CHARACTERIZATION OF THE INTERACTION BETWEEN RHOA AND THE AMINO-TERMINAL REGION OF PKN

The yeast two-hybrid system and in vitro binding assay were carried ou t to characterize the interaction between PKN and a small GTP-binding protein, RhoA. It was revealed that the region corresponding to the am ino acid residues 33-111 in the amino-terminal region of PKN was suffi cient to confer the ability to associate with RhoA, Each synthetic pep tide fragment corresponding to the amino acid residues 74-93 and 94-11 3 of PKN inhibited the interaction between PKN and RhoA in the in vitr o binding assay, suggesting that this region is important in the assoc iation with RhoA. The endogenous and the GAP-stimulated GTPase activit y of RhoA was inhibited by the interaction with PKN, suggesting the pr esence of a regulatory mechanism that sustains the GTP-bound active fo rm of RhoA.