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.

摘要： Cdc42Hs is a Ras-related GTP binding protein which has a tightly regulated GTP binding, GTP hydrolysis cycle. Using Cdc42Hs as a fusion protein with glutathione-s-transferase(GST) attached to glutathione agarose beads as an affinity reagent, we identified a 180 kd protein from rabbit liver cytosol which interacts specifically with Cdc42Hs in the GTPγS bound form. In addition, we have identified a 175 kd protein which interacts with Cdc42Hs in the GTP-j-S. GDP and nucleotide depleted states. Both of these proteins also interact with the closely related GTP binding protein, Had, but not with the RhoA or HaRas proteins. Analysis of the binding of p180 and p175 to Cdc42Hs "effector" mutants showed that these proteins probably interact with some residues in the putative effector loop of Cdc42Hs. In addition, competition experiments indicate that plSO competes with p65 PAK but not with Cdc42Hs-GAP for binding to Cdc42Hs. Furthermore, the 180 kd protein appears to maintain Cdc42 in its GTP bound state. Microsequencing of these proteins identified the 180 kd protein as greater than 90% identical to human IQGAPl in a 24 amino acid peptide and the 175 kd protein as 55% identical to human IQGAPl in a 19 amino acid peptide and 77% identical in a 13 amino acid peptide. This data identifies IQGAPl or an IQGAP related protein as a Cdc42Hs binding partner and potential effector for this GTP binding protein.

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.