Identification of a DOCK180-related Guanine Nucleotide Exchange Factor That Is Capable of Mediating a Positive Feedback Activation of Cdc42
2006; Elsevier BV; Volume: 281; Issue: 46 Linguagem: Inglês
10.1074/jbc.m606248200
ISSN1083-351X
AutoresQiong Lin, Wannian Yang, Daniel Baird, Qiyu Feng, Richard A. Cerione,
Tópico(s)Cellular transport and secretion
ResumoCdc42, a member of the Rho subfamily of small GTPases, influences a wide range of activities including the establishment of cell polarity, migration, and the regulation of cell growth and differentiation. Here we describe the identification of an ∼220-kDa protein that formed a stable complex with activated forms of Cdc42 and thereby showed characteristics of a downstream target/effector for this GTPase. However, molecular cloning of the cDNA encoding this protein (p220) revealed that it was highly related to Zizimin-1 and identical in sequence to a gene product in the data base designated DOCK11, which are members of the DOCK180 family of guanine nucleotide exchange factors (GEFs) for Cdc42 and Rac. Biochemical characterization shows that p220 is a specific GEF for Cdc42, with the GEF activity originating from its DHR2 (for DOCK homology region 2) domain. Nucleotide-depleted Cdc42 forms a stable complex with the DHR2 domain, whereas the binding of activated Cdc42 requires both the DHR2 domain and residues 66-126 within the amino-terminal portion of p220. Moreover, the full-length protein shows markedly higher GEF activity than the isolated DHR2 domain, whereas removal of the amino-terminal 126 amino acids necessary for binding-activated Cdc42 dramatically diminishes the activity. These and other results point to activated Cdc42 providing a positive feedback regulation of the GEF activity of p220. Thus, we refer to p220/DOCK11 as activated Cdc42-associated GEF, befitting its functional activity. Cdc42, a member of the Rho subfamily of small GTPases, influences a wide range of activities including the establishment of cell polarity, migration, and the regulation of cell growth and differentiation. Here we describe the identification of an ∼220-kDa protein that formed a stable complex with activated forms of Cdc42 and thereby showed characteristics of a downstream target/effector for this GTPase. However, molecular cloning of the cDNA encoding this protein (p220) revealed that it was highly related to Zizimin-1 and identical in sequence to a gene product in the data base designated DOCK11, which are members of the DOCK180 family of guanine nucleotide exchange factors (GEFs) for Cdc42 and Rac. Biochemical characterization shows that p220 is a specific GEF for Cdc42, with the GEF activity originating from its DHR2 (for DOCK homology region 2) domain. Nucleotide-depleted Cdc42 forms a stable complex with the DHR2 domain, whereas the binding of activated Cdc42 requires both the DHR2 domain and residues 66-126 within the amino-terminal portion of p220. Moreover, the full-length protein shows markedly higher GEF activity than the isolated DHR2 domain, whereas removal of the amino-terminal 126 amino acids necessary for binding-activated Cdc42 dramatically diminishes the activity. These and other results point to activated Cdc42 providing a positive feedback regulation of the GEF activity of p220. Thus, we refer to p220/DOCK11 as activated Cdc42-associated GEF, befitting its functional activity. Cdc42 has been shown to regulate multiple cellular processes including actin cytoskeletal organization, membrane trafficking, cytokinesis, and cell polarity, as well as cell growth and differentiation (1Johnson D.I. Microbiol. Mol. Biol. Rev. 1999; 63: 54-105Crossref PubMed Google Scholar, 2Nobes C.D. Hall A. Biochem. Soc. Trans. 1995; 23: 456-459Crossref PubMed Scopus (303) Google Scholar, 3Cerione R.A. Trends Cell Biol. 2004; 14: 127-132Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The activation of Cdc42 is triggered by a variety of stimuli including growth factors, cytokines, cell adhesion factors, and conditions leading to cell stress (4Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (883) Google Scholar, 5Shekarabi M. Kennedy T.E. Mol. Cell Neurosci. 2002; 19: 1-17Crossref PubMed Scopus (151) Google Scholar, 6Wong K. Ren X.R. Huang Y.Z. Xie Y. Liu G. Saito H. Tang H. Wen L. Brady-Kalnay S.M. Mei L. Wu J.Y. Xiong W.C. Rao Y. Cell. 2001; 107: 209-221Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 7Driggers P.H. Segars J.H. Rubino D.M. J. Biol. Chem. 2001; 276: 46792-46797Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 8Ku G.M. Yablonski D. Manser E. Lim L. Weiss A. EMBO J. 2001; 20: 457-465Crossref PubMed Scopus (92) Google Scholar, 9Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (652) Google Scholar, 10Puls A. Eliopoulos A.G. Nobes C.D. Bridges T. Young L.S. Hall A. J. Cell Sci. 1999; 112: 2983-2992Crossref PubMed Google Scholar). More than a dozen downstream target/effectors for Cdc42 have been identified, including scaffold proteins like WASP and IQGAP, theγCOP subunit of the COPI complex that coats trafficking vesicles, as well as different serine/threonine and tyrosine kinases including p21-activated kinase, mixed lineage kinase, and activated Cdc42-associated kinase (11Bagrodia S. Taylor S.J. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 12Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 13Manser E. Leung T. Salihuddin H. Tan L. Lim L. Nature. 1993; 363: 364-367Crossref PubMed Scopus (260) Google Scholar, 14Yang W. Cerione R.A. J. Biol. Chem. 1997; 272: 24819-24824Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 15Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar, 16Erickson J.W. Cerione R.A. Hart M.J. J. Biol. Chem. 1997; 272: 24443-24447Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 17Leung T. Chen X.Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (220) Google Scholar, 18Joberty G. Petersen C. Gao L. Macara I.G. Nat. Cell Biol. 2000; 2: 531-539Crossref PubMed Scopus (765) Google Scholar, 19Wu W.J. Erickson J.W. Lin R. Cerione R.A. Nature. 2000; 405: 800-804Crossref PubMed Scopus (186) Google Scholar, 20Joberty G. Perlungher R.R. Macara I.G. Mol. Cell. Biol. 1999; 19: 6585-6597Crossref PubMed Scopus (105) Google Scholar). Thus, Cdc42 serves as a critical signaling switch converting an array of stimulatory inputs into a diversity of cellular responses and biological activities. The activation of Cdc42 is directly catalyzed by guanine nucleotide exchange factors (GEFs). 2The abbreviations used are: GEF, guanine nucleotide exchange factor; ACG, activated Cdc42-associated GEF; DH, Dbl homology; PH, Pleckstrin homology; DHR, DOCK homology region; HA, hemagglutinin; GST, glutathione S-transferase; PBS, phosphate-buffered saline; AMP-PNP, adenosine 5′-(β,γ-iminotriphosphate); GTPγS, guanosine 5′-O-(thiotriphosphate); ACB, activated Cdc42 binding. Dbl, which has potent transforming activity when truncated from its amino-terminal end, is the founding member of a family of GEFs for Cdc42 and related Rho GTPases (21Hart M.J. Eva A. Evans T. Aaronson S.A. Cerione R.A. Nature. 1991; 354: 311-314Crossref PubMed Scopus (337) Google Scholar). All of the members of this family are characterized by a tandem array of Dbl homology (DH) and Pleckstrin homology (PH) domains with the DH domains being primarily responsible for mediating GEF activity (22Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (983) Google Scholar, 23Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar, 24Erickson J.W. Cerione R.A. Biochemistry. 2004; 43: 837-842Crossref PubMed Scopus (115) Google Scholar). In addition to these conserved sequences, most members of the Dbl family of GEFs contain other functional or protein-protein interaction domains that help to connect the activation of Cdc42 or other related Rho GTPases to a number of downstream signaling partners and cellular activities. For example, Intersectin-1L, a Cdc42-specific GEF, uses five SH3 and two EH domains, together with the tandem DH/PH domain, to couple the activation of Cdc42 to the endocytosis of cell surface receptors, presumably by organizing the F-actin structure along the surfaces of endocytic vesicles (25McGavin M.K. Badour K. Hardy L.A. Kubiseski T.J. Zhang J. Siminovitch K.A. J. Exp. Med. 2001; 194: 1777-1787Crossref PubMed Scopus (107) Google Scholar). More recently, a new class of GEFs for Cdc42 and Rac that lack tandem DH and PH domains has been identified. Two examples are Zizimin-1 and DOCK180 (director of cytokinesis 180) (26Meller N. Irani-Tehrani M. Kiosses W.B. Del Pozo M.A. Schwartz M.A. Nat. Cell Biol. 2002; 4: 639-647Crossref PubMed Scopus (147) Google Scholar, 27Cote J.F. Vuori K. J. Cell Sci. 2002; 115: 4901-4913Crossref PubMed Scopus (345) Google Scholar). Members of this family of GEFs have relatively high molecular mass (∼200 kDa) and contain two conserved domains, designated CZH1 (for conserved Zizimin homology domain 1)/DHR1 (for DOCK homology region 1) and CZH2/DHR2. The CZH2/DHR2 domain represents the primary functional unit, because it catalyzes the nucleotide exchange activity of the GTPase substrates (26Meller N. Irani-Tehrani M. Kiosses W.B. Del Pozo M.A. Schwartz M.A. Nat. Cell Biol. 2002; 4: 639-647Crossref PubMed Scopus (147) Google Scholar, 27Cote J.F. Vuori K. J. Cell Sci. 2002; 115: 4901-4913Crossref PubMed Scopus (345) Google Scholar). Although Dbl family GEFs are not found in plants, members of the Zizimin-1/DOCK180 family are conserved throughout yeast, plants, and animals (26Meller N. Irani-Tehrani M. Kiosses W.B. Del Pozo M.A. Schwartz M.A. Nat. Cell Biol. 2002; 4: 639-647Crossref PubMed Scopus (147) Google Scholar, 27Cote J.F. Vuori K. J. Cell Sci. 2002; 115: 4901-4913Crossref PubMed Scopus (345) Google Scholar). Many members of the DOCK180 family, including DOCK180 itself, as well as DOCK2 and DOCK3, activate Rac to regulate cell adhesion, cell spreading, migration, and phagocytosis (28Wu Y.C. Horvitz H.R. Nature. 1998; 392: 501-504Crossref PubMed Scopus (141) Google Scholar, 29Fukui Y. Hashimoto O. Sanui T. Oono T. Koga H. Abe M. Inayoshi A. Noda M. Oike M. Shirai T. Sasazuki T. Nature. 2001; 412: 826-831Crossref PubMed Scopus (368) Google Scholar, 30Namekata K. Enokido Y. Iwasawa K. Kimura H. J. Biol. Chem. 2004; 279: 14331-14337Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). However, the function of Zizimin-1, a specific GEF for Cdc42, is still not known. In the classical model for the activation of small GTPases, the GEF interacts with the GDP-bound form of its substrate GTPase and catalyzes the dissociation of GDP. The relatively high cellular concentrations of GTP (relative to GDP) allow an exchange of the dissociated GDP for GTP, which then induces structural changes within the GTPase that enable it to interact with downstream signaling target/effector proteins. In many cases, the binding of GTP to the GTPase also prompts its dissociation from the GEF, thereby enabling the GEF to act catalytically in promoting GDP-GTP exchange on multiple GTPases. However, there have been some recent reports showing that GEFs can engage in stable complexes with the GTP-bound form of their substrate GTPase. One example is the Cool-2/α-Pix (cloned out of library 2/α p21-activated kinase-interactive exchange factor) protein, which contains the tandem arrangement of DH and PH domains characteristic of Dbl family GEFs but was originally identified through its ability to bind to the Cdc42/Rac-target p21-activated kinase (31Bagrodia S. Taylor S.J. Jordon K.A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 32Manser E. Looh T.H. Koh C.G. Zhao Z.S. Chen X.Q. Tan L. Tan I. Leung T. Lim L. Mol. Cell. 1998; 1: 183-192Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). Cool-2/α-Pix forms a stable complex with activated (GTP-bound) Cdc42, which in turn strongly stimulates the GEF activity of Cool-2/α-Pix toward Rac. This has led to the suggestion that Cool-2/α-Pix is able to mediate a Cdc42-Rac signaling cascade (33Baird D. Feng Q. Cerione R.A. Curr. Biol. 2005; 15: 1-10Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In this report, we have identified a Cdc42-GEF that exhibits an interesting variation on the theme described above. This new GEF is a member of the Zizimin-1/DOCK180 family, and because it was originally isolated through its ability to bind activated forms of Cdc42, we refer to it here as activated Cdc42-associated GEF (ACG). ACG appears to represent a novel example of a GEF that responds to activated Cdc42 in a manner that may allow for positive feedback stimulation of GDP-GTP exchange on additional Cdc42 molecules. Chemicals, Plasmids, and Antibodies—The site-directed mutagenesis kits and Pfu DNA polymerase were purchased from Stratagene. Texas Red-conjugated anti-mouse IgG was obtained from Molecular Probes (Invitrogen). Monoclonal anti-HA antibody (HA.11) was from Covance, and the IMAGE cDNA clones were purchased from ATCC and Open Biosystems. Preparation of GST Fusion Proteins and Their Use in Pull-down Assays—Escherichia coli containing plasmids encoding GST fusion proteins were cultured to a density of A600 = 1.0. Expression of the fusion proteins was induced by adding isopropyl β-d-thiogalactopyranoside (final concentration, 0.5 mm) to the culture medium for 3-5 h. The bacteria were precipitated by centrifugation at 6,000 × g for 10 min, and the pellet was resuspended in a bacterial lysis buffer (40 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.5% Triton X-100, 1 mm EDTA, 1 mm EGTA, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). The suspension was sonicated and centrifuged at 15,000 × g for 10 min. The supernatant was collected and incubated with glutathione-agarose beads (1:1 slurry in the bacterial lysis buffer) for 2 h at 4 °C with rotation. The beads were then washed (three times) with lysis buffer, and the immobilized GST fusion protein beads were resuspended in the same buffer. For large scale pull-down assays, the GST fusion protein beads containing 40-80 μg of protein were incubated with 10 ml of mammalian cell lysates (10-20 mg of protein) at 4 °C for 2-3 h with rotation. The beads were washed (three times) with mammalian cell lysis buffer (40 mm Hepes, pH 7.4, 100 mm NaCl, 1% Triton X-100, 25 mm β-glycerophosphate, 1 mm sodium orthovanadate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) (8-10 ml each time) and resuspended with 2× SDS-PAGE sample buffer. Following SDS-PAGE, the proteins that co-precipitated with the GST fusion proteins were visualized by staining with 0.5-1% Coomassie Blue. The bands of interest were excised and subjected to microsequencing by microcapillary reverse phase high pressure liquid chromatography nano-electrospray tandem mass spectrometry on a Finnigan LCQ DECA quadrupole ion trap mass spectrometer at the Harvard Microchemistry Facility. For small scale pull-down assays, the GST fusion protein beads containing 10-20 μg of protein were incubated with 0.5-1.0 ml of cell lysates (0.5-1.0 mg of protein) at 4 °C for 2-3 h with rotation. The beads were washed (three times) with lysis buffer and resuspended in 2× SDS-PAGE sample buffer. The co-precipitated proteins were separated by SDS-PAGE and identified by immunoblotting. Cell Culture, Immunoprecipitation, and Immunoblots—Mouse embryonic teratoma (P19) cells and COS7 cells were cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum and 5% CO2 at 37 °C. The transfection of plasmid DNA into COS7 cells was performed using the Lipofectamine transfection kit (Invitrogen). The cells were lysed after 48 h of transfection. For the preparation of cell lysates, the cells were rinsed with PBS once and lysed with mammalian cell lysis buffer (0.5 ml/60-mm dish or 1 ml/100-mm dish) with rocking at 4 °C for 20 min. The lysates were collected and cleared by centrifugation at 14,000 rpm for 4 min and then immediately used. For immunoprecipitation, the precleared cell lysates were incubated with primary antibody on ice for 30 min. Protein A beads were then added, and the mixture was incubated at 4 °C for 2 h with rotation. The beads were washed (three times) with mammalian cell lysis buffer, and the immunoprecipitated complexes were used either for GEF assays or directly dissolved in sample buffer for SDS-PAGE. Immunoblots were performed as suggested in the instructions accompanying the ECL immunoblot kits (Amersham Biosciences). Immunofluorescence Staining—The cells were cultured on coverslips to 50-80% confluence. After the culture medium was removed, the cells were rinsed (two times) with PBS, fixed with 3.7% formaldehyde at 25 °C for 10 min, and permeabilized with 0.2% Triton X-100 in PBS at 25 °C for 10 min. Following washing with PBS, the cells were incubated with primary antibody at 37 °C for 30 min. The cells were then washed with PBS (three times) and incubated with secondary antibody that was conjugated with a fluorescent dye at 37 °C for 30 min. Finally, the cells were washed with PBS (three times for 10 min each time). The coverslips were mounted on glass slides, and immunofluorescence staining was monitored with a Leica confocal fluorescent microscope. Guanine Nucleotide Exchange Assays—Nucleotide exchange activity was assayed as previously described (21Hart M.J. Eva A. Evans T. Aaronson S.A. Cerione R.A. Nature. 1991; 354: 311-314Crossref PubMed Scopus (337) Google Scholar). GST-Cdc42 or GST-Rac (typically 4 μg) was incubated with 10 μm [3H]GDP(12.6 Ci/mmol) in 25 μl of loading buffer (20 mm Tris-HCl, pH 7.4, 50 mm NaCl, 3 mm MgCl2, 0.1 mm dithiothreitol, 0.1 mm EDTA, and 0.1 mm AMP-PNP), for 25 min at 25 °C. Immunoprecipitated ACG was added to the exchange assay mixture containing 20 mm Tris-HCl, pH 7.4, 80 mm NaCl, 5 mm MgCl2, 1 mm AMP-PNP, 0.1 mm dithiothreitol, 0.4 mg/ml bovine serum albumin, and 1 mm GTPγSto a final volume of 40 μl and incubated at 25 °C for 15 min. Aliquots (10 μl) from the GST-Cdc42 loading incubation were added to 40 μl of the exchange assay mixture to start the reaction. After incubation at 25 °C for the indicated times, aliquots (10 μl) of the reaction mixture were removed and diluted into 0.5 ml of washing buffer (20 mm Tris-HCl, pH 7.4, 80 mm NaCl, 10 mm MgCl2, and 10 mm β-mercaptoethanol) to stop the reaction. The samples were placed on BA85 filters and washed (three times) with washing buffer. The radioactivity on the filter, representing the amount of [3H]GDP bound to Cdc42 that was not exchanged with GTPγS, was quantified by scintillation counting. Identification of an ∼220-kDa Binding Partner for Activated Cdc42— We have been using the mouse embryonic teratoma cell line P19 as a model system for examining the role of Cdc42 in cellular differentiation. In particular, we have used P19 cell lysates to perform pull-down experiments with GST-Cdc42 fusion proteins as a way of potentially identifying new Cdc42-regulatory proteins and target/effectors. Fig. 1A shows the results of an experiment where lysates were incubated with glutathione-conjugated agarose beads bound to the GTPase-defective GST-Cdc42(Q61L) mutant. The co-precipitated proteins were separated by SDS-PAGE and visualized by staining with Coomassie Blue (lane 2). A number of additional incubations were performed to control for nonspecific binding of bacterial proteins to GST-Cdc42 or for proteins from P19 cell lysates that might either associate directly with the GST moiety or with beads alone. These included incubations of GST-Cdc42(Q61L) with the lysis buffer (Fig. 1A, lane 1), GST with P19 cell lysates (Fig. 1A, lane 3), and glutathione-conjugated agarose beads with the cell lysates (Fig. 1A, lane 4). We found that a number of proteins appeared to selectively co-precipitate with GST-Cdc42(Q61L). The predominant protein was ∼180 kDa (denoted by the white arrow in lane 2 of Fig. 1A) and was determined to be IQGAP1 (34Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar, 35McCallum S.J. Erickson J.W. Cerione R.A. J. Biol. Chem. 1998; 273: 22537-22544Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Among the other most consistently observed bands that specifically bound to GST-Cdc42(Q61L) was one that corresponded to an ∼220 kDa protein (see the black arrow in lane 2 of Fig. 1A). The p220 band was excised and subjected to microsequence analysis. Six peptide sequences were obtained and aligned against the NBCI GenBank™ data base using a Blast search (36Altschul S.F. Madden T.M. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59923) Google Scholar). Four of the six peptides were identical to Zizimin-1, with the remaining two peptides showing ∼90% identity. This indicated that p220 was a member of the Zizimin-1/DOCK180 family of GEFs. We further found that the peptide sequences XP_060056 and NP_653259, derived from computational analysis of the human genomic DNA sequence, completely matched the six peptide sequences of p220. Subsequently, Cote and Vuori (27Cote J.F. Vuori K. J. Cell Sci. 2002; 115: 4901-4913Crossref PubMed Scopus (345) Google Scholar) classified XP_060056 as a member of the DOCK180 superfamily and named it DOCK11. However, for the sake of simplicity, we refer to this protein as ACG, because this more accurately depicts its functional properties (see below). To obtain the full-length cDNA encoding ACG, we used the derived genomic DNA sequences NM_144658 and XP_060056 to design primers to perform PCR, with a human brain cDNA library serving as a template. We also used Image clones 2329541, 2021991, 4671357, 5582516, and 5583671 to help assemble the cDNA encoding ACG. The full-length cDNA contains 6650 base pairs (GenBank™ accession number AY692226), corresponding to 2073 amino acid residues (Fig. 1B). The sequence for ACG was analyzed by a NCBI Blast search against GenBank™ and was found to contain three distinct domains. These are an amino-terminal PH domain (Fig. 1B, in bold), a DHR1/CZH1 domain located approximately in the middle of the ACG molecule (boxed), and a DHR2/CZH2 domain found near the carboxyl-terminal end of the protein (boxed) (also see the schematic depiction of full-length ACG at the top of Fig. 3A). Given the presence of the DHR1 and DHR2 domains, ACG shows the typical earmarks of a member of the Zizimin-1/DOCK180 family of proteins. To further confirm and characterize the interaction of ACG with activated forms of Cdc42, we overexpressed HA-tagged ACG in COS7 cells and examined its ability to be pulled down by GST-Cdc42 (wild type); GST-Cdc42(Q61L); GST-Cdc42(Q61L,K183S,K184S), which represents a GTPase-defective and γCOP binding-defective double mutant; and GST-Cdc42(Q61L,Y40C), which is GTPase-defective as well as defective for binding target/effectors like p21-activated kinase, mixed lineage kinase, and activated Cdc42-associated kinase. As shown in Fig. 1C, HA-tagged ACG was co-precipitated with Cdc42(Q61L), as well as with Cdc42(Q-61L,K183S,K184S) (lanes 2 and 3), but not with wild-type Cdc42 nor with Cdc42(Q61L,Y40C) (lanes 1 and 4). Interestingly, the binding of Cdc42(Q61L,K183S,K184S) to ACG was much stronger than that of Cdc42(Q61L) (compare lanes 3 and 2), suggesting that ACG may compete with γCOP for Cdc42. Overall, these results confirmed that ACG forms stable complexes with activated forms of Cdc42. ACG Is a GEF for Cdc42—DOCK180 has been shown to exhibit GEF activity toward Rac (37Kiyokawa E. Hashimoto Y. Kobayashi S. Sugimura H. Kurata T. Matsuda M. Genes Dev. 1998; 12: 3331-3336Crossref PubMed Scopus (384) Google Scholar), whereas Zizimin-1 (also known as DOCK9) acts as a specific GEF for Cdc42 (26Meller N. Irani-Tehrani M. Kiosses W.B. Del Pozo M.A. Schwartz M.A. Nat. Cell Biol. 2002; 4: 639-647Crossref PubMed Scopus (147) Google Scholar). In each of these cases, the GEF activity is mediated through a highly conserved region designated as the DHR2 or the CZH2 domain (26Meller N. Irani-Tehrani M. Kiosses W.B. Del Pozo M.A. Schwartz M.A. Nat. Cell Biol. 2002; 4: 639-647Crossref PubMed Scopus (147) Google Scholar, 27Cote J.F. Vuori K. J. Cell Sci. 2002; 115: 4901-4913Crossref PubMed Scopus (345) Google Scholar). Thus, we were interested in seeing whether ACG exhibited GEF activity. We were unable to express either full-length ACG or its DHR2 domain in a soluble form in E. coli or to obtain sufficient levels in insect cells to allow biochemical characterization. However, we were able to express the DHR2 domain as an HA-tagged protein in COS7 cells and then assay its interactions with different GST-linked GTPases through pull-down assays. We found that unlike the case for full-length ACG, which binds preferentially to activated forms of Cdc42 (e.g. Cdc42(Q61L)), the recombinant HA-tagged DHR2 domain bound selectively to the nucleotide binding-defective Cdc42(T17N) mutant (Fig. 2A), as is typically the case for GEFs. The DHR2 domain of ACG did not bind to the nucleotide binding-defective mutant of Rac (Rac(T17N)) and only bound very weakly to the corresponding mutant for the related GTPase TC10 (TC10(T31N)). Thus, these data suggested that the DHR2 domain of ACG may exhibit specific GEF activity toward Cdc42. To further examine this possibility, we transfected HA-tagged full-length ACG or the HA-tagged DHR2 domain in COS7 cells, immunoprecipitated these proteins with anti-HA antibody, and then assayed their GEF activity. We used anti-HA antibody that was immunoprecipitated from vector-transfected cell lysates as a negative control, and EDTA, which chelates Mg2+ and strongly stimulates GDP-GTP exchange on small Ras-related GTPases, as a positive control. As shown in Fig. 2B, full-length ACG was an effective GEF for Cdc42, such that its activity was comparable with that obtained upon treatment with excess EDTA, whereas the recombinant DHR2 domain caused only a minor increase in the rate of [3H]GDP dissociation from Cdc42, compared with the control condition (i.e. Cdc42 alone). These results were somewhat surprising, because often the isolated limit-functional domains of GEFs show much stronger activity than their full-length parental proteins, because of the need for regulatory events to activate the GEF activity of the latter. This suggested that full-length ACG can be effectively activated in cells. Fig. 2C further demonstrates that the GEF activity for ACG is specific for Cdc42. As previously reported, insect cell recombinant Cool-2/α-Pix shows Rac-GEF activity and stimulates the rate of [3H]GDP dissociation from Rac (38Feng Q. Baird D. Cerione R.A. EMBO J. 2004; 23: 3492-3504Crossref PubMed Scopus (74) Google Scholar), whereas ACG is absolutely ineffective. Characterization of the Interaction of ACG with Activated Forms of Cdc42—The preference shown by ACG in binding to activated forms of Cdc42 (Fig. 1) differs from what has been reported for full-length Zizimin-1, which selectively interacts with nucleotide-depleted forms of Cdc42 (26Meller N. Irani-Tehrani M. Kiosses W.B. Del Pozo M.A. Schwartz M.A. Nat. Cell Biol. 2002; 4: 639-647Crossref PubMed Scopus (147) Google Scholar), as is the usual case for Cdc42-GEFs. This distinctive feature of ACG raised the possibility that activated forms of Cdc42 might exert regulatory effects on its Cdc42-GEF activity. To examine this possibility, we first needed to better define how activated Cdc42 binds to full-length ACG. A series of HA-tagged truncation mutants of ACG were prepared (Fig. 3A) and expressed in COS7 cells, and then their abilities to bind to either GST-Cdc42(Q61L), Cdc42(T17N), or wild-type GST-Cdc42 were determined through pull-down assays. Full-length ACG served as a positive control for binding to activated Cdc42(Q61L), whereas the isolated DHR2 domain was used as a positive control for binding to Cdc42(T17N). Interestingly, when the amino-terminal 272 amino acid residues of ACG were removed, its binding preference switched from activated forms of Cdc42 (e.g. Cdc42(Q61L)) to the nucleotide-depleted Cdc42(T17N) (Fig. 3B, fourth panel from top). This suggested that the amino-terminal portion of ACG was not only necessary for binding to activated forms of Cdc42 but also interfered with and/or weakened the binding of nucleotide-depleted Cdc42 and thereby repressed GEF activity. Surprisingly, when the carboxyl-terminal DHR2 domain was deleted, binding to Cdc42(Q61L) was reduced (Fig. 3B, second panel from the top). Furthermore, a more substantial truncation that deleted residues 671-2073 resulted in the complete loss of binding to both activated and nucleotide-depleted forms of Cdc42 (Fig. 3B, third panel from the top). Taken together, these data suggest that the DHR2 domain is required for binding to activated as well as nucleotide-depleted forms of Cdc42. Apparently, the DHR2 domain represents the sole binding site for the latter, whereas the amino-terminal region of ACG together with DHR2 are necessary for optimal binding to activated Cdc42. Because the truncation of the first 272 amino acid residues resulted in the loss of binding to Cdc42(Q61L) and an accompanying increase in the binding to Cdc42(T17N) (Fig. 3B), and because the PH domain of ACG is located within this region (Fig. 3A), we wondered whether the PH domain might play a role in regulating the binding of different forms of Cdc42. Thus, we generated two more amino-terminal truncation mutants of ACG, designated as ΔN65 and ΔN126 (Fig. 4A). Both of these truncation mutants still contained the PH domain. The HA-tagged ΔN65 and ΔN126 were transfected into COS7 cells, and their abilities to bind
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