PROTEIN-KINASE-A PHOSPHORYLATION OF RHOA MEDIATES THE MORPHOLOGICAL AND FUNCTIONAL-EFFECTS OF CYCLIC-AMP IN CYTOTOXIC LYMPHOCYTES

We have observed that stimulation of human natural killer cells with d ibutyryl cAMP (Bt(2)cAMP) reproduced the effects of ADP ribosylation o f the GTP binding protein RhoA by Clostridium botulinum C3 transferase : both agents induced similar morphological changes, inhibited cell mo tility and blocked the cytolytic function, We demonstrate here that cA MP-dependent protein kinase A (PKA) phosphorylates RhoA in its C-termi nal region, on serine residue 188, This phosphorylation does not affec t the ability of recombinant RhoA to bind guanine nucleotides, nor doe s it modify its intrinsic GTPase activity, However, treatment of cells with Bt(2)cAMP results in the translocation of membrane-associated Rh oA towards the cytosol, Experiments using purified membrane preparatio ns indicated that Rho-GDP dissociation inhibitor, which can complex ph osphorylated RhoA in its GTP-bound state, was the effector of this tra nslocation, Taken together, these data suggest that PKA phosphorylatio n of RhoA is a central event in mediating the cellular effects of cAMP , and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP-bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP c ycling.