Dependence of activated G alpha 12-induced G(1) to S phase cell cycle progression on both Ras/mitogen-activated protein kinase and Ras/Ras1/Jun N-terminal kinase cascades in NIH3T3 fibroblasts

We evaluated the roles of mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK) signaling cascades in G alpha 12-induced G(1) to S phase cell cycle progression in NIH3T3(M17) fibroblasts. Transient expression of a constitutively active mutant of G alpha 12, G alpha 12(R203C), resulted in a a-fold increase in the number of bromodeoxyuridine-positive S phase cells over vector control level under serum-deprived conditions. Consistent with the ability of G alpha 12(R203C) to induce G(1)/S transition, its expression led to a a-fold increase in cyclin A promoter activity, which showed a marked synergism with a low concentration of serum, resulting in up to a 15-fold elevation over the basal level. In addition, G alpha 12(R203C) caused a a-fold stimulation in E2F-mediated transactivation. Wild type G alpha 12 showed similar stimulatory effects on cyclin A promoter activity and E2F-mediated transactivation, although of lesser magnitude. We observed a modest but constitutive activation of MAPK in cells transfected with G alpha 12(R203C), which was abolished by a dominant negative form of Ras, G alpha 12(R203C) also induced a 3-fold increase in JNK activity, which was abolished by dominant negative forms of either Rad or Ras. The expression of dominant negative forms of Ras, MAPK, Rac1, or JNK inhibited G alpha 12(R203C)-induced increases in bromodeoxyuridine-positive cells. Also, the dominant negative forms of Ras, MAPK, and JNK strongly inhibited G alpha 12(R203C)-induced stimulation of cyclin A promoter activity. These results demonstrate that both the Ras/MAPK and Ras/Rac1/JNK pathways convey necessary, if not sufficient, mitogenic signals induced by G alpha 12 activation.