G(i)-dependent activation of c-Jun N-terminal kinase in human embryonal kidney 293 cells

Abstract

Heterotrimeric G proteins stimulate the activities of two stress- activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen- activated protein kinase in mammalian cells. In this study, we examined whether α subunits of G(i) family activate JNK using transient expression system in human embryonal kidney 293 cells. Constitutively activated mutants of Gα(i2), Gα(i2), and Gα(i3) increased JNK activity. In contrast, constitutively activated Gα(o) and Gα(z) mutants did not stimulate JNK activity. To examine the mechanism of JNK activation by Gα(i), kinase- deficient mutants of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), which are known to be JNK activators, were transfected into the cells. However, Gα(i)-induced JNK activation was not blocked effectively by kinase-deficient MKK4 and MKK7. In addition, activated Gα(i) mutant failed to stimulate MKK4 and MKK7 activities. Furthermore, JNK activation by Gα(i) was inhibited by dominant-negative Rho and Cdc42 and tyrosine kinase inhibitors, but not dominant-negative Rac and phosphatidylinositol 3-kinase inhibitors. These results indicate that Gα(i) regulates JNK activity dependent on small GTPases Rho and Cdc42 and on tyrosine kinase but not on MKK4 and MKK7.