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• W2023807898 abstract "The Ras-GAP related protein IQGAP1 binds several proteins, including actin, calmodulin, E-cadherin and the Rho family GTPase Cdc42. To gain insight into its in vivo function, IQGAP1 was overexpressed in mammalian cells. Transfection of IQGAP1 significantly increased the levels of active, GTP-bound Cdc42, resulting in the formation of peripheral actin microspikes. By contrast, transfection of an IQGAP1 mutant lacking part of theGAP-related domain (IQGAP1ΔGRD) substantially decreased the amount of GTP-bound Cdc42 in cell lysates. Consistent with these findings, IQGAP1ΔGRD blocked Cdc42 function in cells that stably overexpress constitutively active Cdc42 and abrogated the effect of bradykinin on Cdc42. In cells transfected with IQGAP1ΔGRD, bradykinin was unable to activate Cdc42, translocate Cdc42 to the membrane fraction, or induce filopodia production. IQGAP1ΔGRD transfection altered cellular morphology, producing small, round cells that closely resemble Cdc42−/− cells. Some insight into the mechanism was provided by in vitroanalysis, which revealed that IQGAP1ΔGRD increased the intrinsic GTPase activity of Cdc42, thereby increasing the amount of inactive, GDP-bound Cdc42. These data imply that IQGAP1 has a crucial role in transducing Cdc42 signaling to the cytoskeleton. The Ras-GAP related protein IQGAP1 binds several proteins, including actin, calmodulin, E-cadherin and the Rho family GTPase Cdc42. To gain insight into its in vivo function, IQGAP1 was overexpressed in mammalian cells. Transfection of IQGAP1 significantly increased the levels of active, GTP-bound Cdc42, resulting in the formation of peripheral actin microspikes. By contrast, transfection of an IQGAP1 mutant lacking part of theGAP-related domain (IQGAP1ΔGRD) substantially decreased the amount of GTP-bound Cdc42 in cell lysates. Consistent with these findings, IQGAP1ΔGRD blocked Cdc42 function in cells that stably overexpress constitutively active Cdc42 and abrogated the effect of bradykinin on Cdc42. In cells transfected with IQGAP1ΔGRD, bradykinin was unable to activate Cdc42, translocate Cdc42 to the membrane fraction, or induce filopodia production. IQGAP1ΔGRD transfection altered cellular morphology, producing small, round cells that closely resemble Cdc42−/− cells. Some insight into the mechanism was provided by in vitroanalysis, which revealed that IQGAP1ΔGRD increased the intrinsic GTPase activity of Cdc42, thereby increasing the amount of inactive, GDP-bound Cdc42. These data imply that IQGAP1 has a crucial role in transducing Cdc42 signaling to the cytoskeleton. Rho guanine nucleotide dissociation inhibitor GTPase-activating protein phosphate- buffered saline guanine nucleotide exchange factor Wiskott-Aldrich syndrome protein maltose-binding protein glutathione S-transferase fluorescein isothiocyanate tetramethyl rhodamine isothicyanate guanylyl-5′-imidodiphosphate Ras-GAP-related domain guanine nucleotide dissociation inhibitor Dulbecco's modified Eagle's medium polyvinylidene difluoride green fluorescence protein guanosine 5′-3-O-(thio)triphosphate Rho family GTPases, members of the Ras superfamily, act as key molecular switches in intracellular signaling pathways that lead to alterations in cellular morphology, gene transcription, cell cycle progression, and programmed cell death (1Mackay D.J.G. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 2Kaibuchi K. Kuroda S. Fukata M. Nakagawa M. Curr. Opin. Cell Biol. 1999; 11: 591-596Crossref PubMed Scopus (170) Google Scholar). Rho, Rac, and Cdc42, the most extensively characterized members of the Rho family, exist in both inactive GDP-bound and active GTP-bound states (3Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1682) Google Scholar). Inactive Rho proteins are found associated with the Rho guanine nucleotide dissociation inhibitor, RhoGDI.1 This complex is targeted by guanine nucleotide exchange factors (GEFs), which activate Rho GTPases by promoting a GDP to GTP exchange. Inactivation is accelerated by GTPase-activating proteins (GAPs), which increase the intrinsic hydrolysis of bound GTP to GDP (4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). Rho proteins mediate cytoskeletal rearrangement (4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar); in particular, Cdc42 induces the formation of peripheral actin microspikes and filopodia (5Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar, 6Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (883) Google Scholar). In its active, GTP-bound form, Cdc42 binds a number of target proteins, including the Wiskott-Aldrich syndrome protein (WASP) (7Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (576) Google Scholar, 8Aspenstrom P. Lindberg U. Hall A. Curr. Biol. 1996; 6: 70-75Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), Par-6 (9Joberty G. Petersen C. Gao L. Macara I.G. Nat. Cell Biol. 2000; 2: 531-539Crossref PubMed Scopus (767) Google Scholar), p21-activated kinases (10Manser E. Leung T. Salihuddin H. Zhao Z. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1305) Google Scholar), and IQGAP1 (11Joyal 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, 12Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). IQGAP1, a 190-kDa protein, derives its name from the IQ domain and a region with sequence similarity to the catalytic domain of Ras-GAPs (13Weissbach L. Settleman J. Kalady M.F. Snijders A.J. Murthy A.E. Yan Y.X. Bernards A. J. Biol. Chem. 1994; 269: 20517-20521Abstract Full Text PDF PubMed Google Scholar). IQGAP1 contains several protein recognition motifs, including four IQ motifs, a calponin homology domain, a WW domain and a Ras-GAP-related domain (GRD) (14Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar). Several proteins, such as Cdc42 (11Joyal 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, 12Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), Rac (14Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar), actin (15Ho 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, 16Bashour A.-M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (214) Google Scholar), calmodulin (11Joyal 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, 15Ho 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), E-cadherin (17Li Z. Kim S.H. Higgins J.M.G. Brenner M.B. Sacks D.B. J. Biol. Chem. 1999; 274: 37885-37892Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 18Kuroda S. Fukata M. Nakagawa M. Fujii K. Nakamura T. Ookubo T. Izawa I. Nagase T. Nomura N. Tani H. Shoji I. Matsuura Y. Yonehard S. Kaibuchi K. Science. 1998; 281: 832-835Crossref PubMed Scopus (429) Google Scholar), and β-catenin (18Kuroda S. Fukata M. Nakagawa M. Fujii K. Nakamura T. Ookubo T. Izawa I. Nagase T. Nomura N. Tani H. Shoji I. Matsuura Y. Yonehard S. Kaibuchi K. Science. 1998; 281: 832-835Crossref PubMed Scopus (429) Google Scholar) bind to IQGAP1. Recent studies have suggested a role for IQGAP1 as a molecular link between active Cdc42 and the actin cytoskeleton (16Bashour A.-M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (214) Google Scholar,19Erickson 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). For example, upon induction of actin polymerization, both IQGAP1 and Cdc42 are incorporated into actin-containing complexes (19Erickson 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). IQGAP1 also has cross-linking activity toward F-actin that is augmented by active, GTP-bound Cdc42 (20Fukata M. Kuroda S. Fujii K. Nakamura T. Shoji I. Matsuura Y.Y. Okawa K. Iwamatsu A. Kikuchi A. Kaibuchi K. J. Biol. Chem. 1997; 272: 29579-29583Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). There is, however, limited information regarding the in vivo function of IQGAP1 in mammalian cells. Recent data reveal that microinjection of human IQGAP1 intoXenopus embryos generated superficial ectodermal lesions in a Cdc42-dependent manner (21Sokol S.Y., Li, Z. Sacks D.B. J. Biol. Chem. 2001; 276: 48425-48430Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). To gain further insight into the function of IQGAP1 in mammalian cells, we overexpressed wild type and mutant IQGAP1 constructs in several cell types. IQGAP1 overexpression significantly increased active Cdc42 in mammalian cell lysates. By contrast, an IQGAP1 mutant lacking part of the GRD (IQGAP1ΔGRD, residues 1122–1324 deleted) increased the intrinsic GTPase activity of Cdc42, thereby reducing the amount of active Cdc42 in cells. Importantly, IQGAP1ΔGRD prevented bradykinin-mediated activation of Cdc42, translocation of Cdc42 to the membrane fraction, and formation of filopodia. Expression of IQGAP1ΔGRD also decreased active Cdc42 and abrogated microspike formation in cells overexpressing constitutively active Cdc42. Together, these data strongly suggest that IQGAP1 is an important link between Cdc42 and the cytoskeleton in cells. Tissue culture reagents were obtained from Invitrogen. Fetal bovine serum was purchased from BioWhittaker. Anti-Cdc42, Rac, and Rho antibodies were obtained from Transduction Laboratories. Anti-myc monoclonal antibodies (9E10.2) were manufactured by Maine Biotechnology and anti-myc polyclonal antibodies were purchased from Upstate Biotechnology. The anti-IQGAP1 polyclonal antibody has been previously characterized (15Ho 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). Secondary antibodies for ECL detection were from Amersham Biosciences. Fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse or anti-rabbit antibodies were from Jackson ImmunoResearch. Radiolabeled reagents were obtained from PerkinElmer Life Sciences. Bradykinin was purchased from Sigma Chemical Co. GMPPNP was obtained from Calbiochem. All other reagents were of standard analytical grade. T47D cells, which stably overexpress either wild type Cdc42 (T47D/WT Cdc42) or constitutively active Cdc42, termed T47D/V12Cdc42 (22Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (653) Google Scholar), were a generous gift of Dr. Patricia Keely (University of Wisconsin). HEK-293H cells were obtained from Invitrogen. T47D cells were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum in a 37 °C humidified incubator containing 5% CO2. MCF-7 breast carcinoma cells, COS-7 cells, and HEK-293H were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. MCF-7 cells and COS-7 cells were transfected using FuGENE 6 (Roche Molecular Biochemicals) as previously described (17Li Z. Kim S.H. Higgins J.M.G. Brenner M.B. Sacks D.B. J. Biol. Chem. 1999; 274: 37885-37892Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). HEK-293H cells and T47D cells were transfected using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. When analyzing dose-dependent effects, an appropriate amount of empty vector was added to each condition so that the total amount of DNA in each transfection was the same. MCF-7 cells were stably transfected with either pcDNA3 or pcDNA3-myc-IQGAP1 using FuGENE6. Clones were isolated and selected in DMEM containing 800 μg/ml Geneticin (G418). Following subcloning of several individual clones, expression of myc-IQGAP1 was analyzed by Western blotting. One clone, termed MCF-7/IQGAP1–3F, which overexpresses IQGAP1 3-fold compared with vector-transfected MCF-7 cells (data not shown), was utilized in this work. A myc-tagged wild type human IQGAP1 in a pcDNA3 vector was used (14Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar, 15Ho 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). Construction of the IQGAP1ΔWW (residues 643–744 deleted), IQGAP1ΔGRD (residues 1122–1324 deleted), IQGAP1ΔC (residues 1502–1657 deleted), and IQGAP1ΔIQ (residues 699–905 deleted) mutants was described previously (21Sokol S.Y., Li, Z. Sacks D.B. J. Biol. Chem. 2001; 276: 48425-48430Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). IQGAP1ΔGRDF, which lacks the entire GRD (residues 982–1248 deleted), was constructed as follows. pcDNA3-myc-IQGAP1 was cut with EaeI and AflII, and the blunt ends were generated with T4 polymerase. Following digestion withEco47III and XhaI, a 487-base pairEco47III-EaeI fragment and a 1836-base pairAflII-XhaI fragment were purified. pcDNA3-myc-IQGAP1 was cut with Eco47III andXhaI, and a 7839-base pairXhaI-Eco47III fragment was purified. All three fragments were then ligated with T4 DNA ligase. A dual promoter vector that co-expresses GFP and wild type IQGAP1 was generated as described (23Briggs M.W., Li, Z. Sacks D.B. J. Biol. Chem. 2002; 277: 7453-7465Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Briefly, pcDNA3-IQGAP1 and pEGFP were digested, and the resultant fragments were made blunt-ended and ligated. A plasmid containing both reading frames in the same orientation was identified by restriction analysis and selected for use. All plasmids were purified using Qiagen DNA Purification kits (Qiagen), according to the manufacturer's instructions and sequenced to verify mutagenesis. MBP-Cdc42 was constructed as follows. pMAL-C2X was digested withPstI, and the blunt ends were generated by T4 polymerase. The Cdc42 gene was excised from pGEX-cdc42 (generously provided by Dr. Matthew Hart) with EcoRI, and the blunt ends were generated with T4 polymerase and digested with BamHI. Fragments were purified from low melting point agarose and inserted into pMAL-C2X at the BamHI and PstI site. Sequence orientation was confirmed by restriction mapping, and the DNA insert region was sequenced to establish that MBP and Cdc42 were cloned in-frame. MBP-Cdc42 was expressed from this plasmid inEscherichia coli strain BL21 grown in 1 liter of Luria broth. Bacteria were induced with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside for 3.5 h and were then lysed by sonication in buffer A (20 mm Tris-HCl, pH 7.4, 0.2 m NaCl, 10 mm β-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, and 1 mm EDTA). Insoluble material was pelleted, and the supernatant was loaded onto amylose resin columns. After washing with buffer A, MBP-Cdc42 was eluted with 10 mm maltose and dialyzed against phosphate-buffered saline (PBS, 145 mmNaCl, 12 mm Na2HPO4, and 4 mm Na2H2PO4, pH 7.2) containing 6 mm β-mercaptoethanol. Analysis by SDS-PAGE revealed that preparations of MBP-Cdc42 were regularly >90% pure (data not shown). The GST-WASP-GBD (GTPase-binding-domain) has been described previously (24Kim S.H., Li, Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 25Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Briefly, GST-WASP-GBD was generated by polymerase chain reaction, digested, and ligated into a pGEX-KG vector (25Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Following expression of the GST fusion protein in E. coli, purification was performed by glutathione-Sepharose affinity chromatography. Production of GST-IQGAP1 and GST-IQGAP1ΔIQ has been described previously (15Ho 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). Detection of active Cdc42 was performed essentially as previously described (24Kim S.H., Li, Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Briefly, MCF-7 or HEK-293H cells were washed twice in PBS and lysed in 500 μl of buffer B (20 mm Hepes, pH 7.4, 150 mm NaCl, 1% (v/v) Nonidet P-40, 20 mm NaF, 1 mmMgCl2, and protease inhibitors) containing 20 μm GTP. Cell lysates were quick-frozen, thawed, sonicated at 4 °C, and subjected to centrifugation at 15,000 ×g for 10 min at 4 °C. Equal amounts of lysate were pre-cleared for 1 h at 4 °C with 40 μl of glutathione-Sepharose. An equal aliquot of each sample (50 μg) was examined directly as whole cell lysate and equal amounts of protein were incubated with 40 μg of GST-WASP-GBD for 2 h at 4 °C. Following collection of complexes with glutathione-Sepharose, the beads were washed six times with buffer B. SDS-PAGE was performed, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Blots were probed with anti-Cdc42 antibodies and horseradish peroxidase-conjugated sheep-anti-mouse antibodies, and enhanced chemiluminescence (ECL) was used for detection. Where indicated, blots were stripped by incubating in 62.5 mm Tris, pH 6.8, 2% (w/v) SDS, and 0.7% (v/v) β-mercaptoethanol for 30 min at 50 °C and re-probed with anti-Rac or anti-Rho antibodies. COS-7 cells were transfected as outlined above and immunoprecipitation was performed essentially as previously described (17Li Z. Kim S.H. Higgins J.M.G. Brenner M.B. Sacks D.B. J. Biol. Chem. 1999; 274: 37885-37892Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Briefly, following preclearing, equal amounts of protein lysate were incubated with anti-IQGAP1 or anti-myc antibodies for 3 h at 4 °C. Anti-IQGAP1 immune complexes were collected for 2 h with 40 μl of Protein A-Sepharose (AmershamBiosciences) and washed five times with buffer B, and Western blotting was carried out as outlined above. Anti-myc immune complexes were collected with 40 μl of Gamma-bind G-Sepharose (Amersham Biosciences) and processed as described above. Determination of Cdc42 GTPase activity was performed essentially as previously described (15Ho 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). Briefly, 2 μg of purified MBP-Cdc42 was incubated for 15 min in buffer C (1 mm EDTA, 25 mm Tris (pH 7.5), 1 mm dithiothreitol, and 125 mm NaCl). Cdc42 was loaded with radiolabeled GTP for 15 min in buffer D (0.1 mm [γ-32P]GTP and 30 mmMgCl2). Purified GST-IQGAP1, GST-IQGAP1ΔIQ, or GST-IQGAP1ΔGRD was added to equal amounts of [γ-32P]GTP-loaded Cdc42, and GTP hydrolysis was initiated by adding 7.5 mm MgCl2 at 22 °C. As a control, samples without added Mg2+ were processed in parallel. After 15 min, assays were terminated by addition of 4 ml of ice-cold PBS containing 5 mm MgCl2. Samples were spotted on Millipore HA 0.45-μm filters, and a vacuum was applied using a Millipore Sampling Manifold Apparatus. Following two washes with ice-cold PBS containing 5 mm MgCl2, the amount of radiolabeled GTP retained on filter membranes was quantified by liquid scintillation spectrometry. GTP hydrolysis was calculated as the difference between the amount of [γ-32P]GTP retained in the absence and presence of Mg2+. In all analyses, wild type and mutant IQGAP1 constructs were processed in parallel in the same assay. HEK-293H cells were washed twice in PBS and lysed in 1 ml of hypotonic lysis buffer E (20 mmTris, pH 7.5, 25 mm NaF, 1 mm EDTA) for 20 min on ice. Lysates were subjected to 30 strokes in a Dounce homogenizer on ice. Crude homogenates were then subjected to centrifugation at 100,000 × g for 30 min at 4 °C. The supernatant (S100) fraction was collected, and the pellet fraction (P100) was resuspended in 1 ml of buffer E. MCF-7 cells were trypsinized, plated on Permanox plastic slides in DMEM, allowed to attach overnight, and transiently transfected with myc-tagged wild type IQGAP1 or IQGAP1ΔGRD. Cells were serum-starved for 24 h and treated with 400 nm bradykinin for 10 min. Slides were then processed for immunocytochemistry as previously described (24Kim S.H., Li, Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Briefly, slides were washed once with PBS, followed by fixation in 3.7% (v/v) formaldehyde for 20 min at 22 °C. Cells were permeabilized in 0.5% (v/v) Triton X-100 for 15 min, and blocked for 1 h in 2% (w/v) bovine serum albumin at 22 °C. Slides were then incubated for 1 h at 22 °C with rhodamine-phalloidin and an anti-myc antibody followed by FITC-labeled goat-anti-rabbit antibody. After four washes with PBS, slides were mounted with Aqua Polymount (Polysciences, Inc.). Specificity of immunostaining was determined by omission of the primary antibody. Confocal laser scanning microscopy was done with a Zeiss Axiovert S100 microscope and the MRC-1024 Confocal Imaging System (Bio-Rad) as described previously (17Li Z. Kim S.H. Higgins J.M.G. Brenner M.B. Sacks D.B. J. Biol. Chem. 1999; 274: 37885-37892Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Images were processed using the Lasersharp 3.0 program (Bio-Rad). For live cell imaging, MCF-7 cells were plated on Lab-Tek II chambered coverglass (Nalge Nunc Int.) as previously described (24Kim S.H., Li, Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Cells were transiently transfected with a dual promoter plasmid co-expressing GFP and wild type IQGAP1. Transfection efficiency was usually between 50 and 60%. Cells were imaged at 5-min intervals for 20 min by time-lapse video microscopy using a Zeiss Axiovert S100 microscope. Images were collected and processed as described above. For binding, MBP-Cdc42 was loaded with either GDP or GMPPNP as previously described (26Thompson G. Owen D. Chalk P.A. Lowe P.N. Biochemistry. 1998; 37: 7885-7891Crossref PubMed Scopus (123) Google Scholar). Briefly, MBP-Cdc42 was incubated at 37 °C in the presence of 4.4 mm GMPPNP, 0.3 m ammonium sulfate, and alkaline phosphatase-agarose for 30 min. MgCl2 was then added to 20 mm. Unbound nucleotide was removed on a Sephadex G-25 centrifuge gel filtration column, equilibrated in 10 mmTris-HCl, pH 7.5, 1 mm dithiothreitol, and 1 mmMgCl2. Nucleotide complexes of MBP-Cdc42 were precleared by incubating with 40 μl of glutathione-Sepharose for 1 h. GST, GST-IQGAP1, or GST-IQGAP1ΔGRD was incubated with loaded MBP-Cdc42 in 500 μl of buffer B for 3 h at 4 °C. Samples were washed six times in buffer B, and complexes were isolated with glutathione-Sepharose and resolved by SDS-PAGE. Proteins were transferred to PVDF membrane and probed with anti-Cdc42 and anti-IQGAP1 antibodies. Densitometry of 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). Previous work published by both our laboratory (15Ho 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) and other investigators (25Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) documented that IQGAP1 inhibits the intrinsic GTPase activity of Cdc42in vitro, suggesting that IQGAP1 may stabilize Cdc42 in the active (GTP-bound) form. Therefore, the effect of IQGAP1 on levels of active Cdc42 in intact cells was examined by a pull-down assay utilizing a GST-WASP-GBD construct. Because GST-WASP-GBD binds only GTP-bound Cdc42, the amount of bound Cdc42 reflects the amount of active Cdc42 (24Kim S.H., Li, Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). HEK-293H cells were transiently transfected with either vector or wild type IQGAP1. Overexpression of IQGAP1 by 2.0- ± 0.05-fold (mean ± S.E., n = 10, p< 0.005) resulted in 1.59- ± 0.02-fold (mean ± S.E.,n = 10, p < 0.005) increase in active Cdc42 (Fig. 1, A andB). The increased IQGAP1 did not change the total amount of Cdc42 in the cell lysate (Fig. 1 A). The positive effect of IQGAP1 on Cdc42 was confirmed in a different system. MCF-7 human breast epithelial cells that stably overexpress IQGAP1 by 3.1- ± 0.25-fold (mean ± S.E., n = 3,p < 0.05) were developed. Compared with vector-transfected cells, overexpression of IQGAP1 increased active Cdc42 in MCF-7 cells by 2.1- ± 0.24-fold (mean ± S.E.,n = 3, p < 0.05), without altering total Cdc42 (Fig. 1, A and B). A panel of mutant IQGAP1 constructs was used to ascertain which domain(s) of IQGAP1 is (are) important in mediating the increase in active Cdc42. Analysis was performed with IQGAP1 mutants lacking the WW domain (IQGAP1ΔWW), the IQ domain (IQGAP1ΔIQ), a portion of the GRD (IQGAP1ΔGRD), the C-terminal tail (IQGAP1ΔC), and the entire GRD (IQGAP1ΔGRDF). (Note that Cdc42 co-immunoprecipitates with IQGAP1ΔWW but not with IQGAP1ΔIQ, IQGAP1ΔC, or IQGAP1ΔGRD (21Sokol S.Y., Li, Z. Sacks D.B. J. Biol. Chem. 2001; 276: 48425-48430Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar).) Probing Western blots with anti-myc antibodies (all IQGAP1 constructs have a myc tag) verified that all IQGAP1 constructs were expressed at approximately equal levels (Fig. 1 A). Analogous to wild type IQGAP1 and consistent with co-immunoprecipitation results, IQGAP1ΔWW significantly increased active Cdc42 (by 1.94- ± 0.08-fold, mean ± S.E., n = 10, p< 0.05) (Fig. 1, A and B). By contrast, neither IQGAP1ΔIQ, IQGAP1ΔC, nor IQGAP1ΔGRDF significantly changed the amount of active Cdc42. Unexpectedly, active Cdc42 was reduced by 0.64- ± 0.01-fold (mean ± S.E., n = 10,p < 0.0005) by expression of IQGAP1ΔGRD (Fig. 1,A and B). The effect of IQGAP1ΔGRD on Cdc42 activity was investigated further. HEK-293H cells were transiently transfected with different amounts of IQGAP1ΔGRD. Where necessary, an appropriate amount of empty vector was added so that the total amount of DNA transfected into all cells was identical. Compared with vector-transfected cells, IQGAP1ΔGRD expression resulted in a dose-dependent decrease in the amount of active Cdc42 (Fig.2 A). Co-transfection with equal amounts of IQGAP1 and IQGAP1ΔGRD abrogated the increase in active Cdc42 produced by wild type IQGAP1 (Fig.2 B). These dual transfected cells exhibited an ∼30% decrease in active Cdc42 compared with cells overexpressing IQGAP1 alone. Together these observations indicate that IQGAP1ΔGRD is a dominant negative inhibitor of IQGAP1 activity. Cdc42 is an important regulator of the cytoskeleton (1Mackay D.J.G. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). Because overexpression of wild type IQGAP1 and IQGAP1ΔGRD modulated active Cdc42 differentially, we examined the effect of these constructs on the cytoskeleton by confocal microscopy. MCF-7 cells were transfected with either vector, IQGAP1, or IQGAP1ΔGRD. Slides were probed with anti-myc antibody to identify transfected cells and stained with rhodamine-phalloidin to visualize F-actin. In MCF-7 cells, transfected wild type IQGAP1 is distributed throughout the cytoplasm, with accumulation at cell-cell junctions (Fig. 3 A,myc). This location is essentially identical to that of endogenous IQGAP1 in these cells (17Li Z. Kim S.H. Higgins J.M.G. Brenner M.B. Sacks D.B. J. Biol. Chem. 1999; 274: 37885-37892Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Following transient transfection, wild type IQGAP1-expressing cells exhibited significantly more microspikes than vector-transfected cells (Fig. 3, A, F-actin, and B). Quantification at 24 h revealed that cells transfected with vector or IQGAP1 had 0.13 ± 0.04 and 10.82 ± 0.43 microspikes per cell (mean ± S.E., n = 100, p < 0.0001), respectively (Fig. 3 B). Live cell imaging was performed to determine whether IQGAP1 induced the formation of true filopodia. A plasmid that co-expresses GFP and wild type IQGAP1 was transiently transfected into MCF-7 cells. Time lapse analysis demonstrated that transfection of IQGAP1 induced the formation of filopodia, which were observed at the 10-, 15-, and 20-min time points (Fig. 3 C). MCF-7 cells transfected with IQGAP1ΔGRD exhibited a morphology markedly different from that seen in cells transfected with wild type IQGAP1. IQGAP1ΔGRD-containing cells were rounded and substantia" @default.
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• W2023807898 date "2002-07-01" @default.
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• W2023807898 title "IQGAP1 Is a Component of Cdc42 Signaling to the Cytoskeleton" @default.
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