Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

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

2005; Elsevier BV; Volume: 280; Issue: 38 Linguagem: Inglês

10.1074/jbc.m501361200

ISSN

1083-351X

Autores

Jasmina Profirovic, M. Gorovoy, Jiaxin Niu, Saša Pavlović, T. Voyno-Yasenetskaya,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

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. 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. Vasodilator-stimulated phosphoprotein (VASP) 4The abbreviations used are: VASP, vasodilator-stimulated phosphoprotein; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PKAc, catalytic PKA; PKAr, PKA regulatory; HUVECs, human umbilical vein endothelial cells; PKI, PKA inhibitor; WT, wild type; HA, hemagglutinin; FBS, fetal bovine serum; l-NAME, N-nitro-l-arginine methyl ester; siRNA, small interfering RNA; CREB, cAMP-response element-binding protein; HBSS, Hanks' balanced salt solution; GAP, GTPase-activating protein; eNOS, endothelial nitric-oxide synthase; IκB, inhibitor of κB; SRE, serum-response element; DN, dominant negative.4The abbreviations used are: VASP, vasodilator-stimulated phosphoprotein; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PKAc, catalytic PKA; PKAr, PKA regulatory; HUVECs, human umbilical vein endothelial cells; PKI, PKA inhibitor; WT, wild type; HA, hemagglutinin; FBS, fetal bovine serum; l-NAME, N-nitro-l-arginine methyl ester; siRNA, small interfering RNA; CREB, cAMP-response element-binding protein; HBSS, Hanks' balanced salt solution; GAP, GTPase-activating protein; eNOS, endothelial nitric-oxide synthase; IκB, inhibitor of κB; SRE, serum-response element; DN, dominant negative. is purified and characterized as a 46-kDa membrane-associated protein that can be phosphorylated by cAMP- and cGMP-dependent protein kinases (PKA and PKG, respectively) (1Halbrugge M. Walter U. J. Chromatogr. 1990; 521: 335-343Crossref PubMed Scopus (19) Google Scholar). VASP is the founding member of the Ena/VASP family of proteins, which consists of VASP, Drosophila Enabled (Ena), a mammalian Ena homolog Mena, and Ena-VASP-like protein EVL (2Haffner C. Jarchau T. Reinhard M. Hoppe J. Lohmann S.M. Walter U. EMBO J. 1995; 14: 19-27Crossref PubMed Scopus (173) Google Scholar, 3Gertler F.B. Comer A.R. Juang J.L. Ahern S.M. Clark M.J. Liebl E.C. Hoffmann F.M. Genes Dev. 1995; 9: 521-533Crossref PubMed Scopus (222) Google Scholar, 4Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). Similarly to other members of the Ena/VASP family of proteins, VASP contains the central proline-rich region flanked with Ena/VASP homology domains 1 and 2 (2Haffner C. Jarchau T. Reinhard M. Hoppe J. Lohmann S.M. Walter U. EMBO J. 1995; 14: 19-27Crossref PubMed Scopus (173) Google Scholar). VASP is highly expressed in vascular endothelial cells, platelets, smooth muscle cells, and fibroblasts where it can be found in focal adhesions, along stress fibers, and in the areas of highly dynamic membrane activity, such as extending lamellipodia and filopodia (2Haffner C. Jarchau T. Reinhard M. Hoppe J. Lohmann S.M. Walter U. EMBO J. 1995; 14: 19-27Crossref PubMed Scopus (173) Google Scholar, 5Reinhard M. Halbrugge M. Scheer U. Wiegand C. Jockusch B.M. Walter U. EMBO J. 1992; 11: 2063-2070Crossref PubMed Scopus (286) Google Scholar, 6Reinhard M. Jarchau T. Walter U. Trends Biochem. Sci. 2001; 26: 243-249Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). VASP plays an important role in physiological and pathophysiological responses of platelets and endothelial cells. Recent studies (7Massberg S. Gruener S. Konrad I. Garcia Arguinzonis M.I. Eigenthaler M. Hemler K. Kersting J. Schulz C. Mueller I. Besta F. Nieswandt B. Heinzmann U. Walter U. Gawaz M. Blood. 2004; 103: 136-142Crossref PubMed Scopus (117) Google Scholar) showed that VASP is an essential factor that negatively regulates platelet adhesion; in VASP knockout mice, platelet adhesion to the vessel wall was enhanced. Moreover, the loss of VASP increases platelet adhesion to the atherosclerotic endothelium and subendothelial matrix (7Massberg S. Gruener S. Konrad I. Garcia Arguinzonis M.I. Eigenthaler M. Hemler K. Kersting J. Schulz C. Mueller I. Besta F. Nieswandt B. Heinzmann U. Walter U. Gawaz M. Blood. 2004; 103: 136-142Crossref PubMed Scopus (117) Google Scholar). More importantly, phosphorylated VASP may participate in the endothelial barrier function and tight junction regulation. Phosphorylated VASP localizes to endothelial cell-cell junctions and may be co-immunoprecipitated with zonula occludens 1 after PKA activation (8Comerford K.M. Lawrence D.W. Synnestvedt K. Levi B.P. Colgan S.P. FASEB J. 2002; 16: 583-585Crossref PubMed Scopus (152) Google Scholar). There is a growing body of evidence that VASP has complex effects on cytoskeletal organization and cell motility by regulating actin network geometry and thereby controlling the protrusive behavior of the cell (9Bear J.E. Loureiro J.J. Libova I. Fässler R. Wehland J. Gertler F.B. Cell. 2000; 101: 717-728Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). It was shown that VASP could induce polymerization of G-actin into F-actin bundles in in vitro assays. VASP was also shown to stabilize F-actin (10Laurent V. Loisel T.P. Harbeck B. Wehman A. Grobe L. Jockusch B.M. Wehland J. Gertler F.B. Carlier M.F. J. Cell Biol. 1999; 144: 1245-1258Crossref PubMed Scopus (291) Google Scholar), to nucleate F-actin assembly (11Lambrechts A. Kwiatkowski A.V. Lanier L.M. Bear J.E. Vandekerckhove J. Ampe C. Gertler F.B. J. Biol. Chem. 2000; 275: 36143-36151Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 12Fradelizi J. Noireaux V. Plastino J. Menichi B. Louvard D. Sykes C. Golsteyn R.M. Friederich E. Nat. Cell Biol. 2001; 3: 699-707Crossref PubMed Scopus (99) Google Scholar), to antagonize capping protein activity and promote actin filament elongation (13Bear J.E. Krause M. Gertler F.B. Curr. Opin. Cell Biol. 2001; 13: 158-166Crossref PubMed Scopus (91) Google Scholar), and to facilitate Arp2/3-dependent actin polymerization (14Loisel T.P. Boujemaa R. Pantaloni D. Carlier M.F. Nature. 1999; 401: 613-616Crossref PubMed Scopus (797) Google Scholar, 15Skoble J. Auerbuch V. Goley E.D. Welch M.D. Portnoy D.A. J. Cell Biol. 2001; 155: 89-100Crossref PubMed Scopus (111) Google Scholar). More importantly, the lamellipodial protrusion rate was shown to correlate positively with the intensity of green fluorescent protein-VASP at the leading edge (16Rottner K. Behrendt B. Small J.V. Wehland J. Nat. Cell Biol. 1999; 1: 321-322Crossref PubMed Scopus (265) Google Scholar). All these molecular events are involved in the regulation of cell motility. VASP phosphorylation is a modulator for VASP-dependent regulation of the actin cytoskeleton. VASP can be phosphorylated at three sites (human VASP), serine 157, serine 239, and threonine 278 (corresponding to serine 153, serine 235, and threonine 274 in murine protein). Phosphorylation of serine 157 leads to a marked retardation in electrophoretic mobility and shift in apparent molecular mass from 46 to 50 kDa in SDS-PAGE (17Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar). PKA is an important effector enzyme that is commonly activated by cAMP in response to cAMP-elevating agents. The established mechanism of PKA activation in response to various agonists involves the stimulatory G protein, Gs, which activates adenylyl cyclase resulting in a production of cAMP. Binding of cAMP to PKA regulatory subunits (PKAr) leads to the release and activation of catalytic PKA subunits (PKAc) (18Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (955) Google Scholar). More importantly, our laboratory reported two novel cAMP-independent mechanisms of PKA activation. One study showed that vasoactive peptides endothelin-1 and angiotensin II activate PKA by inducing phosphorylation and degradation of the inhibitor of κB (IκB), subsequently releasing PKAc from inhibition by IκB (19Dulin N.O. Niu J. Browning D.D. Ye R.D. Voyno-Yasenetskaya T. J. Biol. Chem. 2001; 276: 20827-20830Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). A similar mechanism was reported for PKA activation upon lipopolysaccharide (20Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (726) Google Scholar) and thrombin stimulation (21Zieger M. Tausch S. Henklein P. Nowak G. Kaufmann R. Biochem. Biophys. Res. Commun. 2001; 282: 952-957Crossref PubMed Scopus (20) Google Scholar). In addition, we reported that Gα13, upon interaction with protein kinase A-anchoring protein 110 (AKAP110), induces release of the PKAc from the AKAP110-PKAr complex, resulting in the PKA activation (22Niu J. Vaiskunaite R. Suzuki N. Kozasa T. Carr D.W. Dulin N. Voyno-Yasenetskaya T.A. Curr. Biol. 2001; 11: 1686-1690Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Gα13 protein regulates multiple signaling pathways by transducing signals from the cell surface to activate diverse cellular responses. It regulates the activity of the Na+/H+ exchanger NHE1 (23Voyno-Yasenetskaya T.A. Conklin B.R. Gilbert R.L. Hooley R. Bourne H.R. Barber D.L. J. Biol. Chem. 1994; 269: 4721-4724Abstract Full Text PDF PubMed Google Scholar), apoptosis (24Berestetskaya Y.V. Faure M.P. Ichijo H. Voyno-Yasenetskaya T.A. J. Biol. Chem. 1998; 273: 27816-27823Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), and the extracellular signal-regulated kinase and c-Jun NH2-terminal kinase pathways (25Voyno-Yasenetskaya T.A. Faure M.P. Ahn N.G. Bourne H.R. J. Biol. Chem. 1996; 271: 21081-21087Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and participates in embryonic development (26Offermanns S. Mancino V. Revel J.P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (287) Google Scholar). It also induces mitogenesis and neoplastic transformation (27Voyno-Yasenetskaya T.A. Pace A.M. Bourne H.R. Oncogene. 1994; 9: 2559-2565PubMed Google Scholar, 28Xu N. Voyno-Yasenetskaya T. Gutkind J.S. Biochem. Biophys. Res. Commun. 1994; 201: 603-609Crossref PubMed Scopus (86) Google Scholar). It was shown that important effectors in Gα13-mediated downstream signaling are RhoA, Rac, and Cdc42, members of a Rho family of small GTPases (25Voyno-Yasenetskaya T.A. Faure M.P. Ahn N.G. Bourne H.R. J. Biol. Chem. 1996; 271: 21081-21087Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 29Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Activation of RhoA by Gα13 results in actin stress fiber formation and cell retraction (29Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Rho is activated by Gα13 upon direct interaction of Gα13 with the Rho-specific guanine nucleotide exchange factor, p115 RhoGEF (30Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar, 31Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (672) Google Scholar). Recent studies from our laboratory showed two additional proteins directly interacting with Gα13. One is radixin, the member of ERM (ezrin/radixin/moesin) family of proteins, whose function is regulated by direct interaction with Gα13 (32Vaiskunaite R. Adarichev V. Furthmayr H. Kozasa T. Gudkov A. Voyno-Yasenetskaya T.A. J. Biol. Chem. 2000; 275: 26206-26212Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The other is AKAP110, as described above (22Niu J. Vaiskunaite R. Suzuki N. Kozasa T. Carr D.W. Dulin N. Voyno-Yasenetskaya T.A. Curr. Biol. 2001; 11: 1686-1690Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Multiple receptors are reported to couple to Gα13, including the thrombin PAR-1 receptor (33Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (392) Google Scholar). Here we show that both thrombin and Gα13 can activate PKA and induce VASP phosphorylation in HUVECs and HEK-293 cells. Moreover, we described a novel mechanism of Gα13-induced cAMP-independent PKA activation and VASP phosphorylation that involves activation of RhoA and MEKK1. Activated RhoA and MEKK1, in turn, lead to phosphorylation and subsequent degradation of IκB, thus releasing PKAc from the complex with IκB and NF-κB. PKAc then phosphorylates VASP. As VASP proteins are multifunctional organizers of actin cytoskeleton (34Sechi A.S. Wehland J. Front. Biosci. 2004; 9: 1294-1310Crossref PubMed Scopus (73) Google Scholar), this study opens the possibility that thrombin and Gα13 are involved in subtle regulation of actin cytoskeleton. Reagents—Murine FLAG-tagged VASP cDNA was a gift from D. Browning (see Ref. 68Browning D.D. McShane M. Marty C. Ye R.D. J. Biol. Chem. 2001; 276: 13039-13048Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). SRE.L luciferase reporter plasmid (42Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1201) Google Scholar) was provided by P. Sternweis. The cDNA for the phosphorylation-deficient mutant of mouse IκBα,IκBα-S32A, S36A (IκBαm) was obtained from I. Verma (see Ref. 69Pando M.P. Verma I.M. J. Biol. Chem. 2000; 275: 21278-21286Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). cDNAs for HA-tagged dominant active MEKK1 and dominant negative MEKK1-K432A (MEKK1-DN) were obtained from G. Johnson (see Ref. 70Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar). Plasmid encoding for ROCK-2 (71Nakagawa O. Fujisawa K. Ishizaki T. Saito Y. Nakao K. Narumiya S. FEBS Lett. 1996; 392: 189-193Crossref PubMed Scopus (645) Google Scholar) was obtained from R. Ye. Myc-tagged LIMK-1 was obtained from O. Bernard (see Ref. 72Dan C. Kelly A. Bernard O. Minden A. J. Biol. Chem. 2001; 276: 32115-32121Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Myristoylated PKA inhibitor 14-22 amide (PKI), MG-132 proteasome inhibitor, and polyclonal anti-VASP antibody were purchased from Calbiochem. α-Thrombin was purchased from Enzyme Research Laboratories Inc. Anti-phospho-VASP (Ser-239) antibody was from Cell Signaling. Monoclonal anti-FLAG antibody, PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride (H-89), and eNOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) were purchased from Sigma. Anti-Gα12, -Gα13, -HA, -RhoA, -ROCK-2, and -IκBα antibodies were from Santa Cruz Biotechnology. RPMI 1640, glutamine, antibiotic-antimycotic, fetal bovine serum (FBS), Hanks' balanced salt solution (HBSS), and Lipofectamine-2000 reagent were from Invitrogen. Endothelial growth medium (EGM-2) BulletKit and EBM-2 basal medium were purchased from Cambrex. Superfect was purchased from Qiagen. Cell Culture and DNA Transfection—The human embryonic kidney 293 (HEK-293) cells (ATCC) were maintained in RPMI 1640 medium, supplemented with 2 mm glutamine, 100 units/ml penicillin G, 100 units/ml streptomycin sulfate, 25 μg/ml amphotericin B, and 10% FBS. The human umbilical vein endothelial cells (HUVECs) were cultured for up to 8 passages in EGM-2 BulletKit. Transient transfections of HEK-293 cells and HUVECs were performed using Lipofectamine-2000 and SuperFect, respectively, according to the protocols from the manufacturers. The HEK-293 cells were serum-starved in 0.2% FBS for 16 h. HUVECs were maintained in 1% serum 1–2 h before the experiment. Small Interfering RNA (siRNA)—RhoA-specific siRNA duplexes were purchased from Dharmacon. Nonsilencing control siRNA was from Qiagen. The siRNA transfection was performed using siRNA transfection reagent and siRNA transfection medium (Santa Cruz Biotechnology) according to the manufacturer's protocol. The final siRNA concentration was 60 nm. Immunoblotting—The HEK-293 cells were lysed 24–30 h after transfection. The quiescent HUVECs were lysed after stimulation with α-thrombin. Lysis buffer contained 25 mm HEPES (pH 7.5), 150 mm NaCl, 10% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 5 mm EDTA, 10 mm NaF, 100 μm Na3VO4, 1 mm dithiothreitol, and 5 μl/ml mammalian protease inhibitor mixture (Sigma). The insoluble material was removed from the lysates by centrifugation at 15,000 × g for 10 min, and cleared lysates were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane, and analyzed by immunoblotting with appropriate antibodies. Phosphorylation-dependent electrophoretic mobility shift of FLAG-tagged VASP was detected by immunoblotting with the anti-FLAG antibody, whereas the shift of endogenous VASP was detected by immunoblotting with the anti-VASP antibody. The percentage of phosphorylated VASP was calculated by using densitometry analysis of VASP bands detected with anti-VASP antibody from scanned Western blot images using the ImageJ program. Luciferase Reporter Gene Assays—SRE-, κB-, and CREB-dependent gene expression was determined by the SRE.L reporting system, κB-luciferase reporting system, and CREB "PathDetect" trans-reporting system (Stratagene), respectively. Briefly, HEK-293 cells grown on 24-well plates at 90% confluency were transiently transfected with the following plasmids (per well): 50 ng of SRE.L, luciferase cDNA under the control of the serum-response factor-responsive element from the c-fos promoter (reporter plasmid), for assessment of SRE activation, 50 ng of κB-driven luciferase reporter plasmid, for assessment of NF-κB activation, or 75 ng of pFR-Luciferase (reporter plasmid) and 4 ng of pFA2-CREB (fusion trans-activator plasmid), for assessment of CREB activation, together with 50 ng of pCMV-β-gal (transfection efficiency control plasmid) and 100 ng of empty vector or plasmid as indicated. 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-, κB-, and CREB-dependent expression of firefly luciferase and β-galactosidase activity using corresponding assay kits (Promega). Luciferase activities were measured with a Sirius luminometer (Berthold Detection Systems). Luciferase activity of each sample was normalized to β-galactosidase activity to correct for the differences in the transfection efficiency and expressed as the folds increase over the control. The data represent mean ± S.D. of triplicate determinations. The experiments were repeated two to three times with similar results. Kinase Assay—Kinase activity of Myc-tagged-LIMK1 was determined in the COS-7 cells grown on the 60-mm plates and transfected with Myc-tagged LIMK1. Cells were harvested 48 h after transfection and lysed in the lysis buffer containing 50 mm Tris·HCl (pH 7.5), 100 mm NaCl, 5 mm EDTA, 40 mm Na4P2O7, 1% Triton X-100, 1 mm dithiothreitol, 200 μm Na3VO4, and 5 μl/ml mammalian protease inhibitor mixture (Sigma). The lysates were normalized for protein concentration, and Myc-tagged LIMK1 was immunoprecipitated from 100 μl of cell lysates by incubation with anti-Myc antibody at 4 °C for 2 h and protein A/G-agarose for 30 min. Immunoprecipitates were washed three times in lysis buffer and two times in kinase buffer containing 20 mm HEPES (pH 7.5), 10 mm MgCl2, 150 mm KCl, 10 mm MnCl2, 1 mm dithiothreitol, 50 μm Na3VO4, and 5 μl/ml mammalian protease inhibitor mixture. Thereafter, the immunoprecipitate was incubated with ATP buffer containing 20 μm ATP, and 5 μCi of [γ-32P]ATP, together with 4 μg of purified recombinant glutathione S-transferase-cofilin for 20 min at 30 °C. The reaction was terminated with SDS-PAGE sample buffer, followed by SDS-PAGE, autoradiography, and PhosphorImager analysis. Cyclic AMP Assay—cAMP production was determined using the cAMP Biotrack enzyme immunoassay system according to the manufacturer's protocol (Amersham Biosciences). Briefly, cell lysates were incubated with antiserum, containing anti-cAMP antibody, followed by the incubation with cAMP-peroxidase conjugate, washing, and incubation with peroxidase substrate. The reaction was stopped by the addition of 1 m sulfuric acid, and the absorbance was determined at 450 nm. The results were determined from the standard curve and represent the mean ± S.D. from triplicate determinations. Immunostaining and Confocal Microscopy—HUVECs were transfected for 24 h where indicated and maintained in the EGM-2 medium supplemented with 10% FBS. Two hours before the stimulation with α-thrombin, the cells were maintained in EBM-2 basal medium supplemented with 1% FBS. Thereafter, the cells were washed with HBSS, fixed with 2% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Nonspecific binding was blocked with blocking solution containing 0.2% fish skin gelatin (Sigma) and 1% bovine serum albumin in HBSS followed by incubation with appropriate primary and secondary antibody dissolved in the blocking solution. The coverslips were then mounted using ProLong antifade kit (Molecular Probes). Images were taken by laser-scanning confocal microscopy on a Zeiss LSM 510 microscope equipped with 63× water-immersion objective and laser excitations at 488 and 543 nm. 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. α-Thrombin Stimulation and Gα13Q226L Expression Induce VASP Phosphorylation and Phosphorylation-dependent Translocation—We tested the effect of thrombin stimulation on the phosphorylation of VASP by using HUVECs. We stimulated confluent HUVECs with 25 nm α-thrombin for different periods of time and subjected cleared cell extracts to SDS-PAGE and immunoblotting with anti-VASP antibody. α-Thrombin induced prolonged phosphorylation of VASP (Fig. 1A) that lasted for at least 4 h after stimulation. Targeting Ena/VASP proteins to the membrane and focal adhesions is required for cell adhesion, motility, and formation of filopodia (35Han Y.H. Chung C.Y. Wessels D. Stephens S. Titus M.A. Soll D.R. Firtel R.A. J. Biol. Chem. 2002; 277: 49877-49887Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), indicating that VASP localization in the cell is critical for its function. Therefore, we next analyzed the distribution of VASP in the HUVECs stimulated with α-thrombin. We stimulated confluent and quiescent HUVECs with 25 nm α-thrombin for 5 min followed by fixation, permeabilization, and blocking of the nonspecific binding sites. Thereafter, VASP localization in the cells was detected by staining with polyclonal anti-VASP antibody, whereas F-actin was stained with phalloidin. Our studies showed that in HUVECs endogenous VASP is located at the tips of the actin fibers and along the actin fibers. After α-thrombin stimulation, the cells retracted, rounded up, and formed stress fibers. Most interestingly, in these cells VASP accumulated at the periphery of the cell (Fig. 1B). To test if VASP phosphorylation was required for the α-thrombin-induced translocation, we generated three FLAG-tagged VASP mutants where serine 153 and serine 235 were mutated into alanine, individually and jointly. We transfected HUVECs with FLAG-tagged wild type VASP (WT-VASP), VASP-S153A, VASP-S235A, or VASP-S153A/S235A (PD-VASP) and stimulated either with HBSS or 25 nm α-thrombin for 5 min and fixed them. The transfected cells were detected by anti-FLAG antibody and scored based on the localization of FLAG-tagged VASP in the cell. We calculated the percentage of cells with VASP accumulated at the periphery from at least 200 FLAG-positive cells counted for each transfection. The percent of the cells with VASP accumulated at the periphery of the cell was ∼52% in the case of WT-VASP, whereas it was 20, 23, and 14% for VASP-S153A, VASP-S235A, and PD-VASP (Fig. 1C), respectively. These results suggested that α-thrombin-induced phosphorylation of VASP on both serine 153 and serine 235 was required for VASP translocation. The representative images of localization of WT-VASP and PD-VASP in control and α-thrombin-stimulated cells are shown in Fig. 1D. Both FLAG-tagged WT- and PD-VASP had similar localization to that of endogenous VASP. However, after α-thrombin stimulation only WT-VASP translocated to the periphery of the cells, and localization of PD-VASP was not affected by thrombin. The thrombin receptor is coupled to members of the Gi, Gq, and G12,13 families of proteins (reviewed in Ref. 36Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2107) Google Scholar). To dissect G protein(s) that mediate α-thrombin-induced VASP phosphorylation, we used regulators of G protein signaling (RGS proteins). RGS proteins function as GTPase-activating proteins (GAPs) thus serving as negative regulators of G protein signaling (37Zhong H. Neubg R.R. J. Pharmacol. Exp. Ther. 2001; 297: 837-845PubMed Google Scholar). RGS domain of Rho-specific guanine nucleotide exchange factor p115RhoGEF was shown to have GAP activity specifically for Gα12 and Gα13 (30Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar). RGS3 protein was shown to exhibits GAP activity for Gαi and Gαq (38Scheschonka A. Dessauer C.W. Sinnarajah S. Chidiac P. Shi C.S. Kehrl J.H. Mol. Pharmacol. 2000; 58: 719-728Crossref PubMed Scopus (74) Google Scholar), whereas RGS10 exhibits GAP activity for Gαi (39Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Crossref PubMed Scopus (307) Google Scholar). We tested if transfection of HUVECs with RGS domain of p115RhoGEF, RGS3, or RGS10 could affect α-thrombin-induced VASP phosphorylation. Our results showed that expression of the RGS domain of p115RhoGEF but not RGS3 and RGS10 abolished VASP phosphorylation induced by α-thrombin (Fig. 2A), suggesting that it is likely that Gα12 and/or Gα13 are involved in this pathway. We next transiently transfected constitutively active mutants of Gα12 and Gα13 that lack GTPase activity, Gα12Q229L and Gα13Q226L, into HEK-293 cells along with FLAG-tagged VASP. Our result showed that Gα13Q226L expression induced VASP phosphorylation of both serine 153, as it was detected by electrophoretic mobility shift of VASP, and serine 235, as it was detected by phosphospecific antibody (Fig. 2B). Most interestingly, expression of Gα12Q229L did not induce VASP phosphorylation (Fig. 2B), indicating that this phenomenon is specific for Gα13. It is known that Gα12 regulates gene expression by activation of distinct transcriptional control elements such as the SRE (

Referência(s)