PKN beta interacts with the SH3 domains of Graf and a novel Graf related protein, Graf2, which are GTPase activating proteins for Rho family

PKN beta is a novel isoform of PKN alpha, which is one of the target protei n kinases for the small GTPase Rho, By yeast two-hybrid screening of a huma n embryonic kidney 293 cell cDNA library with the PKN beta linker region co ntaining proline-rich motifs as a bait, clones encoding Graf (GAP for Rho a ssociated with focal adhesion kinase) and a novel Graf-related protein, ter med Graf2, were isolated. The full length of Graf2 contains a putative PH d omain, a RhoGAP domain, and an SH3 domain as well as Graf, Northern and Wes tern blot analyses demonstrated that Graf2 is expressed in several tissues, with the highest expression in skeletal muscle. Recombinant Graf2 exhibite d GTPase-activating activity toward the small GTPase RhoA and Cdc42Hs, but not toward Rad, in vitro, The SH3 domains of Graf and Graf2 purified from E scherichia coil bound directly to PKN beta, Graf or Graf2 was co-immunoprec ipitated with PKN beta in COS-7 cells transiently transfected with Graf or Graf2 and PKN beta expression constructs. The catalytically active form of PKN beta phosphorylated Graf and Graf2 in vitro, The interplay of PKN beta and the GTPase-activating proteins, Graf and Graf2, may offer a novel mecha nism regulating the Rho-mediated signaling.