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

Sasa Pavlovic,Jiaxin Niu,Matvey Gorovoy,Tatyana Voyno-Yasenetskaya,Jasmina Profirovic

doi:10.1074/jbc.m501361200

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) alpha-thrombin induced phosphorylation of VASP. Specific inhibition of Galpha13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Galpha13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-kappaB signaling pathway. alpha-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Galpha13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Galpha13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IkappaB and pretreatment with the proteasome inhibitor MG-132 abolished Galpha13- and alpha-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Galpha13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IkappaB, release of PKA catalytic subunit from the complex with IkappaB and NF-kappaB, and subsequent phosphorylation of VASP.

Highlights

␣-Thrombin- and G␣13-induced Vasodilator-stimulated phosphoprotein (VASP) Phosphorylation Is Mediated by protein kinase A (PKA) Activation—As VASP is a major substrate of PKA [45], we examined if VASP phosphorylation induced by ␣-thrombin, G␣13, and RhoA is mediated by PKA
In this study we present evidence that thrombin and the G␣ subunit of the heterotrimeric G13 protein induce activation of PKA and phosphorylation of VASP, suggesting a novel pathway of cAMP-independent VASP phosphorylation
As VASP localization to the membrane and focal adhesions is required for cell adhesion, motility, and formation of filopodia [35], it is likely that thrombin stimulation and G␣13 activation may modify VASP function by its phosphorylation and translocation

Summary

MATERIALS AND METHODS

Reagents—Murine FLAG-tagged VASP cDNA was a gift from D. Cells were starved for 16 h, washed twice with cold phosphate-buffered saline, and lysed with protein extraction reagent, and the cleared lysates were assayed for SRE-, ␬B-, and CREB-dependent expression of firefly luciferase and ␤-galactosidase activity using corresponding assay kits (Promega). Cells were harvested 48 h after transfection and lysed in the lysis buffer containing 50 mM Tris1⁄7HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 40 mM Na4P2O7, 1% Triton X-100, 1 mM dithiothreitol, 200 ␮M Na3VO4, and 5 ␮l/ml mammalian protease inhibitor mixture (Sigma). The lysates were normalized for protein concentration, and Myc-tagged LIMK1 was immunoprecipitated from 100 ␮l of cell lysates by incubation with anti-Myc antibody at 4 °C for 2 h and protein A/G-agarose for 30 min. For quantitative analysis of the immunofluorescence data, HUVECs expressing FLAG-tagged VASP wild type and mutants were scored based on the localization of VASP in the cell. The percentage of the cells with VASP accumulated at the periphery was calculated from at least 200 FLAG-positive cells counted for each transfection

RESULTS

DISCUSSION