Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility

Hepatocyte growth factor (HGF) induced motility of cultured mouse keratinocytes (308R cells). This HGF-induced cell motility was inhibited by microinjection of either rho GDI, an inhibitory GDP/GTP exchange protein for rho p21 small GTP-binding protein, or a botulinum exoenzyme C3 which is known to selectively impair the function of rho p21 by ADP-ribosylating its effector domain. The rho GDI action was prevented by comicroinjection with the guanosine 5'-(3-0-thio)triphosphate (GTPγS)-bound active form of rhoA p21, and the C3 action was prevented by comicroinjection with a rhoA p21 mutant (rhoA(Ile41) p21) which is resistant to the C3 action. The HGF-induced cell motility was not inhibited by microinjection of a dominant negative rac1 p21 mutant (rac1(Asn17) p 21) or a dominant negative Ki-ras p21 mutant (Ki-ras(Asn17) p21). Microinjection of the GTPγS-bound form of rac1 p21 or a dominant active Ki-ras p21 mutant (Ki-ras(Val12) p21) did not induce cell motility. These results indicate that both rho p21 and rho GDI, but neither rac p21 nor ras p21, are involved in the HGF-induced cell motility. However, microinjection of the GTPγS-bound form of rhoA p21 alone did not induce cell motility in the absence of HGF, suggesting that activation of rho p21 is necessary but not sufficient for the HGF-induced cell motility. The HGF-induced cell motility was mimicked by 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C-activating phorbol ester, but not by Ca²⁺ ionophore. The phorbol esterinduced cell motility was also inhibited by microinjection of rho GDI or C3. These results indicate that both rho p21 and rho GDI are also involved in the phorbol esterinduced cell motility.