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W4231491489 abstract "Calmodulin regulates diverse Ca2+-dependent cellular processes, including cell cycle progression and cytoskeletal rearrangement. A recently identified calmodulin-binding protein, IQGAP1, interacts with both actin and Cdc42. In this study, evidence is presented that, in the absence of Ca2+, IQGAP1 bound to Cdc42, which maintained Cdc42 in the active GTP-bound state. Addition of Ca2+ both directly abrogated the effect of IQGAP1 on the intrinsic GTPase activity of Cdc42 and, in the presence of calmodulin, dissociated Cdc42 from IQGAP1. In addition, in vitro binding assays revealed that calmodulin associated with both the calponin homology domain and the IQ motifs of IQGAP1. Moreover, F-actin competed with Ca2+/calmodulin for binding to the calponin homology domain, but not the IQ motifs, of IQGAP1. Analysis of cell lysates revealed that calmodulin bound to IQGAP1 in a ternary complex with Cdc42. Increasing the Ca2+ concentration enhanced the interaction between calmodulin and IQGAP1, with a concomitant decrease in the association of IQGAP1 with Cdc42. Our data suggest that IQGAP1 functions as a scaffolding protein, providing a molecular link between Ca2+/calmodulin and Cdc42 signaling. Calmodulin regulates diverse Ca2+-dependent cellular processes, including cell cycle progression and cytoskeletal rearrangement. A recently identified calmodulin-binding protein, IQGAP1, interacts with both actin and Cdc42. In this study, evidence is presented that, in the absence of Ca2+, IQGAP1 bound to Cdc42, which maintained Cdc42 in the active GTP-bound state. Addition of Ca2+ both directly abrogated the effect of IQGAP1 on the intrinsic GTPase activity of Cdc42 and, in the presence of calmodulin, dissociated Cdc42 from IQGAP1. In addition, in vitro binding assays revealed that calmodulin associated with both the calponin homology domain and the IQ motifs of IQGAP1. Moreover, F-actin competed with Ca2+/calmodulin for binding to the calponin homology domain, but not the IQ motifs, of IQGAP1. Analysis of cell lysates revealed that calmodulin bound to IQGAP1 in a ternary complex with Cdc42. Increasing the Ca2+ concentration enhanced the interaction between calmodulin and IQGAP1, with a concomitant decrease in the association of IQGAP1 with Cdc42. Our data suggest that IQGAP1 functions as a scaffolding protein, providing a molecular link between Ca2+/calmodulin and Cdc42 signaling. GTPase-activating protein glutathione S-transferase guanosine 5′-O-(3-thiotriphosphate) polyacrylamide gel electrophoresis polyvinylidene difluoride enhanced chemiluminescence phosphate-buffered saline calponin homology domain. The Ras superfamily comprises a group of small GTPases that function in intracellular signaling cascades, affecting cell growth and differentiation as well as cytoskeletal organization, vesicle trafficking, and nuclear transport (1Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1752) Google Scholar). These proteins act as molecular switches, alternating between an active GTP bound form and an inactive GDP bound form. Regulators include GTPase-activating proteins (GAPs)1 that facilitate conversion from active to inactive states, guanine nucleotide exchange factors that catalyze release of GDP from the GTPase, and GDP-dissociation inhibitory factors that inhibit both GAP-mediated and intrinsic GTP hydrolysis. Deregulation of both GTPases and their regulators may be involved in cell cycle derangement and oncogenesis (2Barbacid M. Eur. J. Clin. Invest. 1990; 20: 225-235Crossref PubMed Scopus (252) Google Scholar).The Rho family GTPase Cdc42 is involved in cytoskeletal rearrangement and cell cycle progression (3Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 4Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). Specifically, active Cdc42 stimulates filopodium and microspike formation in fibroblasts (5Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar, 6Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar) and polarization of actin and microtubules in T cells (7Stowers L. Yelon D. Berg L.J. Chant J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5027-5031Crossref PubMed Scopus (344) Google Scholar). In addition, microinjection of Cdc42 into Swiss 3T3 fibroblasts stimulates DNA synthesis (4Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). It is therefore not surprising that Cdc42 has been implicated in carcinogenesis. Transforming potential has been confirmed by overexpression studies in which dominant negative mutants of Cdc42 inhibit Ras-mediated transformation, whereas constitutively active mutants promote anchorage-independent growth in Rat1 fibroblasts (8Qiu R.G. Abo A. McCormick F. Symons M. Mol. Cell. Biol. 1997; 17: 3449-3458Crossref PubMed Scopus (265) Google Scholar).Downstream effectors of Cdc42 are being elucidated and may include phosphatidylinositol 3-kinase (9Zheng Y. Bagrodia S. Cerione R.A. J. Biol. Chem. 1994; 269: 18727-18730Abstract Full Text PDF PubMed Google Scholar) and pp70S6K (10Chow M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), which are involved in cell cycle progression, and the Wiskott-Aldrich syndrome protein (11Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar) and n-chimaerin (12Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1996; 16: 5069-5080Crossref PubMed Scopus (130) Google Scholar), which may regulate actin polymerization. Cdc42 also stimulates p21-activated Ser/Thr kinases, which in turn regulate the activation of the nuclear mitogenic protein kinases, c-Jun kinase and p38 (13Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 14Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1444) Google Scholar). Interestingly, another potential downstream effector of Cdc42, IQGAP1, displays significant sequence similarity to Sar1 and the tumor suppressor neurofibromin (15Weissbach 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), and is the major calmodulin-binding protein in Ca2+-free breast cell lysates (16Joyal 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 (124) Google Scholar).Calmodulin is a highly conserved, ubiquitous protein involved in diverse Ca2+-dependent cellular processes, including cell cycle progression and proliferation, cyclic nucleotide metabolism, glycogen metabolism, cytoskeletal arrangement, and smooth muscle contraction (17Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishing Co., Inc., New York1988Google Scholar). It possesses four Ca2+-binding sites, occupation of which effects a conformational change that facilitates association with multiple target proteins. Binding to calmodulin occurs via either basic amphiphilic α-helices or IQ motifs, 23 amino acid sequences with the consensus IQXXXRGXXXR (18Houdusse A. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10644-10647Crossref PubMed Scopus (99) Google Scholar). A “complete” IQ motif contains a C-terminal arginine in its consensus sequence, dictating no Ca2+ requirement for calmodulin binding. Alternatively, an “incomplete” IQ motif without arginine requires Ca2+for binding (18Houdusse A. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10644-10647Crossref PubMed Scopus (99) Google Scholar).A substantial body of evidence implicates calmodulin in carcinogenesis. For example, the level of calmodulin is significantly increased in malignant tissue (19Van Eldik L.J. Burgess W.H. J. Biol. Chem. 1983; 258: 4539-4547Abstract Full Text PDF PubMed Google Scholar), including breast carcinoma (20Singer A.L. Sherwin R.P. Dunn A.S. Appleman M.M. Cancer Res. 1976; 36: 60-66PubMed Google Scholar), and overexpression of calmodulin alters cell morphology and shortens the cell cycle (21Rasmussen C.D. Means A.R. Cell. Motil. Cytoskel. 1992; 21: 45-57Crossref PubMed Scopus (12) Google Scholar). Although a causal relationship between calmodulin concentration and malignancy has not been demonstrated, it is hypothesized that increased concentrations of calmodulin may contribute to neoplastic transformation.The downstream effectors of such transformation are unknown. One candidate is the recently isolated 189-kDa protein, IQGAP1. IQGAP1 contains three complete IQ motifs, one incomplete IQ, and a N-terminal region homologous to the actin and calmodulin-binding domain of calponin (22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar, 23Mezgueldi M. Mendre C. Calas B. Kassab R. Fattoum A. J. Biol. Chem. 1995; 270: 8867-8876Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) (Fig. 1). In addition, IQGAP1 contains a region with significant sequence similarity to the catalytic domain of Ras-GAPs (15Weissbach 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 binds Rac and Cdc42 (22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar) and also cross-links microfilaments (24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar).Recently, it was demonstrated that calmodulin binds the N-terminal region of IQGAP1 (22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar), which contains both the calponin homology domain (CHD) and the IQ motifs. Ca2+/calmodulin attenuates the association of IQGAP1 with Cdc42 (16Joyal 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 (124) Google Scholar) and F-actin (24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar). IQGAP1 thus appears to be an actin-associated protein that can transduce Ca2+/calmodulin signals to Cdc42 at the cytoskeleton. To further explore the functional sequelae of the interaction between Ca2+/calmodulin and IQGAP1, we isolated both full-length endogenous human IQGAP1 and glutathione S-transferase (GST) fusion constructs containing selected regions of IQGAP1. We show here that Ca2+ binds directly to IQGAP1 and modulates the IQGAP1-mediated inhibition of Cdc42-catalyzed GTP hydrolysis. We also present evidence that Ca2+/calmodulin competes with F-actin for binding to the CHD of IQGAP1. Finally, we demonstrate that Ca2+ enhances the binding of calmodulin to IQGAP1 thereby inducing the release of Cdc42 from IQGAP1. We conclude that IQGAP1 may provide a molecular link between Ca2+/calmodulin signaling pathways and Cdc42-mediated processes.DISCUSSIONCdc42 facilitates cell cycle progression and cytoskeletal rearrangement (3Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 4Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). However, the regulators that control Cdc42 function and the downstream effectors that link activated Cdc42 to actin reorganization have remained obscure. In this paper, we characterize the regulatory pathways connecting Ca2+, calmodulin, IQGAP1, and Cdc42.Previous in vitro data support a model in which Ca2+/calmodulin regulates Cdc42-mediated GTPase activity through the intermediary protein IQGAP1 (16Joyal 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 (124) Google Scholar, 22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar). The C-terminal region of IQGAP1 has significant sequence similarity to the catalytic domain of all Ras-GAPs and has been hypothesized to act as a GAP (15Weissbach 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). However, data from Hart et al. (22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar) indicate that recombinant IQGAP1 actually stabilizes the GTP-bound state of Cdc42. To elucidate this interaction, the effect of purified, full-length, endogenous IQGAP1 on Cdc42 activity was examined. We demonstrate that Ca2+, independently of calmodulin, modulates the regulation of Cdc42 activity by IQGAP1. Specifically, while IQGAP1 alone maintains Cdc42 in its active GTP-bound state, Ca2+/IQGAP1 fails to inhibit the intrinsic GTPase activity of Cdc42. As Ca2+alone does not impair the binding of IQGAP1 to Cdc42 (16Joyal 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 (124) Google Scholar), this Ca2+ effect is not secondary to dissociation of IQGAP1 from Cdc42.We theorized that the direct effect of Ca2+ on IQGAP1 may be due to direct association of Ca2+ with IQGAP1. Notably, amino acids 48–161 in the N-terminal region of IQGAP1 display significant sequence similarity to MP-20, a putative Ca2+-binding Drosophila muscle protein (15Weissbach 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, 26Ayme-Southgate A. Lasko P. French C. Pardue M.L. J. Cell Biol. 1989; 108: 521-531Crossref PubMed Scopus (67) Google Scholar). Indeed, the N-terminal region and CHD (which includes amino acids 48–161) of IQGAP1 were shown to bind Ca2+ directly by45Ca2+ overlay. As the GAP homology region that associates with Cdc42 is in the C-terminal half of IQGAP1 (see Fig. 1), Ca2+ binding presumably promotes a conformational change in IQGAP1.Our previous work revealed that calmodulin in the presence of Ca2+ effects the dissociation of IQGAP1 from Cdc42 (16Joyal 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 (124) Google Scholar).In vitro, Ca2+/calmodulin appears then to be a negative regulator of Cdc42 activity on two levels. In the absence of Ca2+, IQGAP1 binds Cdc42 and maintains it in its active GTP bound state. An increase in Ca2+ concentration both abolishes the inhibition of Cdc42 GTPase activity by IQGAP1 and, in the presence of calmodulin, dissociates IQGAP1 from Cdc42. Although we have not demonstrated this thesis in vivo, the data in Figs. 6and 7 provide support for this model in the normal cell milieu. The results presented in Fig. 8 further substantiate a physiological role for calmodulin in IQGAP1 function, as a substantial proportion of endogenous IQGAP1 is bound to Ca2+/calmodulin.A second mechanism for Ca2+/calmodulin regulation of Cdc42 via IQGAP1 involves subcellular localization. Bashour et al.(24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar) have shown that IQGAP1 interacts with and cross-links microfilaments. Calmodulin inhibits this interaction. In addition, immunohistochemical studies colocalize IQGAP1 with cytochalasin D-sensitive microfilaments in lamellipodia and membrane ruffles (24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar). Here, we demonstrate that Ca2+/calmodulin binds to the CHD of IQGAP1 and F-actin diminishes this association. Since F-actin does not bind directly to calmodulin (17Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishing Co., Inc., New York1988Google Scholar), our data indicate that F-actin binds to the CHD of IQGAP1. Because Ca2+/calmodulin and F-actin compete for binding to the CHD, IQGAP1 may couple Cdc42 to microfilaments in the absence of Ca2+. Indeed, GTP-bound Cdc42 co-immunoprecipitated with IQGAP1 and F-actin (27Erickson 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) and enhanced F-actin cross-linking by IQGAP1 (28Fukata 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 (163) Google Scholar). In the presence of Ca2+, calmodulin may dissociate IQGAP1 from not only Cdc42 but also from F-actin, insuring a separation of Cdc42 from microfilaments.However, if the results of our in vitro GTPase analysis are replicated in intact cells and IQGAP1 does indeed inhibit the intrinsic GTPase activity of Cdc42 in the absence of Ca2+, an alternative possibility exists. Active GTP-bound Cdc42 may act upstream of IQGAP1 and may inhibit a function of IQGAP1 at the cytoskeleton. In the absence of Ca2+, IQGAP1 may bind activated Cdc42, stabilize its active GTP-bound state, and localize it to cytoskeletal structures. There, active Cdc42 may inhibit the effect of IQGAP1 on cytoskeletal rearrangement. Increased intracellular Ca2+may abolish the inhibition of Cdc42 GTPase activity by IQGAP1 and may dissociate IQGAP1 from both Cdc42 and microfilaments. In this model, IQGAP1 is both a regulator of Cdc42 localization and activity, and a downstream target of Cdc42 function. The N-terminal of IQGAP1 binds Ca2+/calmodulin and actin, while the C-terminal interacts with Cdc42. As such, IQGAP1 may serve as a scaffold for a multimeric actin complex, providing a molecular link between the cytoskeleton and Cdc42 and mediating a regulatory role by Ca2+/calmodulin.The functional sequelae of the interaction of Ca2+/calmodulin with IQGAP1 remain under investigation. The interaction between calmodulin and IQGAP1 is likely physiologic, as IQGAP1 is the predominant calmodulin-binding protein in Ca2+-free breast carcinoma cell lysates (16Joyal 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 (124) Google Scholar) and a substantial fraction of endogenous IQGAP1 is bound to Ca2+/calmodulin in the normal cellular milieu. As Cdc42 participates in cell proliferation and regulation of the cytoskeleton, involvement of Ca2+ and calmodulin in such processes may involve IQGAP1 as an intermediary. The Ras superfamily comprises a group of small GTPases that function in intracellular signaling cascades, affecting cell growth and differentiation as well as cytoskeletal organization, vesicle trafficking, and nuclear transport (1Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1752) Google Scholar). These proteins act as molecular switches, alternating between an active GTP bound form and an inactive GDP bound form. Regulators include GTPase-activating proteins (GAPs)1 that facilitate conversion from active to inactive states, guanine nucleotide exchange factors that catalyze release of GDP from the GTPase, and GDP-dissociation inhibitory factors that inhibit both GAP-mediated and intrinsic GTP hydrolysis. Deregulation of both GTPases and their regulators may be involved in cell cycle derangement and oncogenesis (2Barbacid M. Eur. J. Clin. Invest. 1990; 20: 225-235Crossref PubMed Scopus (252) Google Scholar). The Rho family GTPase Cdc42 is involved in cytoskeletal rearrangement and cell cycle progression (3Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 4Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). Specifically, active Cdc42 stimulates filopodium and microspike formation in fibroblasts (5Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar, 6Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar) and polarization of actin and microtubules in T cells (7Stowers L. Yelon D. Berg L.J. Chant J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5027-5031Crossref PubMed Scopus (344) Google Scholar). In addition, microinjection of Cdc42 into Swiss 3T3 fibroblasts stimulates DNA synthesis (4Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). It is therefore not surprising that Cdc42 has been implicated in carcinogenesis. Transforming potential has been confirmed by overexpression studies in which dominant negative mutants of Cdc42 inhibit Ras-mediated transformation, whereas constitutively active mutants promote anchorage-independent growth in Rat1 fibroblasts (8Qiu R.G. Abo A. McCormick F. Symons M. Mol. Cell. Biol. 1997; 17: 3449-3458Crossref PubMed Scopus (265) Google Scholar). Downstream effectors of Cdc42 are being elucidated and may include phosphatidylinositol 3-kinase (9Zheng Y. Bagrodia S. Cerione R.A. J. Biol. Chem. 1994; 269: 18727-18730Abstract Full Text PDF PubMed Google Scholar) and pp70S6K (10Chow M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), which are involved in cell cycle progression, and the Wiskott-Aldrich syndrome protein (11Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar) and n-chimaerin (12Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1996; 16: 5069-5080Crossref PubMed Scopus (130) Google Scholar), which may regulate actin polymerization. Cdc42 also stimulates p21-activated Ser/Thr kinases, which in turn regulate the activation of the nuclear mitogenic protein kinases, c-Jun kinase and p38 (13Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 14Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1444) Google Scholar). Interestingly, another potential downstream effector of Cdc42, IQGAP1, displays significant sequence similarity to Sar1 and the tumor suppressor neurofibromin (15Weissbach 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), and is the major calmodulin-binding protein in Ca2+-free breast cell lysates (16Joyal 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 (124) Google Scholar). Calmodulin is a highly conserved, ubiquitous protein involved in diverse Ca2+-dependent cellular processes, including cell cycle progression and proliferation, cyclic nucleotide metabolism, glycogen metabolism, cytoskeletal arrangement, and smooth muscle contraction (17Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishing Co., Inc., New York1988Google Scholar). It possesses four Ca2+-binding sites, occupation of which effects a conformational change that facilitates association with multiple target proteins. Binding to calmodulin occurs via either basic amphiphilic α-helices or IQ motifs, 23 amino acid sequences with the consensus IQXXXRGXXXR (18Houdusse A. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10644-10647Crossref PubMed Scopus (99) Google Scholar). A “complete” IQ motif contains a C-terminal arginine in its consensus sequence, dictating no Ca2+ requirement for calmodulin binding. Alternatively, an “incomplete” IQ motif without arginine requires Ca2+for binding (18Houdusse A. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10644-10647Crossref PubMed Scopus (99) Google Scholar). A substantial body of evidence implicates calmodulin in carcinogenesis. For example, the level of calmodulin is significantly increased in malignant tissue (19Van Eldik L.J. Burgess W.H. J. Biol. Chem. 1983; 258: 4539-4547Abstract Full Text PDF PubMed Google Scholar), including breast carcinoma (20Singer A.L. Sherwin R.P. Dunn A.S. Appleman M.M. Cancer Res. 1976; 36: 60-66PubMed Google Scholar), and overexpression of calmodulin alters cell morphology and shortens the cell cycle (21Rasmussen C.D. Means A.R. Cell. Motil. Cytoskel. 1992; 21: 45-57Crossref PubMed Scopus (12) Google Scholar). Although a causal relationship between calmodulin concentration and malignancy has not been demonstrated, it is hypothesized that increased concentrations of calmodulin may contribute to neoplastic transformation. The downstream effectors of such transformation are unknown. One candidate is the recently isolated 189-kDa protein, IQGAP1. IQGAP1 contains three complete IQ motifs, one incomplete IQ, and a N-terminal region homologous to the actin and calmodulin-binding domain of calponin (22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar, 23Mezgueldi M. Mendre C. Calas B. Kassab R. Fattoum A. J. Biol. Chem. 1995; 270: 8867-8876Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) (Fig. 1). In addition, IQGAP1 contains a region with significant sequence similarity to the catalytic domain of Ras-GAPs (15Weissbach 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 binds Rac and Cdc42 (22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar) and also cross-links microfilaments (24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar). Recently, it was demonstrated that calmodulin binds the N-terminal region of IQGAP1 (22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar), which contains both the calponin homology domain (CHD) and the IQ motifs. Ca2+/calmodulin attenuates the association of IQGAP1 with Cdc42 (16Joyal 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 (124) Google Scholar) and F-actin (24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar). IQGAP1 thus appears to be an actin-associated protein that can transduce Ca2+/calmodulin signals to Cdc42 at the cytoskeleton. To further explore the functional sequelae of the interaction between Ca2+/calmodulin and IQGAP1, we isolated both full-length endogenous human IQGAP1 and glutathione S-transferase (GST) fusion constructs containing selected regions of IQGAP1. We show here that Ca2+ binds directly to IQGAP1 and modulates the IQGAP1-mediated inhibition of Cdc42-catalyzed GTP hydrolysis. We also present evidence that Ca2+/calmodulin competes with F-actin for binding to the CHD of IQGAP1. Finally, we demonstrate that Ca2+ enhances the binding of calmodulin to IQGAP1 thereby inducing the release of Cdc42 from IQGAP1. We conclude that IQGAP1 may provide a molecular link between Ca2+/calmodulin signaling pathways and Cdc42-mediated processes. DISCUSSIONCdc42 facilitates cell cycle progression and cytoskeletal rearrangement (3Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 4Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). However, the regulators that control Cdc42 function and the downstream effectors that link activated Cdc42 to actin reorganization have remained obscure. In this paper, we characterize the regulatory pathways connecting Ca2+, calmodulin, IQGAP1, and Cdc42.Previous in vitro data support a model in which Ca2+/calmodulin regulates Cdc42-mediated GTPase activity through the intermediary protein IQGAP1 (16Joyal 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 (124) Google Scholar, 22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar). The C-terminal region of IQGAP1 has significant sequence similarity to the catalytic domain of all Ras-GAPs and has been hypothesized to act as a GAP (15Weissbach 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). However, data from Hart et al. (22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar) indicate that recombinant IQGAP1 actually stabilizes the GTP-bound state of Cdc42. To elucidate this interaction, the effect of purified, full-length, endogenous IQGAP1 on Cdc42 activity was examined. We demonstrate that Ca2+, independently of calmodulin, modulates the regulation of Cdc42 activity by IQGAP1. Specifically, while IQGAP1 alone maintains Cdc42 in its active GTP-bound state, Ca2+/IQGAP1 fails to inhibit the intrinsic GTPase activity of Cdc42. As Ca2+alone does not impair the binding of IQGAP1 to Cdc42 (16Joyal 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 (124) Google Scholar), this Ca2+ effect is not secondary to dissociation of IQGAP1 from Cdc42.We theorized that the direct effect of Ca2+ on IQGAP1 may be due to direct association of Ca2+ with IQGAP1. Notably, amino acids 48–161 in the N-terminal region of IQGAP1 display significant sequence similarity to MP-20, a putative Ca2+-binding Drosophila muscle protein (15Weissbach 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, 26Ayme-Southgate A. Lasko P. French C. Pardue M.L. J. Cell Biol. 1989; 108: 521-531Crossref PubMed Scopus (67) Google Scholar). Indeed, the N-terminal region and CHD (which includes amino acids 48–161) of IQGAP1 were shown to bind Ca2+ directly by45Ca2+ overlay. As the GAP homology region that associates with Cdc42 is in the C-terminal half of IQGAP1 (see Fig. 1), Ca2+ binding presumably promotes a conformational change in IQGAP1.Our previous work revealed that calmodulin in the presence of Ca2+ effects the dissociation of IQGAP1 from Cdc42 (16Joyal 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 (124) Google Scholar).In vitro, Ca2+/calmodulin appears then to be a negative regulator of Cdc42 activity on two levels. In the absence of Ca2+, IQGAP1 binds Cdc42 and maintains it in its active GTP bound state. An increase in Ca2+ concentration both abolishes the inhibition of Cdc42 GTPase activity by IQGAP1 and, in the presence of calmodulin, dissociates IQGAP1 from Cdc42. Although we have not demonstrated this thesis in vivo, the data in Figs. 6and 7 provide support for this model in the normal cell milieu. The results presented in Fig. 8 further substantiate a physiological role for calmodulin in IQGAP1 function, as a substantial proportion of endogenous IQGAP1 is bound to Ca2+/calmodulin.A second mechanism for Ca2+/calmodulin regulation of Cdc42 via IQGAP1 involves subcellular localization. Bashour et al.(24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar) have shown that IQGAP1 interacts with and cross-links microfilaments. Calmodulin inhibits this interaction. In addition, immunohistochemical studies colocalize IQGAP1 with cytochalasin D-sensitive microfilaments in lamellipodia and membrane ruffles (24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar). Here, we demonstrate that Ca2+/calmodulin binds to the CHD of IQGAP1 and F-actin diminishes this association. Since F-actin does not bind directly to calmodulin (17Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishing Co., Inc., New York1988Google Scholar), our data indicate that F-actin binds to the CHD of IQGAP1. Because Ca2+/calmodulin and F-actin compete for binding to the CHD, IQGAP1 may couple Cdc42 to microfilaments in the absence of Ca2+. Indeed, GTP-bound Cdc42 co-immunoprecipitated with IQGAP1 and F-actin (27Erickson 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) and enhanced F-actin cross-linking by IQGAP1 (28Fukata 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 (163) Google Scholar). In the presence of Ca2+, calmodulin may dissociate IQGAP1 from not only Cdc42 but also from F-actin, insuring a separation of Cdc42 from microfilaments.However, if the results of our in vitro GTPase analysis are replicated in intact cells and IQGAP1 does indeed inhibit the intrinsic GTPase activity of Cdc42 in the absence of Ca2+, an alternative possibility exists. Active GTP-bound Cdc42 may act upstream of IQGAP1 and may inhibit a function of IQGAP1 at the cytoskeleton. In the absence of Ca2+, IQGAP1 may bind activated Cdc42, stabilize its active GTP-bound state, and localize it to cytoskeletal structures. There, active Cdc42 may inhibit the effect of IQGAP1 on cytoskeletal rearrangement. Increased intracellular Ca2+may abolish the inhibition of Cdc42 GTPase activity by IQGAP1 and may dissociate IQGAP1 from both Cdc42 and microfilaments. In this model, IQGAP1 is both a regulator of Cdc42 localization and activity, and a downstream target of Cdc42 function. The N-terminal of IQGAP1 binds Ca2+/calmodulin and actin, while the C-terminal interacts with Cdc42. As such, IQGAP1 may serve as a scaffold for a multimeric actin complex, providing a molecular link between the cytoskeleton and Cdc42 and mediating a regulatory role by Ca2+/calmodulin.The functional sequelae of the interaction of Ca2+/calmodulin with IQGAP1 remain under investigation. The interaction between calmodulin and IQGAP1 is likely physiologic, as IQGAP1 is the predominant calmodulin-binding protein in Ca2+-free breast carcinoma cell lysates (16Joyal 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 (124) Google Scholar) and a substantial fraction of endogenous IQGAP1 is bound to Ca2+/calmodulin in the normal cellular milieu. As Cdc42 participates in cell proliferation and regulation of the cytoskeleton, involvement of Ca2+ and calmodulin in such processes may involve IQGAP1 as an intermediary. Cdc42 facilitates cell cycle progression and cytoskeletal rearrangement (3Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 4Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar). However, the regulators that control Cdc42 function and the downstream effectors that link activated Cdc42 to actin reorganization have remained obscure. In this paper, we characterize the regulatory pathways connecting Ca2+, calmodulin, IQGAP1, and Cdc42. Previous in vitro data support a model in which Ca2+/calmodulin regulates Cdc42-mediated GTPase activity through the intermediary protein IQGAP1 (16Joyal 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 (124) Google Scholar, 22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar). The C-terminal region of IQGAP1 has significant sequence similarity to the catalytic domain of all Ras-GAPs and has been hypothesized to act as a GAP (15Weissbach 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). However, data from Hart et al. (22Hart M.F. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2887-3005Crossref Scopus (324) Google Scholar) indicate that recombinant IQGAP1 actually stabilizes the GTP-bound state of Cdc42. To elucidate this interaction, the effect of purified, full-length, endogenous IQGAP1 on Cdc42 activity was examined. We demonstrate that Ca2+, independently of calmodulin, modulates the regulation of Cdc42 activity by IQGAP1. Specifically, while IQGAP1 alone maintains Cdc42 in its active GTP-bound state, Ca2+/IQGAP1 fails to inhibit the intrinsic GTPase activity of Cdc42. As Ca2+alone does not impair the binding of IQGAP1 to Cdc42 (16Joyal 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 (124) Google Scholar), this Ca2+ effect is not secondary to dissociation of IQGAP1 from Cdc42. We theorized that the direct effect of Ca2+ on IQGAP1 may be due to direct association of Ca2+ with IQGAP1. Notably, amino acids 48–161 in the N-terminal region of IQGAP1 display significant sequence similarity to MP-20, a putative Ca2+-binding Drosophila muscle protein (15Weissbach 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, 26Ayme-Southgate A. Lasko P. French C. Pardue M.L. J. Cell Biol. 1989; 108: 521-531Crossref PubMed Scopus (67) Google Scholar). Indeed, the N-terminal region and CHD (which includes amino acids 48–161) of IQGAP1 were shown to bind Ca2+ directly by45Ca2+ overlay. As the GAP homology region that associates with Cdc42 is in the C-terminal half of IQGAP1 (see Fig. 1), Ca2+ binding presumably promotes a conformational change in IQGAP1. Our previous work revealed that calmodulin in the presence of Ca2+ effects the dissociation of IQGAP1 from Cdc42 (16Joyal 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 (124) Google Scholar).In vitro, Ca2+/calmodulin appears then to be a negative regulator of Cdc42 activity on two levels. In the absence of Ca2+, IQGAP1 binds Cdc42 and maintains it in its active GTP bound state. An increase in Ca2+ concentration both abolishes the inhibition of Cdc42 GTPase activity by IQGAP1 and, in the presence of calmodulin, dissociates IQGAP1 from Cdc42. Although we have not demonstrated this thesis in vivo, the data in Figs. 6and 7 provide support for this model in the normal cell milieu. The results presented in Fig. 8 further substantiate a physiological role for calmodulin in IQGAP1 function, as a substantial proportion of endogenous IQGAP1 is bound to Ca2+/calmodulin. A second mechanism for Ca2+/calmodulin regulation of Cdc42 via IQGAP1 involves subcellular localization. Bashour et al.(24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar) have shown that IQGAP1 interacts with and cross-links microfilaments. Calmodulin inhibits this interaction. In addition, immunohistochemical studies colocalize IQGAP1 with cytochalasin D-sensitive microfilaments in lamellipodia and membrane ruffles (24Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (208) Google Scholar). Here, we demonstrate that Ca2+/calmodulin binds to the CHD of IQGAP1 and F-actin diminishes this association. Since F-actin does not bind directly to calmodulin (17Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishing Co., Inc., New York1988Google Scholar), our data indicate that F-actin binds to the CHD of IQGAP1. Because Ca2+/calmodulin and F-actin compete for binding to the CHD, IQGAP1 may couple Cdc42 to microfilaments in the absence of Ca2+. Indeed, GTP-bound Cdc42 co-immunoprecipitated with IQGAP1 and F-actin (27Erickson 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) and enhanced F-actin cross-linking by IQGAP1 (28Fukata 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 (163) Google Scholar). In the presence of Ca2+, calmodulin may dissociate IQGAP1 from not only Cdc42 but also from F-actin, insuring a separation of Cdc42 from microfilaments. However, if the results of our in vitro GTPase analysis are replicated in intact cells and IQGAP1 does indeed inhibit the intrinsic GTPase activity of Cdc42 in the absence of Ca2+, an alternative possibility exists. Active GTP-bound Cdc42 may act upstream of IQGAP1 and may inhibit a function of IQGAP1 at the cytoskeleton. In the absence of Ca2+, IQGAP1 may bind activated Cdc42, stabilize its active GTP-bound state, and localize it to cytoskeletal structures. There, active Cdc42 may inhibit the effect of IQGAP1 on cytoskeletal rearrangement. Increased intracellular Ca2+may abolish the inhibition of Cdc42 GTPase activity by IQGAP1 and may dissociate IQGAP1 from both Cdc42 and microfilaments. In this model, IQGAP1 is both a regulator of Cdc42 localization and activity, and a downstream target of Cdc42 function. The N-terminal of IQGAP1 binds Ca2+/calmodulin and actin, while the C-terminal interacts with Cdc42. As such, IQGAP1 may serve as a scaffold for a multimeric actin complex, providing a molecular link between the cytoskeleton and Cdc42 and mediating a regulatory role by Ca2+/calmodulin. The functional sequelae of the interaction of Ca2+/calmodulin with IQGAP1 remain under investigation. The interaction between calmodulin and IQGAP1 is likely physiologic, as IQGAP1 is the predominant calmodulin-binding protein in Ca2+-free breast carcinoma cell lysates (16Joyal 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 (124) Google Scholar) and a substantial fraction of endogenous IQGAP1 is bound to Ca2+/calmodulin in the normal cellular milieu. As Cdc42 participates in cell proliferation and regulation of the cytoskeleton, involvement of Ca2+ and calmodulin in such processes may involve IQGAP1 as an intermediary. We thank Dr. Matthew Hart (Onyx Pharmaceuticals Richmond, CA) for generously donating reagents, Sharon Porter (Washington University Medical Center, St. Louis, MO) for preparing the anti-calmodulin antibody, and Dr. Jack Ladenson (Washington University Medical Center, St. Louis, MO) for kindly providing the anti-myoglobin antibody." @default.
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