IQGAP1 Binds Rap1 and Modulates Its Activity
2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês
10.1074/jbc.m700487200
ISSN1083-351X
AutoresHa‐Won Jeong, Zhigang Li, Matthew D. Brown, David B. Sacks,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoIQGAP1 is a scaffolding protein involved in multiple fundamental cellular activities, including transcription, cell-cell attachment, and regulation of the cytoskeleton. To function in these pathways, IQGAP1 associates with numerous proteins such as actin, calmodulin, E-cadherin, β-catenin, CLIP-170, and components of the mitogen-activated protein kinase pathway. Moreover, IQGAP1 binds to active Cdc42 and Rac1 but not RhoA or Ras. Here we show that IQGAP1 also binds to the small GTPase Rap1. In vitro analysis demonstrates a direct interaction between Rap1 and IQGAP1, which is augmented by activation (GTP loading) of Rap1. Cdc42 does not modulate the interaction between Rap1 and IQGAP1. In contrast, the association is eliminated by calmodulin both in the absence and presence of Ca2+. The binding of Rap1 to a point mutant IQGAP1 construct that is unable to interact with calmodulin is 2.5-fold more than to wild type IQGAP1. Consistent with these findings, Rap1 binds to the IQ region of IQGAP1. Confocal microscopy demonstrates that Rap1 and IQGAP1 co-localize at the periphery of human epithelial cells but not in the cytoplasm. The interaction has functional sequelae. Overexpression of IQGAP1 substantially reduces adhesion-mediated activation of Rap1. In addition, Rap1 activation by cAMP is attenuated in cells that overexpress IQGAP1 and enhanced in cells lacking IQGAP1. These findings reveal that the interaction of IQGAP1 with Rap1 differs in several respects from its interaction with other small GTPases. Furthermore, our data suggest that IQGAP1 may link the calmodulin and Rap1 signaling pathways. IQGAP1 is a scaffolding protein involved in multiple fundamental cellular activities, including transcription, cell-cell attachment, and regulation of the cytoskeleton. To function in these pathways, IQGAP1 associates with numerous proteins such as actin, calmodulin, E-cadherin, β-catenin, CLIP-170, and components of the mitogen-activated protein kinase pathway. Moreover, IQGAP1 binds to active Cdc42 and Rac1 but not RhoA or Ras. Here we show that IQGAP1 also binds to the small GTPase Rap1. In vitro analysis demonstrates a direct interaction between Rap1 and IQGAP1, which is augmented by activation (GTP loading) of Rap1. Cdc42 does not modulate the interaction between Rap1 and IQGAP1. In contrast, the association is eliminated by calmodulin both in the absence and presence of Ca2+. The binding of Rap1 to a point mutant IQGAP1 construct that is unable to interact with calmodulin is 2.5-fold more than to wild type IQGAP1. Consistent with these findings, Rap1 binds to the IQ region of IQGAP1. Confocal microscopy demonstrates that Rap1 and IQGAP1 co-localize at the periphery of human epithelial cells but not in the cytoplasm. The interaction has functional sequelae. Overexpression of IQGAP1 substantially reduces adhesion-mediated activation of Rap1. In addition, Rap1 activation by cAMP is attenuated in cells that overexpress IQGAP1 and enhanced in cells lacking IQGAP1. These findings reveal that the interaction of IQGAP1 with Rap1 differs in several respects from its interaction with other small GTPases. Furthermore, our data suggest that IQGAP1 may link the calmodulin and Rap1 signaling pathways. The Ras superfamily of small GTPases comprises ∼150 members in humans (1Wennerberg K. Rossman K.L. Der C.J. J. Cell Sci. 2005; 118: 843-846Crossref PubMed Scopus (1017) Google Scholar, 2Mitin N. Rossman K.L. Der C.J. Curr. Biol. 2005; 15: 563-574Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Members of the Ras family, which contains 36 proteins, include the Ras proteins (H-Ras, K-Ras, and N-Ras) and several related proteins such as R-Ras proteins (R-Ras, R-Ras2, and M-Ras), Ral and Rap (2Mitin N. Rossman K.L. Der C.J. Curr. Biol. 2005; 15: 563-574Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Like Ras, Rap1 cycles between an active GTP-bound form and an inactive GDP-bound form. Guanine nucleotide exchange factors (GEFs), 2The abbreviations used are: GEF, guanine nucleotide exchange factor; 8CPT-2Me-cAMP, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate; Ni2+-NTA, nickel nitrilotriacetic acid; GST, glutathione S-transferase; GAP, GTPase-activating protein; Epac, exchange protein directly activated by cAMP; BSA, bovine serum albumin; MBP, maltose-binding protein; MEF, mouse embryo fibroblast; GFP, green fluorescent protein; RFP, red fluorescent protein; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-3-O-(thio)triphosphate. such as Epac (exchange protein directly activated by cAMP), C3G, and CD-GEF, mediate Rap1 activation through exchange of GDP for GTP (3Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. Mol. Cell Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar). Rap1 is inactivated by GTPase-activating proteins (GAPs), which catalyze the hydrolysis of bound GTP to GDP. A wide variety of extracellular stimuli, for example thrombin, growth factors, and interferon, activate Rap1. These effects are mediated by second messengers, including Ca2+, diacylglycerol, and cAMP, which directly stimulate Rap1 GEFs. Ras and Rap1 are very similar at the amino acid level, with identical effector regions (4Caron E. J. Cell Sci. 2003; 116: 435-440Crossref PubMed Scopus (169) Google Scholar). Nevertheless, the proteins have distinct functions. Rap1 has been implicated in the modulation of a number of cellular responses ranging from Ca2+ signaling and secretion to neurite outgrowth and cell proliferation (3Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. Mol. Cell Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar). A well characterized function of Rap1 is in cell adhesion, both integrin-mediated adhesion and cadherin-mediated cell junction formation (5Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (395) Google Scholar). Rap1 interacts with a large number of proteins that contribute to regulating these aspects of cell function (5Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (395) Google Scholar). IQGAP1 is a scaffolding protein composed of multiple protein recognition motifs through which it interacts with a wide spectrum of binding partners (for review, see Refs. 6Brown M.D. Sacks D.B. Trends Cell Biol. 2006; 16: 242-249Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 7Briggs M.W. Sacks D.B. EMBO Rep. 2003; 4: 571-574Crossref PubMed Scopus (250) Google Scholar, 8Noritake J. Watanabe T. Sato K. Wang S. Kaibuchi K. J. Cell Sci. 2005; 118: 2085-2092Crossref PubMed Scopus (293) Google Scholar, 9Mateer S.C. Wang N. Bloom G.S. Cell Motil. Cytoskeleton. 2003; 55: 147-155Crossref PubMed Scopus (78) Google Scholar). In the N-terminal half, the calponin homology domain binds actin (10Ho Y.D. Joyal J.L. Li Z. Sacks D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 11Mateer S.C. McDaniel A.E. Nicolas V. Habermacher G.M. Lin M.J. Cromer D.A. King M.E. Bloom G.S. J. Biol. Chem. 2002; 277: 12324-12333Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), the WW domain binds extracellular signal-regulated kinase 2 (12Roy M. Li Z. Sacks D.B. J. Biol. Chem. 2004; 279: 17329-17337Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), and the IQ domain mediates interactions with calmodulin (10Ho Y.D. Joyal J.L. Li Z. Sacks D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Joyal J.L. Annan R.S. Ho Y.D. Huddleston M.E. Carr S.A. Hart M.J. Sacks D.B. J. Biol. Chem. 1997; 272: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 14Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar) and calmodulin-related proteins (15Briggs M.W. Sacks D.B. FEBS Lett. 2003; 542: 7-11Crossref PubMed Scopus (119) Google Scholar). The C-terminal half-of IQGAP1 is reported to bind E-cadherin (16Kuroda S. Fukata M. Nakagawa M. Fujii K. Nakamura T. Ookubo T. Izawa I. Nagase T. Nomura N. Tani H. Shoji I. Matsuura Y. Yonehara S. Kaibuchi K. Science. 1998; 281: 832-835Crossref PubMed Scopus (429) Google Scholar, 17Li Z. Kim S.H. Higgins J.M. Brenner M.B. Sacks D.B. J. Biol. Chem. 1999; 274: 37885-37892Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), β-catenin (16Kuroda S. Fukata M. Nakagawa M. Fujii K. Nakamura T. Ookubo T. Izawa I. Nagase T. Nomura N. Tani H. Shoji I. Matsuura Y. Yonehara S. Kaibuchi K. Science. 1998; 281: 832-835Crossref PubMed Scopus (429) Google Scholar, 18Briggs M.W. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 7453-7465Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), CLIP-170 (19Fukata M. Watanabe T. Noritake J. Nakagawa M. Yamaga M. Kuroda S. Matsuura Y. Iwamatsu A. Perez F. Kaibuchi K. Cell. 2002; 109: 873-885Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar) and adenomatous polyposis coli (20Watanabe T. Wang S. Noritake J. Sato K. Fukata M. Takefuji M. Nakagawa M. Izumi N. Akiyama T. Kaibuchi K. Dev. Cell. 2004; 7: 871-883Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). Through binding to diverse proteins, IQGAP1 functions in many physiological processes in cells, including cell-cell attachment, cell polarization, transcription, regulation of actin and microtubule function, the mitogen-activated protein kinase cascade, and Ca2+/calmodulin signaling (6Brown M.D. Sacks D.B. Trends Cell Biol. 2006; 16: 242-249Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 7Briggs M.W. Sacks D.B. EMBO Rep. 2003; 4: 571-574Crossref PubMed Scopus (250) Google Scholar). In addition, IQGAP1 has a role in cell motility, cell invasion, neuronal migration, and neurite outgrowth (21Li Z. McNulty D.E. Marler K.J.M. Lim L. Hall C. Annan R.S. Sacks D.B. J. Biol. Chem. 2005; 280: 13871-13878Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 22Kholmanskikh S.S. Koeller H.B. Wynshaw-Boris A. Gomez T. Letourneau P.C. Ross M.E. Nat. Neurosci. 2006; 9: 50-57Crossref PubMed Scopus (141) Google Scholar, 23Mataraza J.M. Briggs M.W. Li Z. Entwistle A. Ridley A.J. Sacks D.B. J. Biol. Chem. 2003; 278: 41237-41245Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). In addition to the binding partners mentioned above, IQGAP1 also interacts with selected small GTPases. Direct binding of IQGAP1 to active (GTP-bound) Cdc42 (13Joyal J.L. Annan R.S. Ho Y.D. Huddleston M.E. Carr S.A. Hart M.J. Sacks D.B. J. Biol. Chem. 1997; 272: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 14Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar), Rac1 (14Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar), and TC10 (24Neudauer C.L. Joberty G. Tatsis N. Macara I.G. Curr. Biol. 1998; 8: 1151-1160Abstract Full Text Full Text PDF PubMed Google Scholar) has been documented. However, IQGAP1 does not bind RhoA or H-Ras (14Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar, 25McCallum S.J. Wu W.J. Cerione R.A. J. Biol. Chem. 1996; 271: 21732-21737Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In this study we show for the first time that IQGAP1 interacts directly with a GTPase that is not a member of the Rho subfamily. The association of IQGAP1 with Rap1, which differs in several respects from its interaction with other small GTPases, alters the activation of Rap1. Reagents—Cell culture reagents were from Invitrogen. The anti-IQGAP1 polyclonal and monoclonal antibodies have been previously characterized (10Ho Y.D. Joyal J.L. Li Z. Sacks D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 26Roy M. Li Z. Sacks D.B. Mol. Cell. Biol. 2005; 25: 9740-9752Google Scholar). Anti-Myc monoclonal antibody (9E10.2) was manufactured by Maine Biotechnology. Antibodies were purchased as follows; monoclonal anti-Cdc42 from BD Transduction Laboratories, polyclonal anti-Rap1 from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal anti-β tubulin from Sigma, and horseradish peroxidase-conjugated secondary antibodies from GE Healthcare. Enhanced chemiluminescence (ECL) reagents were from Millipore (Billerica, MA). 8-(4-Chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8CPT-2Me-cAMP) was purchased from Tocris (Ellisville, MO). All other reagents were of standard analytical grade. Plasmid Construction and Preparation of Fusion Proteins—The construction of Myc-tagged IQGAP1, IQGAP1-C (C-terminal region, amino acids 864-1657), IQGAP1-N (N-terminal region, amino acids 2-863), IQGAP1ΔWW (amino acids 643-744 deleted), IQGAP1ΔIQ (amino acids 699-905 deleted), and IQGAP1 IQ3,4R (selected Arg residues in the third and fourth IQ motifs substituted by Gln) have been described previously (10Ho Y.D. Joyal J.L. Li Z. Sacks D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 12Roy M. Li Z. Sacks D.B. J. Biol. Chem. 2004; 279: 17329-17337Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 27Sokol S.Y. Li Z. Sacks D.B. J. Biol. Chem. 2001; 276: 48425-48430Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 28Li Z. Sacks D.B. J. Biol. Chem. 2003; 278: 4347-4352Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). All plasmids were purified using Qiagen DNA purification kits (Qiagen) according to the manufacturer's instruction. pET His6-Rap1A, His6-Rap1A-63E, and His6-Rap1A-17N were kindly provided by Xavier Cullere and Tanya Mayadas (Brigham and Women's Hospital and Harvard Medical School, Boston, MA), and GFP-Rap1A was a kind gift of Yasuyuki Fujita (University College London, London, UK). pGEX-Cdc42-Q61L, pGEX-Rap1B, and pGEX-Ral-GDS were generously donated by Darerca Owen (University of Cambridge, Cambridge, UK), Andreas Püschel (Westfalische Wilhelms-Universitat, Munster, Germany), and Johannes Bos (University Medical Centre, Utrecht, The Netherlands), respectively. pET His6-Rap1 constructs were expressed in Escherichia coli and purified by nickel affinity chromatography using Ni2+-NTA-agarose (Qiagen). GST-IQGAP1 and pGEX-Cdc42-Q61L were expressed in E. coli and purified by glutathione-Sepharose chromatography (GE Healthcare) essentially as described (10Ho Y.D. Joyal J.L. Li Z. Sacks D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Where indicated, the GST tag was removed from IQGAP1 by tobacco etch virus protease as previously described (29Ren J.G. Li Z. Crimmins D.L. Sacks D.B. J. Biol. Chem. 2005; 280: 34548-34557Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). We generated maltose-binding protein (MBP) fusion constructs of wild type Rap1A, Rap1A-63E, and Rap1A-17N. The BamHI-HindIII fragment containing Rap1A was cut from pRSET-Rap1A and inserted into pMAL-c2X at BamHI and HindIII sites. The proteins migrated to the expected position on SDS-PAGE. GST-Rap1A was generated by PCR on full-length Rap1A using the primers 5′-CGGGATCCCCGTGAGTTACAAGTCTAGTGG-3′ and 5′-CGGAATTCTCAGAGCAGCAGACATGATTTC-3′. The PCR product was cut with BamHI and EcoRI and inserted into pGEX4T-1 at the same restriction sites. IQGAP1 was tagged with red fluorescent protein (RFP) using monomeric mRFP which was generously donated by Roger Tsien (University of California, San Diego, CA). A PCR product of mRFP was generated using primers 5′-GCTCTAGAATGGCCTCCTCCGAGGACG-3′ and 5′-GAAGATCTGGCGCCGGTGGAGTGGCGTCT-3′ with pRSETB-mRFP1 as template. The PCR product was cut with XbaI and BglII, and the insert was replaced with a NheI-BglII fragment bearing the DsRed gene on pDsRed2-C1 (Clontech). To generate RFP-wild type IQGAP1, pcDNA-Myc-IQGAP1 was cut with XbaI and made blunt end with T4 polymerase then partially digested with BamHI. The BamHI-XbaI fragment bearing the whole IQGAP1 gene was inserted into mRFP-C1 at BglII-SmaI site. The RFP tag was attached to IQGAP1ΔIQ and IQGAP1 IQ3,4R using RFP-tagged wild type IQGAP1. The PacI-SpeI fragment was cut from mRFP-IQGAP1 (wild type) and replaced with the same fragment from pcDNA-myc-IQGAP1ΔIQ or pcDNA-Myc-IQGAP1 IQ3,4R. Cell Culture and Transfection—MCF-7 human breast epithelial cells and 293H human embryonic kidney cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum. MCF-7 cells, which stably overexpress pcDNA3-Myc-IQGAP1 (termed MCF/I cells), and MCF-7 cells with stable integration of small interfering RNA for IQGAP1 (termed MCF-siIQ8 cells) have been described previously (18Briggs M.W. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 7453-7465Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 23Mataraza J.M. Briggs M.W. Li Z. Entwistle A. Ridley A.J. Sacks D.B. J. Biol. Chem. 2003; 278: 41237-41245Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 30Swart-Mataraza J.M. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 24753-24763Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), MCF/I cells have 3-fold more IQGAP1 than MCF-7 cells, whereas IQGAP1 protein is reduced by 80% in MCF-siIQ8 cells. Mouse embryo fibroblast (MEF) cells, isolated from IQGAP1-/- mice and normal littermate controls, were immortalized with SV40 large T antigen. 3Ren, J. G., Li, Z., and Sacks, D. B. (2007) Proc. Natl. Acad. Sci. U. S. A., in press. Transient transfections were performed with FuGENE 6 (Roche Diagnostics) or Lipofectamine 2000 (Invitrogen) essentially as described (18Briggs M.W. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 7453-7465Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 21Li Z. McNulty D.E. Marler K.J.M. Lim L. Hall C. Annan R.S. Sacks D.B. J. Biol. Chem. 2005; 280: 13871-13878Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 30Swart-Mataraza J.M. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 24753-24763Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Cells were transfected with 5 μg Myc-IQGAP1, Myc-IQGAP1ΔIQ, Myc-IQGAP1 IQ3,4R, RFP-IQGAP1, RFP-IQGAP1ΔIQ, or RFP-IQGAP1 IQ3,4R and 48 h later were either evaluated in adhesion assays, lysed, or fixed for immunocytochemistry. Gel Filtration Chromatography—MCF-7 and MCF-siIQ8 cells (8 dishes/10 cm each) were lysed in 2 ml of buffer composed of 50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EGTA, 1% Nonidet P-40, 20% glycerol, and protease inhibitors. The lysate was loaded onto a Superose 6 column (Amersham Biosciences) pre-equilibrated with the same buffer. The sample was fractionated at 1 ml/min (0.5-ml fractions were collected) by fast protein liquid chromatography separation performed on an AKTATMFPLC system (Amersham Biosciences) equipped with a UPC-900 monitor and a P-920 pump. The system was monitored and controlled by methods run by the UNICORN control system (Version 5.01). The column was calibrated using apoferritin (443 kDa), β-amylase (200 kDa), and bovine serum albumin (66 kDa) as standards. Forty-microliter aliquots of selected fractions were resolved by Western blotting, then probed for IQGAP1 and Rap1. Adhesion Assays—To analyze cell adhesion, cells were trypsinized, suspended in complete medium, and plated onto collagen I- or fibronectin-coated culture dishes (BD Bioscience) at 37 °C for the times indicated in Figs. 7 and 8. After washing with PBS (to remove nonadherent cells), adherent cells were lysed with buffer A (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 2 mm MgCl2, 10% glycerol, 1% Nonidet P-40, 2 mm Na3VO4, 10 mm NaF, and 5 mm EGTA). Equal amounts of protein lysates were subjected to pulldown assays.FIGURE 8Extracellular matrix-induced activation of Rap1 in cells transfected with mutant IQGAP1. A, MCF-7 cells were transiently transfected with 5 μg of empty pcDNA3 vector (V) or IQGAP1ΔIQ (ΔIQ). Cells were plated on collagen I-coated culture dishes for the times indicated. Adherent cells were harvested with lysis buffer and analyzed by Western blotting. Equal amounts of protein lysate were also used in a GST-RalGDS (for active Rap1) pulldown followed by Western blotting with anti-Rap1 antibody. Protein lysates were also blotted for Myc (all IQGAP1 constructs are Myc-tagged), IQGAP1, and β-tubulin (as loading control). B, the relative amount of GTP-Rap1 in each sample was quantified by densitometry and corrected for total Rap1 in the corresponding cell lysate. Data, expressed relative to MCF-7 cells transfected with vector at time 0, are the means ± S.D. of two independent experiments. C, MCF-7 cells were transiently transfected with empty vector (V), wild type IQGAP1 (WT), or IQGAP1 IQ3,4R (IQ3,4R). Cells were plated on culture dishes coated with fibronectin. After 90 min, cells were processed for Rap1 as described for A. D, the relative amount of GTP-Rap1 in each sample was quantified by densitometry and corrected for total Rap1 in the corresponding lysate. Data, expressed relative to MCF-7 cells transfected with vector at time 0, are representative of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Assay for Activation of Rap1 and Cdc42—Active Cdc42 (31Kim S.H. Li Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) and active Rap1 (32Franke B. Akkerman J.W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar) were assessed essentially as described previously. Briefly, cells were washed with ice-cold PBS and lysed with buffer A. Lysates were cleared by centrifugation, and active Rap1 and Cdc42 were precipitated with glutathione-Sepharose beads precoupled to a GST fusion protein of the Rap1 binding domain of RalGDS for Rap1 and the GTPase binding domain of WASP (Wiskott-Aldrich syndrome protein) for Cdc42. Samples were incubated for 40 min (for Rap1) or 30 min (for Cdc42). Beads were sedimented by centrifugation, washed three times with buffer A, and solubilized in SDS sample buffer. A portion of the cell lysate was reserved for analysis of total Rap1 or Cdc42 content. Cdc42 and Rap1 were detected by Western blotting. 8CPT-2Me-cAMP Treatment—Equal numbers of MCF-7 or MCF/I cells were serum-starved in Dulbecco's modified Eagle's medium containing 1% bovine serum albumin (BSA). After 24 h, cells were stimulated with 100 μm 8CPT-2Me-cAMP for the times indicated in the legend to Fig. 9. GTP-bound Rap1 was detected by pulldown with GST-RalGDS as described above. Similar analysis was done with MEFs derived from IQGAP1-null mice and normal littermate controls. In Vitro Binding Analyses—For binding assays using GST-tagged proteins, MBP-Rap1A was loaded with either GDP or GTPγS essentially as previously described (13Joyal J.L. Annan R.S. Ho Y.D. Huddleston M.E. Carr S.A. Hart M.J. Sacks D.B. J. Biol. Chem. 1997; 272: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 33Thompson G. Owen D. Chalk P.A. Lowe P.N. Biochemistry. 1998; 37: 7885-7891Crossref PubMed Scopus (121) Google Scholar). Briefly, MBP-Rap1A was incubated in buffer B (50 mm Tris, pH 7.4, 150 mm NaCl, and 1% Triton X-100) with 1 mm EDTA. After 10 min at 22 °C, 3 mm MgCl2 and 140 μm GTPγS or GDP were added, and samples were incubated for 30 min at 22 °C. Samples were precleared with 40 μl of glutathione-Sepharose for 1 h, and equal amounts of GST-IQGAP1 in 500 μl of buffer B containing 1 mm EGTA were added for 3 h at 4 °C. Complexes were isolated with glutathione-Sepharose, washed three times in buffer B, and resolved by SDS-PAGE. Western blots were probed with anti-Rap1 antibodies. For binding assays with MBP-tagged proteins, equal amounts of wild type MBP-Rap1A (loaded with GTPγS or GDP), dominant-negative Rap1A-17N, or constitutively active Rap1A-63E were incubated with purified IQGAP1 in 500 μl of buffer B for 3 h at 4 °C. Complexes were isolated with amylose beads, washed, and resolved by SDS-PAGE, and blots were probed with anti-IQGAP1 antibodies (26Roy M. Li Z. Sacks D.B. Mol. Cell. Biol. 2005; 25: 9740-9752Google Scholar). To compare binding of Rap1A and Rap1B to IQGAP1, we used GST-tagged Rap1 proteins. IQGAP1 was cleaved from GST using tobacco etch virus protease as described (29Ren J.G. Li Z. Crimmins D.L. Sacks D.B. J. Biol. Chem. 2005; 280: 34548-34557Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). GST, GST-Rap1A, and GST-Rap1B were loaded with GTPγS as described above. Equal amounts of purified IQGAP1 were incubated with equal amounts of GSTγS-loaded GST, GST-Rap1A, or GTP Rap1B in 20 mm Tris-HCl, pH 8.0, 137 mm NaCl, 0.2% Triton X-100, 10% glycerol, and 1 mm EGTA for 2 h at 4 °C. Complexes were isolated with glutathione-Sepharose, washed, and resolved by SDS-PAGE. The gel was cut into two pieces; the top portion was transferred to polyvinylidene difluoride membranes, and blots were probed with anti-IQGAP1 antibody. The lower half was stained with Coomassie Blue to visualize GST-tagged proteins. [35S]Methionine-labeled transcription and translation (TNT) products were generated with the TNT Quick Coupled Transcription/Translation system (Promega) according to the manufacturer's instructions. Briefly, 0.5 μg of the IQGAP1 plasmids was incubated with 40 μl of TNT Quick Master mix and 20 μCi of [35S]methionine (PerkinElmer Life Sciences) at 30 °C for 1 h. Products were confirmed by SDS-PAGE and autoradiography. Equal amounts of radiolabeled peptide were incubated with His-Rap1A-63E. Complexes were isolated with Ni2+-NTA agarose, resolved by SDS-PAGE, and processed by autoradiography. Competitive Inhibition Analysis—[35S]Methionine-labeled IQGAP1 was preincubated with 5 μg of calmodulin, 5 μg of constitutively active Cdc42 (Cdc42-Q61L), or 5 μg of BSA in 500 μl of buffer B containing 1 mm Ca2+ or 1 mm EGTA for 1 h at 4 °C. Equal amounts of His-Rap1A-63E protein were added, and samples were incubated another 1 h. The complexes were washed and isolated with Ni2+-NTA agarose. Samples were resolved by SDS-PAGE, and bands were analyzed by autoradiography of the dried gel. For calmodulin-Sepharose chromatography, [35S]methionine-labeled IQGAP1 was preincubated with 5 μg of His-Rap1A-63E or 5 μg of BSA in the presence of Ca2+ or EGTA for 1 h at 4 °C. Equal amounts of calmodulin-Sepharose or empty Sepharose were added. After 1 h beads were isolated and washed three times in buffer B containing Ca2+ or EGTA. Proteins bound to calmodulin were resolved by SDS-PAGE and processed by autoradiography of the dried gel. Immunofluorescence Staining and Confocal Microscopy—MCF-7 or MCF-siIQ8 cells were transiently transfected with GFP-Rap1A alone or in combination with RFP-tagged IQGAP1 constructs. Cells were plated on glass coverslips that had been coated overnight with 10 μg/ml type I collagen or 10 μg/ml poly-l-lysine (Sigma-Aldrich) and blocked with 0.5% BSA. Immunocytochemistry was performed as described (23Mataraza J.M. Briggs M.W. Li Z. Entwistle A. Ridley A.J. Sacks D.B. J. Biol. Chem. 2003; 278: 41237-41245Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 31Kim S.H. Li Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) with a few modifications. Cells were washed twice with PBS and incubated in 4% paraformaldehyde-PBS for 20 min at 22 °C. After washing twice in PBS, cells were permeabilized in 0.2% Triton X-100 with 3% BSA in PBS for 1 h at 22 °C. Polyclonal anti-IQGAP1 antibody was added in 0.2% Triton, 1% BSA for 16 h at 4 °C followed by Alexa-Fluor 543-labeled anti-rabbit IgG secondary antibody (Molecular Probes) for 1 h. Where appropriate, actin was visualized with Alexa-Fluor 543-conjugated phalloidin. Rabbit IgG was used as a control for the corresponding primary antibody. The stained cells were analyzed using a Zeiss LSM 510 confocal microscope and analyzed with Zeiss LSM software. Miscellaneous—Densitometry of enhanced chemiluminescence (ECL) signals was analyzed with UN-SCAN-IT software (Silk Scientific Corp.). Statistical analysis was assessed by Student's t test with Instat software (GraphPad Software, Inc.). Protein concentrations were determined with the DC protein assay (Bio-Rad). Direct Interaction between IQGAP1 and Rap1—In vitro analysis was conducted to examine a possible association between IQGAP1 and Rap1. Purified MBP-Rap1A was loaded with either non-hydrolyzable GTPγS to form active Rap1A or with GDP to produce inactive Rap1A. Equal amounts of the Rap1A constructs were incubated with purified GST-IQGAP1. Pulldown with glutathione-Sepharose beads revealed that Rap1A binds to IQGAP1. IQGAP1 binds more efficiently to GTP-Rap1A than to the inactive form of Rap1A (Fig. 1A). Binding was specific, as no Rap1A associated with GST alone. Coomassie staining reveals that equal amounts of GST-IQGAP1 were present in the assay (Fig. 1A). To confirm these findings, the reverse experiment was done. MBP-Rap1A was incubated with purified IQGAP1. Pulldown with amylose beads reveals that more IQGAP1 binds to GTP-loaded Rap1A than to GDP-Rap1A (Fig. 1B). Similar analysis was conducted with MBP-tagged forms of the constitutively active Rap1A-63E and dominant negative Rap1A-17N. Consistent with the data obtained with guanine nucleotide loading, IQGAP1 associates with Rap1A-63E but not with Rap1A-17N (Fig. 1B). The amounts of MBP-Rap1A present in the assay were equivalent (Fig. 1B). Collectively, these data reveal that Rap1A interacts directly with IQGAP1 in a GTP-regulated
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