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 rho A p21, and the C3 action was prevented by comicroinjection with a rho A 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 p21) 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 12-0-tetradecanoyl-phorbol-13-acetate, a protein kinase C-activating phorbol ester, but not by Ca²⁺ ionophore. The phorbol ester-induced 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 ester-induced cell motility.