083-351X
AutoresRhonda A. Newman, Kenneth E. Prehoda,
Tópico(s)Cellular Mechanics and Interactions
ResumoMembrane-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), dlgsw, 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 dlgsw 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 Dlgsw. 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. 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), dlgsw, 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 dlgsw 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 Dlgsw. 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) 2The abbreviations used are: MAGUK, membrane-associated guanylate kinase; ACD, asymmetric cell division; aPKC, atypical protein kinase C; CaVβ, β subunit of voltage-gated calcium channels; Cript, cysteine-rich interactor of PDZ; Dlg, discs large tumor suppressor protein; dlgsw,m/z, dlgsw mutant in the absence of maternal and zygotic contributions of WT Dlg; Eve, Even-skipped; GFP-MUDp, a green fluorescent protein-peptide fusion derived from the mushroom body defect protein; GLC, germline clone; GukH, GukHolder, isoform C; Lgl, lethal giant larvae; Mira, Miranda; PDZ, PSD-95/Dlg/ZO-1; PDZ1, the first PDZ domain of Dlg; PDZ3, the third PDZ domain of Dlg; Pins, Partner of Inscutable; Scrib, Scribble; SH3, Src homology 3; WT, wild type; GFP, green fluorescent protein; HA, hemagglutinin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Pipes, 1,4-piperazinediethanesulfonic acid. 2The abbreviations used are: MAGUK, membrane-associated guanylate kinase; ACD, asymmetric cell division; aPKC, atypical protein kinase C; CaVβ, β subunit of voltage-gated calcium channels; Cript, cysteine-rich interactor of PDZ; Dlg, discs large tumor suppressor protein; dlgsw,m/z, dlgsw mutant in the absence of maternal and zygotic contributions of WT Dlg; Eve, Even-skipped; GFP-MUDp, a green fluorescent protein-peptide fusion derived from the mushroom body defect protein; GLC, germline clone; GukH, GukHolder, isoform C; Lgl, lethal giant larvae; Mira, Miranda; PDZ, PSD-95/Dlg/ZO-1; PDZ1, the first PDZ domain of Dlg; PDZ3, the third PDZ domain of Dlg; Pins, Partner of Inscutable; Scrib, Scribble; SH3, Src homology 3; WT, wild type; GFP, green fluorescent protein; HA, hemagglutinin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Pipes, 1,4-piperazinediethanesulfonic acid. 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 cell-cell contact (1Dimitratos S.D. Woods D.F. Stathakis D.G. Bryant P.J. Bioessays. 1999; 21: 912-921Crossref PubMed Scopus (197) Google Scholar) and organize a variety of cell adhesion molecules, cytoskeletal proteins, receptors, ion channels, and associated signaling molecules at specialized regions of the membrane (2Funke L. Dakoji S. Bredt D.S. Annu. Rev. Biochem. 2005; 74: 219-245Crossref PubMed Scopus (370) Google Scholar). 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 (3Qian Y. Prehoda K.E. J. Biol. Chem. 2006; 281: 35757-35763Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 4Wu H. Reissner C. Kuhlendahl S. Coblentz B. Reuver S. Kindler S. Gundelfinger E.D. Garner C.C. EMBO J. 2000; 19: 5740-5751Crossref PubMed Scopus (94) Google Scholar, 5Brenman J.E. Topinka J.R. Cooper E.C. McGee A.W. Rosen J. Milroy T. Ralston H.J. Bredt D.S. J. Neurosci. 1998; 18: 8805-8813Crossref PubMed Google Scholar). However, whereas the SH3-GK interaction has been extensively characterized biochemically (6McGee A.W. Bredt D.S. J. Biol. Chem. 1999; 274: 17431-17436Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 7Paarmann I. Spangenberg O. Lavie A. Konrad M. J. Biol. Chem. 2002; 277: 40832-40838Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 8Nix S.L. Chishti A.H. Anderson J.M. Walther Z. J. Biol. Chem. 2000; 275: 41192-41200Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), 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 (9Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Abstract Full Text PDF PubMed Scopus (766) Google Scholar). Dlg localizes to septate and neuromuscular junctions and is essential for establishing and maintaining apicobasal polarity of Drosophila epithelia (10De Lorenzo C. Mechler B.M. Bryant P.J. Cancer Metastasis Rev. 1999; 18: 295-311Crossref PubMed Scopus (34) Google Scholar). Dlg also plays an important role in regulating the process of asymmetric cell division (ACD) (11Peng C.Y. Manning L. Albertson R. Doe C.Q. Nature. 2000; 408: 596-600Crossref PubMed Scopus (287) Google Scholar, 12Ohshiro T. Yagami T. Zhang C. Matsuzaki F. Nature. 2000; 408: 593-596Crossref PubMed Scopus (269) Google Scholar, 13Siegrist S.E. Doe C.Q. Cell. 2005; 123: 1323-1335Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). ACD is a mechanism for generating cellular diversity via unequal mitotic divisions of progenitor cells. For instance, in wild-type Drosophila neuroblasts, cell signaling networks interact to allow for asymmetric segregation of basal cell fate determinants, followed by alignment of the mitotic spindle along the apical-basal cortical polarity axis (see Refs. 14Gonczy P. Nat. Rev. Mol. Cell Biol. 2008; 9: 355-366Crossref PubMed Scopus (389) Google Scholar and 15Knoblich J.A. Cell. 2008; 132: 583-597Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar) for review). ACD results in the formation of a self-renewing, stem-cell like neuroblast and a smaller ganglion mother cell, which has neuronal or glial fate. In dlg germline clone (dlgGLC) embryonic metaphase neuroblasts, ganglion mother cell fate determinants are not restricted to the basal cortex (11Peng C.Y. Manning L. Albertson R. Doe C.Q. Nature. 2000; 408: 596-600Crossref PubMed Scopus (287) Google Scholar, 12Ohshiro T. Yagami T. Zhang C. Matsuzaki F. Nature. 2000; 408: 593-596Crossref PubMed Scopus (269) Google Scholar) and the mitotic spindle does not reliably align with the apical-basal cortical polarity axis (13Siegrist S.E. Doe C.Q. Cell. 2005; 123: 1323-1335Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Defects in neurogenesis have also been observed for dlgGLC embryos (16Perrimon N. Dev. Biol. 1988; 127: 392-407Crossref PubMed Scopus (69) Google Scholar), as well as embryos treated with RNA interference against an alternatively spliced isoform of Dlg (17Mendoza C. Olguin P. Lafferte G. Thomas U. Ebitsch S. Gundelfinger E.D. Kukuljan M. Sierralta J. J. Neurosci. 2003; 23: 2093-2101Crossref PubMed Google Scholar), providing evidence for the function of Dlg in neuronal differentiation and axon guidance. Such defects in neurogenesis are thought to be attributed to defective localization of basal cell fate determinants during ACD (17Mendoza C. Olguin P. Lafferte G. Thomas U. Ebitsch S. Gundelfinger E.D. Kukuljan M. Sierralta J. J. Neurosci. 2003; 23: 2093-2101Crossref PubMed Google Scholar). Although Dlg function is important in a broad range of dynamic cellular processes, the role of the Dlg SH3-GK intramolecular interaction in Dlg activity is poorly understood. One potential role for the SH3-GK intramolecular association is to regulate MAGUK complex assembly. In vitro binding assays demonstrated that mutations disrupting this intramolecular interaction allowed mutant SH3-GK modules to associate with SH3 or GK domains of various MAGUK proteins in trans, providing a mechanism of regulating oligomerization of MAGUKs in vivo (6McGee A.W. Bredt D.S. J. Biol. Chem. 1999; 274: 17431-17436Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 8Nix S.L. Chishti A.H. Anderson J.M. Walther Z. J. Biol. Chem. 2000; 275: 41192-41200Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A role in clustering of ion channels was also observed as mutations that disrupted the intramolecular association, whereas having no effect on association with the potassium channel KV1.4 or homo-oligomerization of PSD-95, resulted in loss of channel clustering in vivo (18Shin H. Hsueh Y.P. Yang F.C. Kim E. Sheng M. J. Neurosci. 2000; 20: 3580-3587Crossref PubMed Google Scholar). Furthermore, multiple in vitro studies support its regulation of binding of protein ligands with the GK domain of MAGUKs: examples include interaction of GK domains of Dlg with GukHolder (GukH) (3Qian Y. Prehoda K.E. J. Biol. Chem. 2006; 281: 35757-35763Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), SAP97 with guanylate kinase-associated protein (4Wu H. Reissner C. Kuhlendahl S. Coblentz B. Reuver S. Kindler S. Gundelfinger E.D. Garner C.C. EMBO J. 2000; 19: 5740-5751Crossref PubMed Scopus (94) Google Scholar), and PSD-93 with the microtubule-associated protein 1A (5Brenman J.E. Topinka J.R. Cooper E.C. McGee A.W. Rosen J. Milroy T. Ralston H.J. Bredt D.S. J. Neurosci. 1998; 18: 8805-8813Crossref PubMed Google Scholar). These studies suggest that allosteric modulation of the SH3-GK intramolecular interaction is important for regulation of complex assembly, yet little evidence exists that such regulation of MAGUKs is required for their function in vivo. In crystal structures of the SH3-GK module of PSD-95, two β-strands that emerge from the SH3 and GK domains appear to mediate the interaction between the domains (Fig. 1A) (19McGee A.W. Dakoji S.R. Olsen O. Bredt D.S. Lim W.A. Prehoda K.E. Mol. Cell. 2001; 8: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 20Tavares G.A. Panepucci E.H. Brunger A.T. Mol. Cell. 2001; 8: 1313-1325Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Functional studies of the interaction of these β-strands demonstrate that a COOH-terminal truncation of the strand following the GK domain abrogates the intramolecular association of the SH3-GK module. This 13-amino acid truncation of PSD-95 is comparable with a dlg allele that had previously been identified in a genetic screen, dlgsw (21Woods D.F. Hough C. Peel D. Callaini G. Bryant P.J. J. Cell Biol. 1996; 134: 1469-1482Crossref PubMed Scopus (353) Google Scholar). Drosophila containing only the sw form of Dlg (refer to COOH-terminal sequences in Fig. 1B) die in the latter stages of embryonic development due to failure of dorsal closure and terminal defects (21Woods D.F. Hough C. Peel D. Callaini G. Bryant P.J. J. Cell Biol. 1996; 134: 1469-1482Crossref PubMed Scopus (353) Google Scholar). We hypothesized that this mutation disrupts the intramolecular interaction while leaving the SH3 and GK domains largely intact, making the dlgsw allele an excellent model system for assessing the role of this interaction in MAGUK function. In the studies presented here, we utilized the dlgsw mutant allele to explore the role of the intramolecular interaction in regulating complex assembly and furthermore, to assess the in vivo, physiological significance of this interaction for Dlg function in neuroblast asymmetric cell division. We find that disruption of the SH3-GK intramolecular interaction results in unregulated complex assembly. Such a loss in regulated complex assembly results in loss of Dlg function in the process of asymmetric cell division as assessed by localization of cell fate determinants in a dividing neuroblast and alignment of the mitotic spindle along the apical-basal cortical polarity axis, ultimately resulting in defects in neurogenesis. Thus, these data contribute to our understanding of how the SH3-GK intramolecular interaction regulates MAGUK function in formation and regulation of membrane specializations. Cloning, Overexpression, and Purification—DNA encoding the G isoform of wild-type (WT) and the sw mutant of Dlg were cloned from cDNA libraries that were created by reverse transcription of mRNAs extracted from yw and dlgsw Drosophila melanogaster, respectively. DNA encoding full-length Cript was cloned from a cDNA library created by reverse transcription of mRNAs extracted from 3T1 Mus musculus cells. For bacterial expression, WT Dlg PDZ-SH3-GK (amino acids 474–975), SH3-GK-(598–975), SH3-(581–681), E-GK-F-(771–975), E-GK-(771–962), and corresponding sw mutant fragments were subcloned and ligated into the pET-19b derivative pBH and/or pGEX vectors. Recombinant His-tagged fusions of Dlg proteins were purified using nickel-nitrilotriacetic acid (Qiagen) and Q-Sepharose anion exchange (Sigma) chromatography. In some instances, these proteins required further purification using HiLoad 16/60 Superdex 200 (GE Healthcare) chromatography. For bacterial expression, WT Cript was subcloned and ligated into the pET-19b derivative pBH containing an N-terminal green fluorescent protein fusion. Recombinant His-tagged fusions of GFP-Cript required single step purification using nickel-nitrilotriacetic acid (Qiagen) chromatography. Purity of recombinantly expressed proteins was judged to be >90% using SDS-PAGE and high pressure liquid chromatography. Protein concentrations were determined by Bradford assays and UV absorbance in the presence of 0.1 n NaOH using the extinction coefficient for Trp (22Beaven G.H. Holiday E.R. Adv. Protein Chem. 1952; 7: 319-386Crossref PubMed Scopus (615) Google Scholar). For S2 cell expression of a hemagglutinin (HA)-tagged GukH fragment, the sequence coding for amino acids 800–1044 of the isoform C of GukH was subcloned and ligated into a pMT-/V5-HisA vector (Invitrogen). Drosophila embryo-derived Schneider cells (S2 cells) grown in Schneider's insect media supplemented with 10% fetal bovine serum were transiently transfected using 0.8 μg of pMT-HA-(800–1044) GukH DNA and Effectene reagent (Qiagen). Protein expression was induced after 24 h with 0.5 mm CuSO4 and cells were collected 48 h post-induction. Cells were pelleted by centrifugation at 1,000 × g for 5 min, washed twice in ice-cold phosphate-buffered saline (PBS), and incubated in lysis buffer (50 mm Tris, pH 8, 150 mm NaCl, 1% Nonidet P-40, and a complete protease inhibitor mixture from Roche; 200 μl/transfection) for 30 min on ice. Following incubation, transfected S2 cells were passaged through a 23-guage needle and cell debris was removed by centrifugation at 12,000 × g for 15 min. In Vitro Binding Assays—For qualitative in trans GST pull-downs (Fig. 1C), Escherichia coli cell lysates containing the GST fusion protein of interest were incubated with glutathione-agarose beads and washed three times with binding buffer (100 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm dithiothreitol, 0.5% Triton X-100). A His-tagged fusion of the SH3 domain of Dlg was added to a concentration of 25 μm and incubated with the beads at room temperature for 15 min. The reactions were then washed three times with binding buffer to remove unassociated proteins. Bound proteins were eluted from the glutathione-agarose beads by the addition of SDS loading buffer and were screened by Western blot analyses using a mouse monoclonal anti-His antibody (1:1000; Qiagen). For qualitative GST pull-downs to assess HA-(800–1044) GukH association with Dlg in the absence and presence of GFP-Cript (Fig. 6C), E. coli cell lysates containing GST fusions of WT PDZ-SH3-GK or PDZ-SH3-GKsw were incubated with glutathione-agarose beads and then washed three times with PBS. Subsequently, 150 μl of S2 cell extract, containing HA-(800–1044) GukH, was added in the absence or presence of 2.5 or 5 μm GFP-Cript or a GFP-peptide fusion derived from the mushroom body defect protein (GFP-MUDp) and incubated at 4 °C for 1 h. The reactions were then washed three times with 10 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm dithiothreitol, and 0.002% Triton X-100. Bound proteins were eluted using SDS loading buffer and screened by Western blot analysis using a mouse monoclonal anti-HA (1:1000; Covance) and Ponceau stain. Circular Dichroism Scans—Circular dichroism (CD) spectra were collected using a Jasco J-720 CD spectrophotometer equipped with a temperature controlled cell holder, using a 1-nm bandpass and scanning from 260 to 190 in 0.5-nm increments. Three scans in a 0.1-cm path length quartz cuvette were recorded and averaged. Spectra were collected in 10 mm NaPO4, pH 7.5, 100 mm NaCl at 22 °C. Spectra were corrected for contributions from buffer alone by subtraction (23Andrade M.A. Chacon P. Merelo J.J. Moran F. Protein Eng. 1993; 6: 383-390Crossref PubMed Scopus (949) Google Scholar). Analytical Ultracentrifugation—Sedimentation velocity studies of WT PDZ-SH3-GK and PDZ-SH3-GKsw were conducted on a Beckman XL-I analytical ultracentrifuge at 50,000 × g at 20 °C. Proteins were equilibrated in 20 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm Tris(2-carboxyethyl)phosphine. Sedimentation was continuously monitored for 5 h using an absorbance of 280 nm. Sedimentation velocity data were subsequently analyzed using Sedfit (24Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3045) Google Scholar, 25Schuck P. Perugini M.A. Gonzales N.R. Howlett G.J. Schubert D. Biophys. J. 2002; 82: 1096-1111Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Fly Stocks and Collection of Embryos—Flies of the genotype yw,dlgsw/Basc,y+ were kindly provided by Francois Schweisguth (Ecole Normale Supêrieure, Paris, France). Flies were raised at 18 °C and virgin homozygous yw,dlgsw females, and heterozygous yw,dlgsw/Y males were sorted from flies containing the Basc balancer. These flies were crossed to allow for collection of embryos with no maternal or zygotic contribution of WT Dlg (i.e. dlgsw,m/z). Fixation and Antibody Staining of Drosophila Embryos and Dissociated Neuroblasts—Drosophila embryos were dechorinated by rinsing embryos in bleach solution for 1–2 min and then fixed using a biphasic solution of heptane over 4% formaldehyde in PEM buffer (100 mm Pipes, pH 6.9, 1 mm EGTA, 1 mm MgSO4) for 25 min. Upon fixation, embryos were blocked in PBS, 0.3% Triton X-100, and 1% bovine serum albumin. In vitro neuroblast cultures from yw and dlgsw,m/z embryos were prepared using previously published methods (26Grosskortenhaus R. Pearson B.J. Marusich A. Doe C.Q. Dev. Cell. 2005; 8: 193-202Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Cells were settled onto a coverslip for 30 min and fixed using 4% formaldehyde in PBS for 20 min. Upon fixation, cells were blocked in PBS, 0.1% saponin, and 1% bovine serum albumin. Embryos and dissociated neuroblasts were then stained using various primary antibodies, including rabbit anti-phospho-histone H3 (1:500–1:1000; Upstate), mouse anti-α-tubulin (1:1000; Sigma), mouse anti-γ-tubulin (1:1000; Sigma), mouse anti-Dlg 4F3E3 (1:100 (27Parnas D. Haghighi A.P. Fetter R.D. Kim S.W. Goodman C.S. Neuron. 2001; 32: 415-424Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar)), rabbit anti-Scribble (1:1000 (37Albertson R. Doe C.Q. Nat. Cell Biol. 2003; 5: 166-170Crossref PubMed Scopus (201) Google Scholar)), rabbit anti-aPKCζ (1:100–1:500; Santa Cruz Biotechnology Inc.), rat anti-Pins (1:500 (28Yu F. Morin X. Cai Y. Yang X. Chia W. Cell. 2000; 100: 399-409Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar)), guinea pig anti-Miranda (1:500 (29Lee C.Y. Wilkinson B.D. Siegrist S.E. Wharton R.P. Doe C.Q. Dev. Cell. 2006; 10: 441-449Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar)), mouse anti-Prospero (1:100 (30Spana E.P. Doe C.Q. Development. 1995; 121: 3187-3195PubMed Google Scholar)), rabbit anti-zipper (1:500 (31Liu S.L. Fewkes N. Ricketson D. Penkert R.R. Prehoda K.E. J. Biol. Chem. 2008; 283: 380-387Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar)), rabbit anti-Insc (1:1000; W. Chia), rabbit anti-HB9 (1:500–1:1000 (32Odden J.P. Holbrook S. Doe C.Q. J. Neurosci. 2002; 22: 9143-9149Crossref PubMed Google Scholar)), guinea pig anti-Eve (1:1000; East Asian Distribution Center for Segmentation Antibodies), and mouse anti-FasII (1:100; C. Goodman) followed by treatment with fluorescent-conjugated secondary antibodies from Jackson Immunoresearch Laboratories and Invitrogen. Images were collected using a Leica TCS SP2 confocal using a ×63/1.4NA objective. ImageJ, Photoshop, and Illustrator software were used for subsequent data analysis and preparation of figures. Yeast Two-hybrid—For yeast two-hybrid, pGADT7 and pGBKT7 vectors (Clontech, Palo Alto, CA) were used to monitor association reactions. GUKHolder (amino acids 749–1044) was cloned into the DNA-binding domain vector (pGBKT7), whereas all Dlg constructs were cloned into the Gal4 activation domain vector (pGADT7). The Saccharomyces cerevisiae AH109 strain (Clontech) was cotransformed with pGADT7 and pGBKT7 plasmids and plated on SD medium (–Leu/–Trp). Colonies were subsequently screened for interaction by screening for growth on SD medium (–His/–Leu/–Trp + 20 mm 3-amino-1,2,4-triazole) and for β-galactosidase activity using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) as a substrate. The Dlgsw Mutant Selectively Disrupts the SH3-GK Intramolecular Interaction While Leaving the Domains Intact—As alternative splicing and multiple promoters lead to numerous Dlg isoforms (17Mendoza C. Olguin P. Lafferte G. Thomas U. Ebitsch S. Gundelfinger E.D. Kukuljan M. Sierralta J. J. Neurosci. 2003; 23: 2093-2101Crossref PubMed Google Scholar, 21Woods D.F. Hough C. Peel D. Callaini G. Bryant P.J. J. Cell Biol. 1996; 134: 1469-1482Crossref PubMed Scopus (353) Google Scholar), 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. Although the truncation of the sequence after the GK domain caused by the frameshift is predicted to disrupt the intramolecular interaction, this has not been confirmed. Furthermore, this mutation could cause domain unfolding. We used in trans, 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. This analysis (Fig. 1C) revealed that a GST fusion of the WT GK domain construct that includes the F strand (i.e. E-GK-F) associates with the SH3 domain construct that contains β-strands A-E (i.e. SH3(A-E)), whereas constructs that lack the F strand (i.e. E-GK or E-GK-sw) are unable to associate with the SH3 domain. These results are consistent with previous reports that indicate the role of interaction of E and F β-strands in formation of the SH3-GK intramolecular interaction (19McGee A.W. Dakoji S.R. Olsen O. Bredt D.S. Lim W.A. Prehoda K.E. Mol. Cell. 2001; 8: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 20Tavares G.A. Panepucci E.H. Brunger A.T. Mol. Cell. 2001; 8: 1313-1325Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Although the intramolecular interaction is disrupted in Dlgsw, it is possible that the truncation encoded by this allele disrupts the integrity of the SH3 and GK domains. To ensure that the Dlgsw protein is correctly folded, we assessed the overall secondary structure of WT PDZ-SH3-GK and mutant PDZ-SH3-GKsw using circular dichroism spectroscopy. Circular dichroism-monitored wavelength scans (Fig. 1D) indicate that the Dlgsw mutations do not compromise the overall fold of the GK domain, as no significant change in α-helical content (i.e. Δ molar ellipticity at 222 nm) was observed. Dlg is known to oligomerize through a region NH2-terminal to the PDZ1 domain (33Hsueh Y.P. Kim E. Sheng M. Neuron. 1997; 18: 803-814Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 34Marfatia S.M. Byron O. Campbell G. Liu S.C. Chishti A.H. J. Biol. Chem. 2000; 275: 13759-13770Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). However, intermolecular interactions have also been proposed to occur between MAGUK SH3-GK modules. To determine whether the sw mutation affects the oligomerization state of the SH3 and GK domains, we monitored the oligomerization state of WT PDZ-SH3-GK and mutant PDZ-SH3-GKsw using analytical ultracentrifugation sedimentation velocity experiments. Results from these experiments indicate that both the sw and wild-type proteins are monomeric in solution (Fig. 1E). Thus, we conclude that the oligomerization state of Dlg is unlikely to be affected in the context of full-length Dlgsw. This conclusion is also supported by analysis of a PSD-95 mutation, PSD-95ΔC, which lacks the 13 COOH-terminal residues, which does not disrupt its ability to associate with this NH2-terminal multimerization domain (18Shin H. Hsueh Y.P. Yang F.C. Kim E. Sheng M. J. Neurosci. 2000; 20: 3580-3587Crossref PubMed Google Scholar). Together, these results support the use of Dlgsw as a model for understanding the role of the SH3-GK intramolecular interaction in Dlg function, as this mutant specifically disrupts the interaction while leaving the SH3 and GK domains largely intact. Localization of Discs Large and Scribble Are Not Regulated by the SH3-GK Intramolecular Interaction—To assess the role of the SH3-GK intramolecular interaction in Dlg function, we examined neuroblasts from dlgsw,m/z embryos (those lacking both maternal and zygotic wild-type dlg activity) for defects in asymmetric cell division. In wild-type embryos, Dlg localizes to the neuroblast cortex with enrichment at the apical region of the cell, and is required for cortical polarization and spindle positioning (11Peng C.Y. Manning L. Albertson R. Doe C.Q. Nature. 2000; 408: 596-600Crossref PubMed Scopus (287) Google Scholar, 12Ohshiro T. Yagami T. Zhang C. Matsuzaki F. Nature. 2000; 408: 593-596Crossref PubMed Scopus (269) Google Scholar, 13Siegrist S.E. Doe C.Q. Cell. 2005; 123: 1323-1335Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). We first determined if the SH3-GK intramolecular interaction is required for Dlg targeting by examining Dlgsw protein localization. Localization of Dlg to membrane specializations such as the postsynapse requires interaction of the HOOK domain (see Fig. 1A), as well as the first two PDZ domains of Dlg with cytoskeletal-binding partners (35Thomas U. Ebitsch S. Gorczyca M. Koh Y.H. Hough C.D. Woods D. Gundelfinger E.D. Budnik V. Curr. Biol. 2000; 10: 1108-1117Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Given the intervening position of the HOOK between the SH3 and GK domains of Dlg we determined whether the localization of Dlg in Drosophila neuroblasts is affected by disruption of the SH3-GK intramolecular interaction. However, as shown in Fig. 2D, localization of Dlgsw is indistinguishable from wild-type (Fig. 2A). We also assessed localization of Scribble (Scrib), which forms a tripartite complex with GukHolder and Dlg (36Mathew D. Gramates L.S. Packard M. Thomas U. Bilder D. Perrimon N. Gorczyca M. Budnik V. Curr. Biol. 2002; 12: 531-539Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Previous studies of Scri
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