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J. Biol. Chem. 280 (38): 32866-32876

© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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) 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-{kappa}B 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 I{kappa}B 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 I{kappa}B, release of PKA catalytic subunit from the complex with I{kappa}B and NF-{kappa}B, and subsequent phosphorylation of VASP.


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

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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