Characterization and functional analysis of the calmodulin-binding domain of Rac1 GTPase.
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 mutant forms was not due to an inability of GDP-GTP exchange or a change in subcelllular distribution. Moreover, Rac1 activation was decreased in cells where endogenous level of calmodulin was reduced using shRNA knockdown and increased in cells where calmodulin was overexpressed. Docking analysis and modeling demonstrated that K153 in Rac1 interacts with Q41 in calmodulin. These results suggest an important role for calmodulin in the activation of Rac1 and thus, in cytoskeleton reorganization and cell migration.
Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba, Canada.
Bing
B
Xu
Prashen
P
Chelikani
Rajinder P
RP
Bhullar
D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002465:Cell Movement; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013917:Thrombin / Q000737:chemistry; D020830:rac1 GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism
D002147:Calmodulin; D004815:Epidermal Growth Factor; D005982:Glutathione Transferase; D013917:Thrombin; D020830:rac1 GTP-Binding Protein