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) 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.
Profirovic
Jasmina
J
Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
Gorovoy
Matvey
M
Niu
Jiaxin
J
Pavlovic
Sasa
S
Voyno-Yasenetskaya
Tatyana
T
D000199:Actins / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002460:Cell Line; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005799:Genes, Dominant; D017930:Genes, Reporter; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D007546:Isoquinolines / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D048728:MAP Kinase Kinase Kinase 1 / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal; D008954:Models, Biological; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology; D054460:rho-Associated Kinases; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D020672:I-kappa B Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007546:Isoquinolines; D008840:Microfilament Proteins; D016328:NF-kappa B; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D064067:Rho Guanine Nucleotide Exchange Factors; D013449:Sulfonamides; D000098662:Vasodilator-Stimulated Phosphoprotein; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D013917:Thrombin; D019204:GTP-Binding Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide