摘要： The 190 kD human IQGAP1 protein, by virtue of its N-terminal calponin-homology domain, is found associated with the actin cytoskeleton, and is capable of cross-linking actin filaments. IQGAP1 complexes with several proteins, including the Rho family GTPases Cdc42 and Rac, as well as calmodulin. It was previously noted that one of the IQ motifs of IQGAP1 displays significant similarity to a myosin heavy chain IQ motif responsible for binding the calmodulin-related myosin essential light chain (ELC). Employing the yeast two-hybrid methodology as well as in vitro binding experiments, we present evidence that a truncated version of IQGAP1 can interact with the myosin ELC. This interaction may have significant consequences for various cellular processes that involve actomyosin contractility, and suggests that the biological targets of the ELC may not be restricted to the myosin heavy chain. (C) 1998 Academic Press.

摘要： The small guanosine triphosphatases (GTPases) Cdc42 and Rad regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target: of Cdc42 and Rac1,was localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L Fibroblasts expressing E-cadherin (EL cells), and interacted with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induced the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, resulted in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway.

摘要： Ca²⁺calmodulin induces dissociation of Cdc42 from IQGAP1 (J Biol Chem 1997, 272, 15419). In this study we characterize the interactions among Ca²⁺, calmodulin, IQGAP1 and Cdc42. IQGAP1 inhibited by 38 t 13% (mean ±SE, n-4) the intrinsic GTPase activity of Cdc42. Both Ca²⁺ alone and Ca²⁺/calmodulin abrogated the effect of IQGAP1 on Cdc42 activity. Analysis with GST fusion proteins revealed that Ca²⁺, Ca²⁺/calmodulin and apocalmodulin bound to the N-terminal region of IQGAP1. Further investigation disclosed that Ca²⁺/calmodulin bound to the IQ and calponin homology domains of IQGAP1, with F-actin competing for the latter binding site. Immunodepletion of MCF-7 cell lysates with anticalmodulin monoclonal antibody demonstrated that a substantial proportion of endogenous IQGAP1 was bound to endogenous Ca²⁺calmodulin. Immunocytochemistry indicated that Cdc42 and IQGAP1 exhibit a predominantly perinuclear distribution in MCF-7 cells. Incubation with A23187, a Ca²⁺ ionophore, or bradykimn induced translocation of Cdc42 to the nucleus without altering the localization of IQGAP1. Together these data suggest that IQGAP1 functions as a scaffolding protein, providing a molecular link between Ca²⁺/calmodulin and Cdc42 signalling.

摘要： We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rad small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1(val12) (Kuroda, S., Fukata, M,, Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 28363-23367). Here we identified an IQGAP1-interacting molecule with molecular mass of 43 kDa (p43) from bovine brain cytosol, using glutathione S-transferase (GST)-IQGAP1 affinity column chromatography. The amino acid sequencing of the protein revealed that p43 was identical to beta- and gamma-actin. IQGAP1 was cosedimentated with filamentous actin (F-actin). The amino-terminal domain (amino acids 1-216) of IQGAP1 was responsible for the interaction with F-actin. Falling ball viscometry assay revealed that IQGAP1 cross-linked the F-actin. This IQGAP1 activity was further enhanced by guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) GST-Cdc42 but not by GDP-GST-Cdc42. The gel filtration analysis of IQGAP1 revealed that IQGAP1 appeared as oligomers and that GTP gamma S-GST-Cdc42 but not GDP GST-Cdc42 enhanced the oligomerization of IQGAP1. These results strongly suggest that IQGAP1, acting downstream of Cdc42, can cross-link the actin filament through its oligomerization.

摘要： Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich Iamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two similar to 190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Enogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D-sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.

摘要： Calmodulin regulates numerous fundamental metabolic pathways by binding to and modulating diverse target proteins. In this study, calmodulin-binding proteins were isolated from normal (Hs578Bst) and malignant (MCF-7) human breast cell lines with calmodulin-Sepharose and analyzed by SDS-polyacrylamide gel electrophoresis. A protein that migrated at approximately 190 kDa bound to calmodulin in the presence of Ca2+ and was the only calmodulin-binding protein detected in the absence of Ca2+. This 190-kDa protein was identified as IQGAP1 by nanoelectrospray mass spectrometry and collision-induced dissociation tandem mass spectrometry. IQGAP1 coimmunoprecipitated with calmodulin from lysates of MCF-7 cells. Moreover, overlay with I-125-calmodulin confirmed that IQGAP1 binds directly to calmodulin. Analysis of the functional effects of the interaction revealed that Ca2+/calmodulin disrupted the binding of purified IQGAP1 to the Ras-related protein Cdc42 in a concentration-dependent manner. These data clearly identify IQGAP1 as the predominant calmodulin-binding protein in Ca2+-free breast cell lysates and reveal that calmodulin modulates the interaction between IQGAP1 and Cdc42.

摘要： Cdc42 is a Ras-related GTP-binding protein that has been implicated in the regulation of the actin cytoskeleton and cell morphology. In this study, we have identified a protein with a molecular mass similar to 180 kDa from rabbit Liver cytosol (designated p180), which binds preferentially to the GTP- and guanosine 5'-3-O-(thio)triphosphate-bound forms of Cdc42. Binding of p180 to GTP-bound Cdc42 maintains it in the GTP-bound state. Another cytosolic protein, with an apparent molecular mass of 175 kDa (p175), was also found to interact with Cdc42, but this association showed less dependence on guanine nucleotides. Both p180 and p175 were capable of binding to Rad but not to RhoA or Ha-Ras. The limit functional domain of the Cdc42 GAP protein did not compete with p180 or p175 for binding 60 Cdc42. However, the Cdc42-binding domain from mPAK-3, a member of the PAK (p21 activated kinase) family of serine/threonine kinases, competed with both proteins. The binding of p180 or p175 was inhibited by mutations of the putative effector loop of Cdc42. p180 and p175 also bound less effectively to a Cdc42/Ras chimera in which loop 8 from Ras was substituted for the predicted loop 8 in Cdc42 that includes a 13-amino acid insert present in all Rho family members but absent in Ras. Microsequencing of a p180 peptide revealed 92% identity with the human IQGAP1 protein, while two peptides derived from p175 were 89 and 100% identical to human IQGAP2, These findings identify IQGAP1 and IQGAP2 as a new class of target/effectors that utilize both regions of the switch I domain and an insert region distinct to Rho proteins for binding to Cdc42.