关键词： Rasgap-related protein   Rho-family gtpases   Binding protein   Putative target   Alpha-actinin   F-actin   Kinase   Identification   Calmodulin   Effector  

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.

关键词： Actin stress fibers   Cdc42 gtpases   Calmodulin   Rho   Identification   Purification   Activation   Filopodia   Contain   Kinase  

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.

关键词： Binding   Calcium   Protein   Identification   Antibodies   Domain   Myosin   Ras  

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.

关键词： ras GTPase-Activating Proteins   Amino Acid Sequence   Animals   Base Sequence   Binding Sites   Binding   Competitive   Carrier Proteins   Cell Cycle Proteins   Comparative Study   Cytosol   Electrophoresis   Agar Gel   GTP-Binding Proteins   Genes   ras   Guanosine 5'-O-(3-Thiotriphosphate)   Guanosine Triphosphate   Humans   Liver   Molecular Sequence Data   Molecular Weight   Rabbits   Research Support   U.S. Gov't   P.H.S.   Sequence Homology   Amino Acid   Sequence Homology   Nucleic Acid   cdc42 GTP-Binding Protein   rac GTP-Binding Proteins   rhoA GTP-Binding Protein  

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.

关键词： calmodulin   cdc42Hs   cytoskeleton   IQGAP1   rac  

摘要： Proteins that associate with the GTP-bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches, A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs-GTP and a corresponding cDNA was isolated, Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins acid to the rasGAP-related domain (GRD) contained in IRA, NF-1, SAR1 and rasGAP, p195 was found to be identical to IQGAP1, a protein previously reported to bind ras. Purified recombinant p195/QGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected, The C-terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD-dependent fashion, but a smaller fragment containing only the GRD did not, Cdc42 was also co-immunoprecipitated from cell lysates with antibody specific to pl95/IQGAP1, Calmodulin also co-immunoprecipitated with pl95/IQGAP1 and was found to associate with fragments containing the IQ domain, Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed, In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where colocalization with actin was apparent, These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.

关键词： CARRIER proteins  

摘要： Reports on the purification and characterization of a protein, IQGAP1. Possibility that the protein mediate effects of these GTPases on microfilaments; Information on native IQGAP1 purified from bovine adrenal; Reaction of purified IQGAP1 bound directly to F-actin.