PROTEIN KINASE A A New Route to PKA Activation

Profirovic et al. uncovered a pathway from -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). -Thrombin stimulation of cultured human vascular endothelial cells (HUVECs) elicited phosphorylation-dependent translocation of VASP to the cell periphery. -Thrombin-dependent VASP phosphorylation was inhibited by expression of the regulators of G protein signaling (RGS) domain of p115RhoGEF, implicating G₁₂ or G₁₃ in the response. Moreover, constitutively active G₁₃ 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 -thrombin, and expression of the botulinum C3 exotoxin (which inactivates RhoA) inhibited G₁₃-mediated VASP phosphorylation, whereas expression of constitutively active RhoA promoted VASP phosphorylation. -Thrombin-, G₁₃-_, and RhoA-mediated VASP phosphorylation were all sensitive to inhibition of PKA, and constitutively active G₁₃ or RhoA stimulated PKA-dependent transcriptional activation of a reporter. However, neither -thrombin nor constitutively active G₁₃ 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 B (IB) and nuclear factor B (NF-B). Constitutively active G₁₃ or RhoA promoted NF-B activation, whereas a dominant-negative IB mutant (IBm, which inhibits IB phosphorylation and degradation and the release of PKAc) abolished VASP phosphorylation in response to G₁₃ or RhoA. G₁₃-dependent VASP phosphorylation was blocked by a dominant-negative mutant of the mitogen-activated protein kinase kinase MEKK1, whereas activated MEKK1 stimulated IBm-sensitive VASP phosphorylation. Thus the authors conclude that -thrombin triggers a novel pathway to PKA-mediated VASP phosphorylation that involves RhoA and MEKK1 activation and degradation of IB.

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]

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