PKN and PKN-related Protein Rhophilin as Targets of Small GTPase Rho

• Published in: Japanese Journal of Pharmacology 1996, Vol.71 (suppl.1), p.165-165
• Main Authors: Watanabe, Go, Saito, Yuji, Madaule, Pascal, Ishizaki, Toshimasa, Morii, Narito, Mukaj, Hideyuki, Ono, Yoshitaka, Imamura, Masayuki, Kakizuka, Akira, Narumiya, Shuh
• Format: Article
• Language: eng ; jpn
• ISSN: 0021-5198
• DOI: 10.1016/S0021-5198(19)36900-8

To identify a target molecule of rho, we used a yeast two hybrid system. A clone obtained, "clone 79", interacted strongly with rho A, little with rho C and less with rho B. Among the rho A mutants, the strongest interaction was obtained with Val^14 -rho A, and a weak signal with Asn^19 -rho A, suggesting that clone 79 interacted with rho A in a GTP-dependent manner. Furthermore, a ligand overlay assay using a recombinant clone 79 protein revealed that it bound rho A loaded with GTPγS but not that with GDPβS. CDC42 or rac I with GTPγS. A full length cDNA of this clone encoded about 71Kda protein, termed rhophilin. The rho p21 binding domain localized in N-terminal fragment of rhophilin showed the striking similarity to a regulatory domain of PKN , a 120 KDa Ser/Tbr kinase. The expressed N-terminal region of PKN homologous to the rho binding domain of rhophilin was bound to rho by an overlay assay. Consistently, PKN was precipitated by incubation with GST-rho coupled to GSH-agarose. This precipitation was dependent on prior incubation of GTPγS. Furthermore, PKN was activated by this binding in vitro and in vivo. Finally, LPA stimulated phosphorylation of endogenous PKN and this phosphorylation was inhibited by C3treatment. These results indicate that PKN and rhophilin can serve as target molecules for rho p21 through binding of their PKN-motif to its GTP-bound form.