A Novel Mechanism of G Protein-dependent Phosphorylation of Vasodilator-stimulated Phosphoprotein

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) α-thrombin induced phosphorylation of VASP. Specific inhibition of Gα13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Gα13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-κB signaling pathway. α-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Gα13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Gα13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IκB and pretreatment with the proteasome inhibitor MG-132 abolished Gα13- and α-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Gα13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IκB, release of PKA catalytic subunit from the complex with IκB and NF-κB, and subsequent phosphorylation of VASP.