Pak6 PROTEIN KINASE IS A NOVEL EFFECTOR OF AN ATYPICAL RHO FAMILY GTPase Chp/RhoV
Chp/RhoV is an atypical Rho GTPase whose functions are far from being fully understood. To date several effector proteins of Chp have been identified, including p21 activated kinases Pak1, Pak2, and Pak4. By using a yeast two hybrid system and co immunoprecipitation, here we show that another p21 activated kinase, Pak6, is a novel Chpbinding protein. The interaction between Chp and Pak6 depends on the activation state of the GTPase, suggesting that Pak6 is an effector protein for Chp. Point mutations in the effector domain of Chp or in the CRIB motif of Pak6 significantly impair the interaction between Chp and Pak6 upon co immunoprecipitation, suggesting that the binding interface involves the effector domain of Chp and the CRIB motif in Pak6. We found that Chp does not affect the phosphorylation status of residue S560 in the catalytic domain of Pak6 when Chp and Pak6 are coexpressed in HEK293 cells. Therefore, similarly to Cdc42, Chp is not likely to activate Pak6. In NCI H1299 cells, Chp colocalized with Pak6 on vesicular structures in activation state dependent manner. From these data taken together, we report here the identification of p21 activated kinase Pak6 as a novel effector of an atypical Rho GTPase Chp. Our data suggest further directions in elucidating biological functions of these proteins.