The Mechanism for Regulation of the F-actin Binding Activity of IQGAP1 by Calcium/Calmodulin
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m109535200
ISSN1083-351X
AutoresScott C. Mateer, Amanda McDaniel, Valérie Nicolas, Geoffrey M. Habermacher, Mei-Jung Lin, Damond A. Cromer, Michelle E. King, George S. Bloom,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoIQGAP1 colocalizes with actin filaments in the cell cortex and binds in vitro to F-actin and several signaling proteins, including calmodulin, Cdc42, Rac1, and β-catenin. It is thought that the F-actin binding activity of IQGAP1 is regulated by its reversible association with these signaling molecules, but the mechanisms have remained obscure. Here we describe the regulatory mechanism for calmodulin. Purified adrenal IQGAP1 was found to consist of two distinct protein pools, one of which bound F-actin and lacked calmodulin, and the other of which did not bind F-actin but was tightly associated with calmodulin. Based on this finding we hypothesized that calmodulin negatively regulates binding of IQGAP1 to F-actin. This hypothesis was tested in vitro using recombinant wild type and mutated IQGAP1s and in live cells that transiently expressed IQGAP1-YFP. In vitro, the affinity of wild type IQGAP1 for F-actin decreased with increasing concentrations of calmodulin, and this effect was dramatically enhanced by Ca2+ and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAP1 much more efficiently in the presence of Ca2+ than EGTA, and all 8 IQ motifs in each IQGAP1 dimer could bind calmodulin simultaneously. In live cells, IQGAP1-YFP localized to the cell cortex, but elevation of intracellular Ca2+ reversibly induced the fluorescent fusion protein to become diffusely distributed. Taken together, these results support a model in which a rise in free intracellular Ca2+ promotes binding of calmodulin to IQGAP1, which in turn inhibits IQGAP1 from binding to cortical actin filaments. IQGAP1 colocalizes with actin filaments in the cell cortex and binds in vitro to F-actin and several signaling proteins, including calmodulin, Cdc42, Rac1, and β-catenin. It is thought that the F-actin binding activity of IQGAP1 is regulated by its reversible association with these signaling molecules, but the mechanisms have remained obscure. Here we describe the regulatory mechanism for calmodulin. Purified adrenal IQGAP1 was found to consist of two distinct protein pools, one of which bound F-actin and lacked calmodulin, and the other of which did not bind F-actin but was tightly associated with calmodulin. Based on this finding we hypothesized that calmodulin negatively regulates binding of IQGAP1 to F-actin. This hypothesis was tested in vitro using recombinant wild type and mutated IQGAP1s and in live cells that transiently expressed IQGAP1-YFP. In vitro, the affinity of wild type IQGAP1 for F-actin decreased with increasing concentrations of calmodulin, and this effect was dramatically enhanced by Ca2+ and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAP1 much more efficiently in the presence of Ca2+ than EGTA, and all 8 IQ motifs in each IQGAP1 dimer could bind calmodulin simultaneously. In live cells, IQGAP1-YFP localized to the cell cortex, but elevation of intracellular Ca2+ reversibly induced the fluorescent fusion protein to become diffusely distributed. Taken together, these results support a model in which a rise in free intracellular Ca2+ promotes binding of calmodulin to IQGAP1, which in turn inhibits IQGAP1 from binding to cortical actin filaments. Actin filament organization in the cell is regulated by a diverse set of factors that collectively control actin polymerization, actin filament length, interfilament cross-links, and interactions of polymerized actin with other cytoskeletal systems and membranes. One such regulatory factor, IQGAP1, is a widely expressed mammalian protein that was first cloned from a human cDNA library using PCR primers to conserved regions within the catalytic domain of Ras GTPase-activating proteins (GAPs) 1The abbreviations used are: GAPsGTPase-activating proteinsCHDcalponin homology domainHBSSHanks' balanced salt solutionUTRuntranslated regionTEMEDN, N, ′, N′-tetramethylethylene- diamine (1.Weissbach 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). Analysis of its predicted amino acid sequence revealed that the IQGAP1 polypeptide does contain a GAP-related domain, as well as four putative calmodulin-binding IQ motifs (1.Weissbach 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). Although the GAP-related domain of IQGAP1 does not seem to have GAP catalytic activity (1.Weissbach 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, 2.Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar), it does form part of a region that preferentially binds to activated forms of the small G proteins, Cdc42 and Rac1 (2.Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar, 3.McCallum 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, 4.Kuroda 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 consists of two identical 190-kDa subunits that associate with cortical actin filaments in cultured mammalian cells and cross-links actin filaments into bundles and gels in vitro (2.Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar, 5.Bashour A.-M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (214) Google Scholar, 6.Fukata M. Kuroda S. Fujii K. Nakamura T. Shoji I. Matsuura 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, 7.Erickson 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). In addition to its association with actin filaments and the small G proteins, IQGAP1 was found to be the major protein in cultured human breast cell lysates to bind immobilized calmodulin in the absence of calcium (8.Joyal 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). Subsequently, a high affinity calmodulin-binding site was mapped to its IQ motifs (9.Ho 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). GTPase-activating proteins calponin homology domain Hanks' balanced salt solution untranslated region N, N, ′, N′-tetramethylethylene- diamine The IQGAP1 polypeptide is characterized by the specific arrangement of several modular motifs. An F-actin-binding calponin homology domain is found near its N terminus and is sequentially followed by six imperfect, putative coiled-coil repeats, a WW domain, four IQ motifs, and a GAP-related domain. Several proteins that are structurally related to IQGAP1 have been described. The most similar proteins in terms of primary sequence and domain organization are IQGAP2, a liver-enriched protein in mammals (3.McCallum 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, 10.Brill S. Li S. Lyman C.W. Church D.M. Wasmuth J.J. Weissbach L. Bernards A. Snijders A. Mol. Cell. Biol. 1996; 16: 4869-4878Crossref PubMed Scopus (223) Google Scholar), and a Hydra protein that has been implicated in tentacle formation (11.Venturelli C.R. Kuznetsov S. Salgado L.M. Bosch T.C. Dev. Genes Evol. 2000; 210: 458-463Crossref PubMed Scopus (6) Google Scholar). Additional IQGAP1-related proteins have been found in Dictyostelium(12.Faix J. Dittrich W. FEBS Lett. 1996; 394: 251-257Crossref PubMed Scopus (64) Google Scholar, 13.Adachi H. Takahashi Y. Hasebe T. Shirouzu M. Yokoyama S. Sutoh K. J. Cell Biol. 1997; 137: 891-898Crossref PubMed Scopus (107) Google Scholar, 14.Lee S. Escalante R. Firtel R.A. Development. 1997; 124: 983-996PubMed Google Scholar), and Saccharomyces cerevisiae (15.Epp J.A. Chant J. Curr. Biol. 1997; 7: 921-929Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 16.Lippincott J. Li R. J. Cell Biol. 1998; 140: 355-366Crossref PubMed Scopus (305) Google Scholar, 17.Osman M.A. Cerione R.A. J. Cell Biol. 1998; 142: 443-455Crossref PubMed Scopus (84) Google Scholar). Although some of these proteins do not contain every major structural motif of IQGAP1, they are sufficiently similar to IQGAP1 to be classified as members of the IQGAP protein family. In addition to binding F-actin, Cdc42, Rac1, and calmodulin, IQGAP1 has also been reported to bind β-catenin (18.Kuroda 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), E-cadherin (18.Kuroda 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, 19.Li 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), and myosin essential light chain (20.Weissbach L. Bernards A. Herion D.W. Biochem. Biophys. Res. Commun. 1998; 251: 269-276Crossref PubMed Scopus (52) Google Scholar). In light of its numerous structural motifs and binding partners, it seems likely that the integration of input from multiple signaling pathways determines which proteins are bound to IQGAP1 at any moment. Supporting this idea is the competitive binding to IQGAP1 that has been described between Ca2+/calmodulin and activated Cdc42 (8.Joyal 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, 9.Ho 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), Ca2+/calmodulin and E-cadherin (19.Li 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), and activated Cdc42 or Rac1 and β-catenin (21.Fukata M. Kuroda S. Nakagawa M. Kawajiri A. Itoh N. Shoji I. Matsuura Y. Yonehara S. Fujisawa H. Kikuchi A. Kaibuchi K. J. Biol. Chem. 1999; 274: 26044-26050Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Similarly, we reported previously (5.Bashour A.-M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (214) Google Scholar) that exogenous calmodulin modestly inhibited binding of F-actin to native IQGAP1 purified from bovine adrenal tissue. In the present report, we describe our more recent efforts to clarify how calmodulin influences the binding of IQGAP1 to F-actin. The protein contents of F-actin-binding and non-binding pools of native IQGAP1 were analyzed in further detail. In addition, recombinant full-length and mutant versions of human IQGAP1 were assayed for interactions with calmodulin in the absence and presence of free Ca2+ and with F-actin in the absence and presence of free Ca2+, calmodulin, and Ca2+/calmodulin. The net results of this study suggest that local rises in free intracellular Ca2+ stimulate binding of calmodulin to the IQ motifs on IQGAP1, which in turn reduces the affinity of IQGAP1 for actin filaments. Biochemical, molecular biological, immunochemical, and tissue culture reagents used for this study and their respective vendors are as follows: A23187 Ca2+ ionophore and bovine calmodulin (Calbiochem); Sf9 cells, insect cell media, insect cell antibiotics, the Bac-to-Bac HT expression system, Elongase Amplification System, and the pFastBAC HT vector (Invitrogen); the bacterial strain, BL21DE3, and the pRSET expression vectors (Invitrogen); Tris, TEMED, 40% acrylamide solution (37.5:1), and 2-mercaptoethanol (Bio-Rad); AlexaFluor 488-phalloidin (Molecular Probes, Eugene, OR); secondary antibodies (Kirkegaard & Perry, Gaithersburg, MD); the pSL1180 vector and calmodulin-Sepharose (Amersham Biosciences); the pBlueScript II SK(+) plasmid (Stratagene, La Jolla, CA); restriction enzymes (New England Biolabs, Beverly, MA); oligonucleotides (Integrated DNA Technologies, Inc., Coralville, IA); the BacPAK Baculovirus Rapid Titer kit and pEYFPN1 mammalian expression vector (CLONTECH, Palo Alto, CA); Centriprep concentrators (Millipore Corp., Bedford, MA); Slide-A-Lyzer dialysis cassettes and GelCode Blue staining reagent (Pierce); nickel-nitrilotriacetic acid-agarose purification resin (Qiagen, Valencia, CA); anti-calmodulin monoclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY); Dulbecco's minimum essential medium, and LipofectAMINE Plus (Invitrogen); cosmic calf serum (HyClone, Logan, UT); [α-32P]GTP (ICN, Irvine, CA); Expand Long Template PCR System (Roche Molecular Biochemicals); pGEM-T Easy vector I (Promega, Madison, WI). Unless otherwise stated, all other reagents and chemicals were acquired from Sigma. A pBlueScript II SK(+) plasmid containing the human cDNA for IQGAP1 (pBSIQG1-MH) was kindly provided by Dr. Matt Hart of Onyx Pharmaceuticals. This plasmid was digested with XbaI, and the 694-bp insert containing a portion of the 3′-UTR sequence was removed, and the remaining cDNA was ligated together to form pBSIQG1-2. Next the 5′-UTR was removed by digesting pBSIQG1-2 with NcoI and XhoI. This produced a 523-bp insert that contained not only the 5′-UTR but also 365 nucleotides of the 5′-coding region. The 523-bp insert was removed, and the remaining vector fragment was ligated to a similarly digested 371-bp PCR fragment (see under "PCR Amplification of DNA") that restored the 365 nucleotides of the coding region and introduced an XhoI restriction site 5′ to the start codon. We took the resulting plasmid (pBSIQG-10) and performed a XhoI/XbaI double digest, purified the ∼5500-bp fragment, and ligated it into a similarly digested pSL1180 vector generating the pSLIQG-1 plasmid. Next, to remove the remaining 3′-UTR, the pSLIQG-1 plasmid was digested with KpnI and XbaI, and the vector was purified away from an ∼1100-bp insert. Because this digest removed not only the 3′-UTR but also 500 nucleotides of C-terminal coding region, the purified vector fragment was ligated onto a similarly digested 500-bp PCR fragment (see under "PCR Amplification of DNA") to restore the missing coding region and to introduce 3 stop codons and an XbaI site (pSLIQG-2). Finally, pSLIQG-2 was digested with XhoI and XbaI; the 6500-bp insert was purified and then ligated into a pFastBAC HT vector that had been digested with SalI and XbaI. The resulting plasmid (pFBIQG1) was then used to generate baculovirus expressing IQGAP1FL, the full-length wild type protein (see under "Generation of IQGAP1-containing Baculovirus Particles"). To generate the IQGAP1ΔIQ mutant, which lacks the four contiguous IQ motifs but is otherwise identical to IQGAP1FL, we first digested the pSL1180 vector with BglII and BamHI, removed the 31-nucleotide insert, and ligated the vector together creating the vector pSLΔBglII/BamHI. Next, pSLIQG-2 was digested with XhoI and XbaI, and the resulting 6500-bp IQGAP1 fragment was purified and ligated into similarly digested pSLΔBglII/BamHI plasmid to generate pSLIQG-5. The pSLIQG-5 plasmid was then digested with BamHI and SacI, and a 1241-bp insert fragment was removed. The BamHI/SacI digest removed the region of IQGAP1 that encoded the IQ domain region (amino acids 747–862). To restore amino acids 447–746, PCR was used to amplify the cDNA encoding this region (see under "PCR Amplification of DNA"). The DNA primers used for this amplification introduced a BamHI site by creating a silent mutation in the codon for Glu-746 (GAA was changed to GAG) and contained a SacI site. The resulting PCR product was digested with BamHI and SacI, gel-purified, and then ligated into the purified pSLIQG-5 vector to generate pSLIQG1-ΔIQ. Finally, pSLIQG1-ΔIQ was digested with XhoI and XbaI, and the ∼6100-bp insert fragment was purified and ligated into the pFastBAC HT vector that had been digested with SalI and XbaI. The resulting plasmid (pFBIQG1-ΔIQ) was then used to generate baculovirus encoding IQGAP1ΔIQ (see under "Generation of IQGAP1-containing Baculovirus Particles"). To generate the IQGAP1-(2–522) protein fragment, we utilized a second plasmid given to us by Matt Hart, in which the IQGAP1-coding region had been N-terminally fused in-frame to the Myc epitope tag. In this plasmid the start codon was changed from ATG to GGA to create a BamHI site. Digestion of this plasmid with BamHI and HindIII generated a 1567-bp fragment that was ligated into the pRSET vector to generate pRSETIQG1-(2–522). This plasmid was then used to express recombinant protein in bacteria (see under "Expression and Purification of Proteins"). By using pBSIQG1-MH as a template and primers 5′-CCGCTCGAGATGTCCGCCGCAGACGAG-3′ (forward primer) and 5′-CTCATCCATGGCATTCAACTGAAT-3′ (reverse primer), PCR was used to generate a DNA fragment that introduced an XhoI site 5′ to the start codon and to amplify the 5′ 365-bp coding region. Plasmid pBSIQG1-MH and primers 5′-CGGAGGTACCGACAGAGGAGAAAGGCC-3′ (forward primer) and 5′-GCTCTAGACTATCATTACTTCCCGTAGAACTT-3′ (reverse primer) were used to amplify 500 nucleotides from the 3′ end of IQGAP1 and to introduce 3 stop codons and an XbaI site 3′ to the coding region. Finally, to restore the region of IQGAP1 encoding amino acids 447–862, plasmid pSLIQG-2 and primers 5′-CACCCAGAGCTCTCTGTCGCAGTGGA-3′ (forward primer) and 5′-CGCGGATCCTCATTGGCCAGCCACAGCTG-3′ (reverse primer) were used to generate a DNA fragment that contained a SacI site at its 5′ end and introduced a BamHI site at its 3′ end by creating a silent mutation in the codon for Glu-746 (GAA was changed to GAG). All PCRs used the reagents and protocols of the Elongase Amplification System. The size of each PCR product was verified by agarose gel electrophoresis and gel-purified. Baculoviruses expressing either IQGAP1FL or IQGAP1ΔIQ were generated following the procedures and protocols of the Bac-to-Bac HT expression system. Protein expression was verified by Western blotting and immunofluorescence using IQGAP1-specific antibodies. The virus titer was determined by the University of Virginia Tissue Culture Facility using the BacPAK Baculovirus Rapid Titer Kit. Actin was purified from rabbit muscle, as described previously (5.Bashour A.-M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (214) Google Scholar), and was stored as G-actin in small aliquots at −80 °C. When needed, G-actin was polymerized to generate actin filaments (5.Bashour A.-M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (214) Google Scholar) and stored on ice at 4 °C. Native IQGAP1 was purified from bovine adrenal glands by a modification of a method described previously (8.Joyal 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). Briefly, 20–30 adrenal glands were harvested and placed in cold TES buffer: 50 mm Tris, pH 7.4, 1 mm EGTA, 300 mmsucrose, 1 mmdl-dithiothreitol, 0.1 mm, phenylmethylsulfonyl fluoride, and a protease inhibitor mixture containing 10 μg/ml each of leupeptin, chymostatin, and pepstatin A. The glands were diced and homogenized, and the resulting slurry was then centrifuged at 10,000 rpm (gmax = 16,374 × g) in a Sorval RC-5B centrifuge using the GSA rotor for 30 min. Supernatants were then centrifuged an additional 90 min at 40,000 rpm (gmax = 186,000 ×g) using the 45Ti rotor and a Beckman L8–80 ultracentrifuge. Next, the resulting supernatant was passed through a 0.45-μm filter, aliquoted, and either snap-frozen for later use or applied to a calmodulin-Sepharose column. Freshly prepared or thawed supernatant was supplemented with NaCl to 150 mm and Triton X-100 to 1% before being applied to the column, which was equilibrated with TENT buffer (TES buffer lacking sucrose, but containing 150 mm NaCl and 1% Triton X-100). The flow-through was discarded; the column was then washed extensively with TEN buffer (TEN, 50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EGTA, 1 mmdl-dithiothreitol, 0.1 mm, phenylmethylsulfonyl fluoride, and a protease inhibitor mixture containing 10 μg/ml each of leupeptin, chymostatin, and pepstatin A) (TES lacking sucrose, but containing 150 mm NaCl), and bound protein was eluted with TENS buffer (TEN supplemented with 1 m NaSCN). Eluted fractions were monitored by immunoblotting for IQGAP1, and fractions containing IQGAP1 were pooled and concentrated using 30,000 molecular weight cut-off Amicon Centriprep concentrators following the manufacturer's protocol. The concentrated material was then placed into 10,000 molecular weight cut-off Slide-A-Lyzer dialysis cassettes, dialyzed extensively against TEN buffer, aliquoted, and stored at −80 °C. Recombinant IQGAP1FL and IQGAP1ΔIQ were produced using the baculovirus expression system. Briefly, exponentially growing Sf9 cells were infected with appropriate viruses (multiplicity of infection of at least 3). Infected cultures were incubated on a orbital shaker at 27 °C for 48–60 h. The cultures were then centrifuged in a Sorval RC-5B centrifuge using the SLA-1500 rotor at 8,500 rpm (gmax = 10,976 × g) for 30 min. The pellets were then either lysed immediately or stored at −80 °C. To lyse cells, pellets were resuspended in lysis buffer (50 mmNaH2PO4 pH 8.0, 10 mm imidazole, 300 mm NaCl, 5 mm 2-mercaptoethanol) and sonicated. Next, cell suspensions were centrifuged in a Sorval RC-5B centrifuge using SA-600 rotor at 10,500 rpm (gmax = 15,960 × g) for 30 min. The supernatant was then batch absorbed onto nickel-nitrilotriacetic acid-agarose resin that had been equilibrated previously with lysis buffer. The resin was then washed with several resin volumes of wash buffer A (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 20 mm imidazole, and 5 mm 2-mercaptoethanol) followed by several washes with wash buffer B (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 20 mm imidazole, 500 mm NaSCN, 5 mm 2-mercaptoethanol). The bound material was then removed with elution buffer (50 mmNaH2PO4, pH 8.0, 300 mm NaCl, 250 mm imidazole, 5 mm 2-mercaptoethanol). The eluate was placed into 10,000 molecular weight cut-off Slide-A-Lyzer cassettes and dialyzed against several changes of dialysis buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, and 5 mm 2-mercaptoethanol). Finally, the protein was removed from the cassettes, aliquoted, and stored at −80 °C. Recombinant IQGAP1-(2–522) was expressed in the BL21DE3 strain of Escherichia coli. 10 ml of overnight LB agar plates with ampicillin (100 μg/ml) cultures were inoculated with bacteria containing the pRSETIQG1-(2–522) plasmid from well isolated colonies. The next morning 1.0 liter of LB agar plates with ampicillin (100 μg/ml) cultures were inoculated from the overnight suspension and allowed to grow in an orbital shaker (300 rpm at 37 °C) until the A600 nm was 0.6–0.8. Protein expression was then induced by the addition of isopropyl β-d-thiogalactopyranoside to 0.5 mm, followed by an additional 4-h incubation. The cultures were then centrifuged for 30 min at 8,000 rpm (gmax = 10,415 ×g) using the GSA rotor and a Sorval RC-5B centrifuge. The pellets were either resuspended in lysis buffer for purification or stored at −80 °C. After the pellet was resuspended in lysis buffer, lysozyme was added to a final concentration of 1 mg/ml. The suspension was then allowed to incubate for an additional hour on ice. After this point, the purification followed the protocol described above for the purification of IQGAP1FL and IQGAP1ΔIQ. Unless otherwise stated, recombinant IQGAP1 proteins were combined in a 1.5-ml centrifuge tube with the stated reagents in TN buffer (TEN buffer lacking EGTA), and incubated for 30 min at 27 °C. F-actin was then added to the reaction mix. The sample was then either allowed to incubate at 27 °C for 30 min for actin pelleting assays or drawn into a 100-μl capillary tube followed by incubation for 30 min at 27 °C for falling ball viscometry. After the 30-min incubation, the actin pelleting samples were centrifuged for 20 min at 40,000 rpm (gmax = 87,000 ×g) using the Beckman Optima TLX ultracentrifuge and the TLA 100.3 rotor. The supernatant fraction was transferred to a fresh centrifuge tube, and the pellet was resuspended to volume with 1× loading buffer for SDS-PAGE. The samples were then analyzed by SDS-PAGE (22.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), followed either by staining with GelCode Blue or by immunoblotting with anti-IQGAP1 antibodies. Falling ball viscometry and negative stain electron microscopy were performed as described earlier (5.Bashour A.-M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (214) Google Scholar). The polyclonal antibody to IQGAP1 was produced by immunizing a rabbit with IQGAP1-(2–522) and was used either as unfractionated diluted serum or as bulk IgG purified from serum using protein A-Sepharose beads. The IQGAP1 monoclonal IgG2a antibody was produced by footpad immunization of Balb/c mice with purified native bovine adrenal IQGAP1 and fusion of popliteal lymph node cells with NS-1 mouse plasmacytoma cells. The fusion and selection protocol was essentially identical to that used earlier for production of monoclonal antibodies to kinesin (23.Pfister K.K. Wagner M.C. Stenoien D.A. Brady S.T. Bloom G.S. J. Cell Biol. 1989; 108: 1453-1463Crossref PubMed Scopus (193) Google Scholar). With one modification, protocols described previously were also used for quantitation of GTP overlay blots (24.Bloom G.S. Richards B.W. Leopold P.L. Ritchey D.M. Brady S.T. J. Cell Biol. 1993; 120: 467-476Crossref PubMed Scopus (46) Google Scholar), gel electrophoresis (5.Bashour A.-M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (214) Google Scholar), and immunoblotting (5.Bashour A.-M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (214) Google Scholar). The modification was to use a Umax (Freemont, CA) Astra 2200 scanner with a transparency adapter and 12-bit grayscale depth, instead of a CCD camera, to capture digital images of gels and chemiluminescent Western blots. Concentration series of the following purified proteins were used as standards for quantitation: native bovine adrenal IQGAP1, recombinant IQGAP1FL, recombinant IQGAP1ΔIQ, native bovine calmodulin, recombinant His6-Cdc42, and recombinant His6-Rac1. IQGAP1 was immunoprecipitated using the polyclonal IQGAP1 antiserum or IgG purified from the antiserum by protein A-Sepharose affinity chromatography. The antiserum or IgG was added to samples containing IQGAP1, and the resulting mixtures were incubated for 1 h at 27 °C. Next, protein A-Sepharose was added and incubated for an additional hour at 27 °C. After their incubation, the samples were centrifuged for 5–10 s, and the supernatants were removed. The remaining resin was then washed several times with TN buffer. Finally, the beads were suspended in 1× SDS-PAGE sample buffer, heated, and then analyzed by SDS-PAGE and Western blotting. By using a pBSIQGAP1-2 template and the oligonucleotides, 5′-GTCGACTATGTCCGCCGCAGAC-3′ and 5′-CCCGGGGGTAGAACTTTTTGTT-3′, the entire IQGAP1 sequence was amplified by PCR using the Expand Long Template PCR System. The size of the PCR product was verified by agarose gel electrophoresis. The PCR fragment was then ligated into the pGEM-T Easy vector to produce pGEM-IQGAP1. Finally the pGEM-IQGAP1 plasmid was sequentially digested with SalI and SmaI, and the IQGAP1 insert was ligated into a similarly digested pEYFP-N1 expression vector to produce pYFP-IQGAP1. NIH-3T3 fibroblasts were transiently transfected for IQGAP1-YFP expression using LipofectAMINE Plus according to the vendor's instructions (Invitrogen). The cells were maintained in Dulbecco's minimum essential medium supplemented with 10% cosmic calf serum and 50 μg/ml gentamicin sulfate. Live transfected cells were observed and recorded by confocal epifluorescence microscopy on an imaging system containing the following components: a Zeiss Axiovert 100 microscope equipped with a CARV spinning disc confocal head, a temperature-regulated stage, automated shutter, an Atto Arc 100-watt mercury illuminator, and a Hamamatsu (Bridgewater, NJ) Orca-ER cooled CCD. Images captured by the camera were imported into a Power Macintosh G4 computer (Apple; Cupertino, CA) and processed and analyzed using Open Lab 3.0.3 software (Improvision; Lexington, MA). Cells were maintained on the microscope stage in Attofluor Cell Chambers (Atto Instruments; Rockville, MD) at 37 °C in an atmosphere of 95% air plus 5% CO2. Time lapse imaging was controlled by a program designed using the Automator feature of Open Lab. For Fig. 8 and the corresponding on-line QuickTime movies (see the Supplemental Material), Ca2+ addition was achieved by replacing the tissue culture medium with Ca2+-free Hanks' balanced salt solution (HBSS) supplemented the Ca2+ ionophore, A23187, at 5 μm, plus 1 mm CaCl2. For Ca2+ removal, the solution bathing the cells was rep
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