characterization and functional analysis of the calmodulin-binding domain of rac1 gtpase特性和功能的分析calmodulin-binding rac1 gtpase的领域.pdfVIP

Characterization and Functional Analysis of the Calmodulin-Binding Domain of Rac1 GTPase 1 1 1,2 Bing Xu , Prashen Chelikani , Rajinder P. Bhullar * 1 Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba, Canada, 2 Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada Abstract Rac1, a member of the Rho family of small GTPases, has been shown to promote formation of lamellipodia at the leading edge of motile cells and affect cell migration. We previously demonstrated that calmodulin can bind to a region in the C- terminal of Rac1 and that this interaction is important in the activation of platelet Rac1. Now, we have analyzed amino acid residue(s) in the Rac1-calmodulin binding domain that are essential for the interaction and assessed their functional contribution in Rac1 activation. The results demonstrated that region 151–164 in Rac1 is essential for calmodulin binding. Within the 151–164 region, positively-charged amino acids K153 and R163 were mutated to alanine to study impact on calmodulin binding. Mutant form of Rac1 (K153A) demonstrated significantly reduced binding to calmodulin while the double mutant K153A/R163A demonstrated complete lack of binding to calmodulin. Thrombin or EGF resulted in activation of Rac1 in CHRF-288-11 or HeLa cells respectively and W7 inhibited this activation. Immunoprecipitation studies demonstrated that higher amount of CaM was associated with Rac1 during EGF dependent activation. In cells expressing mutant forms of Rac1 (K153A or K153A/R163A), activation induced by EGF was significantly decreased in comparison to wild type or the R163A forms of Rac1. The lack of Rac1 activation in mut