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

Jasmina Profirovic1, Matvey Gorovoy2, Jiaxin Niu, Sasa Pavlovic, , and Tatyana Voyno-Yasenetskaya¶, Established Investigator of the American Heart Association3

Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612

Abstract: 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) -thrombin induced phosphorylation of VASP. Specific inhibition of G₁₃ protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, G₁₃-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-B signaling pathway. -Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas G₁₃-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that G₁₃-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IB and pretreatment with the proteasome inhibitor MG-132 abolished G₁₃- and -thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of G₁₃-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IB, release of PKA catalytic subunit from the complex with IB and NF-B, and subsequent phosphorylation of VASP.

Received for publication February 4, 2005. Revision received July 15, 2005.

^* This work was supported in part by National Institutes of Health Grants GM56159, GM65160, and HL06078 (to T. V.-Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by American Heart Association Predoctoral Fellowship 0310030Z.

² Supported by American Heart Association Predoctoral Fellowship 0510133Z.

³ To whom correspondence should be addressed: Dept. of Pharmacology (MC 868), University of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312-996-9823; Fax: 312-996-1225; E-mail: tvy{at}uic.edu.

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