cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKα

Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells fi om responding to increased cAMP levels. A constitutively activated RhoA(V14) mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). We demonstrate that RhoA(V14A188), which cannot be phosphorylated by PKA in vitro, is more effective than RhoA(V14) in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PEA phosphorylation of RhoA impairs its biological activity in vivo. ROK alpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PI(A in vitro decreases the binding of RhoA to ROK alpha. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKa! may be involved in this antagonism.