A New Route to PKA Activation

Abstract

Profirovic et al. uncovered a pathway from α-thrombin to cAMP-dependent protein kinase (PKA)-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a membrane-associated protein implicated in regulation of actin structure and cell motility, that appears to be independent of adenosine 3′,5′-monophosphate (cAMP). α-Thrombin stimulation of cultured human vascular endothelial cells (HUVECs) elicited phosphorylation-dependent translocation of VASP to the cell periphery. α-Thrombin-dependent VASP phosphorylation was inhibited by expression of the regulators of G protein signaling (RGS) domain of p115RhoGEF, implicating Gα 12 or Gα 13 in the response. Moreover, constitutively active Gα 13 stimulated VASP phosphorylation in human embryonic kidney (HEK) 293 cells and phosphorylation-dependent translocation in HUVECs. Depleting RhoA with small interfering RNA (siRNA) abolished VASP phosphorylation in response to α-thrombin, and expression of the botulinum C3 exotoxin (which inactivates RhoA) inhibited Gα 13 -mediated VASP phosphorylation, whereas expression of constitutively active RhoA promoted VASP phosphorylation. α-Thrombin-, Gα 13 - , and RhoA-mediated VASP phosphorylation were all sensitive to inhibition of PKA, and constitutively active Gα 13 or RhoA stimulated PKA-dependent transcriptional activation of a reporter. However, neither α-thrombin nor constitutively active Gα 13 or RhoA produced a detectable increase in intracellular cAMP. Previous research indicates that some PKA catalytic subunits exist in a complex with inhibitor of nuclear factor κB (IκB) and nuclear factor κB (NF-κB). Constitutively active Gα 13 or RhoA promoted NF-κB activation, whereas a dominant-negative IκBα mutant (IκBαm, which inhibits IκB phosphorylation and degradation and the release of PKAc) abolished VASP phosphorylation in response to Gα 13 or RhoA. Gα 13 -dependent VASP phosphorylation was blocked by a dominant-negative mutant of the mitogen-activated protein kinase kinase MEKK1, whereas activated MEKK1 stimulated IκBαm-sensitive VASP phosphorylation. Thus the authors conclude that α-thrombin triggers a novel pathway to PKA-mediated VASP phosphorylation that involves RhoA and MEKK1 activation and degradation of IκB. J. Profirovic, M. Gorovoy, J. Niu, S. Pavlovic, T. Voyno-Yasenetskaya, A novel mechanism of G protein-dependent phosphorylation of vasodilator-stimulated phosphoprotein. J. Biol. Chem . 280 , 32866-32876 (2005). [Abstract] [Full Text]