Protein Kinase G Mediates Vascular Endothelial Growth Factor-induced Raf-1 Activation and Proliferation in Human Endothelial Cells
1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês
10.1074/jbc.273.36.23504
ISSN1083-351X
Autores Tópico(s)Inflammatory mediators and NSAID effects
ResumoVascular endothelial growth factor (VEGF) is an endothelium-specific, secreted protein that acts as a vasodilator, angiogenic peptide, and hyperpermeability factor. Recent reports have shown that nitric oxide synthase inhibitors block proliferation and microvascular hyperpermeability induced by VEGF. This study examined the mechanisms by which nitric oxide and its downstream signals mediate the VEGF-induced proliferative response in human umbilical vein endothelial cells (HUVECs). Nitric oxide synthase blockade byN ω-nitro-l-arginine methyl ester prevented both the proliferative effect of VEGF and Raf-1 activation by VEGF as measured by cell counting and the capacity of immunoprecipitated Raf-1 to phosphorylate syntide 2, a Raf-1-specific synthetic substrate. VEGF-induced proliferation and Raf-1 kinase activity were also inhibited by Rp-8-pCPT-cGMPs and KT5823, inhibitors of the regulatory and catalytic subunits of cGMP-dependent protein kinase (PKG), respectively. The ability of PKG to stimulate proliferation was verified by the observation that the PKG activator, 8-pCPT-cGMPs, stimulated both Raf-1 kinase activity and endothelial proliferation in a dose-dependent manner. Furthermore, recombinant catalytically active PKG phosphorylated and activated Raf-1 in a reconstituted system. Finally, Raf-1 immunoprecipitated from VEGF-stimulated endothelial cells coprecipitated with PKG, indicating a direct protein-protein interaction in activated cells. We conclude that VEGF induces increases in both proliferation and Raf-1 kinase activity in HUVECs and these activities are dependent on NO and its downstream effector, PKG. Vascular endothelial growth factor (VEGF) is an endothelium-specific, secreted protein that acts as a vasodilator, angiogenic peptide, and hyperpermeability factor. Recent reports have shown that nitric oxide synthase inhibitors block proliferation and microvascular hyperpermeability induced by VEGF. This study examined the mechanisms by which nitric oxide and its downstream signals mediate the VEGF-induced proliferative response in human umbilical vein endothelial cells (HUVECs). Nitric oxide synthase blockade byN ω-nitro-l-arginine methyl ester prevented both the proliferative effect of VEGF and Raf-1 activation by VEGF as measured by cell counting and the capacity of immunoprecipitated Raf-1 to phosphorylate syntide 2, a Raf-1-specific synthetic substrate. VEGF-induced proliferation and Raf-1 kinase activity were also inhibited by Rp-8-pCPT-cGMPs and KT5823, inhibitors of the regulatory and catalytic subunits of cGMP-dependent protein kinase (PKG), respectively. The ability of PKG to stimulate proliferation was verified by the observation that the PKG activator, 8-pCPT-cGMPs, stimulated both Raf-1 kinase activity and endothelial proliferation in a dose-dependent manner. Furthermore, recombinant catalytically active PKG phosphorylated and activated Raf-1 in a reconstituted system. Finally, Raf-1 immunoprecipitated from VEGF-stimulated endothelial cells coprecipitated with PKG, indicating a direct protein-protein interaction in activated cells. We conclude that VEGF induces increases in both proliferation and Raf-1 kinase activity in HUVECs and these activities are dependent on NO and its downstream effector, PKG. vascular endothelial growth factor cGMP-dependent protein kinase mitogen-activated protein MAP kinase/ERK kinase extracellular signal-regulated protein kinase human umbilical vein endothelial cell N ω-nitro-l-arginine methyl ester cAMP-dependent protein kinase nitric oxide synthase elevated calcium-nitric oxide synthase. VEGF1 is a endothelium-specific mitogen that potently stimulates angiogenesis, vasodilation, and microvascular hyperpermeability. Hypoxia up-regulates VEGF production and release in a wide variety of cells and organs (for a review, see Refs. 1Thomas K.A. J. Biol. Chem. 1996; 271: 603-606Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar and 2Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). This relationship provides an elegant feedback loop by which decreased nutrient supply elicits the angiogenic signal and directs it to the target vascular endothelium (3Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-845Crossref PubMed Scopus (4177) Google Scholar). NO may play an intimate role in the signaling pathways leading to VEGF-induced endothelial cell proliferation (4Morbidelli L. Chang C.-H. Douglas J.G. Granger H.J. Ledda F. Ziche M. Am. J. Physiol. 1996; 270: H411-H415Crossref PubMed Google Scholar, 5Parenti A. Morbidelli L. Cui X.-L. Douglas J.G. Hood J.D. Granger H.J. Ledda F. Ziche M. J. Biol. Chem. 1998; 273: 4220-4226Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). However, it is not yet clear how nitric oxide mediates these actions. A primary downstream signaling effector of nitric oxide is cyclic GMP-dependent protein kinase (PKG). PKG activation occurs downstream from NO activation of soluble guanylate cyclase and the latter enzyme's ensuing conversion of GTP to cGMP. Cyclic GMP binds to four sites on the regulatory subunit of PKG, thereby activating the catalytic subunit of the enzyme. PKG phosphorylates select serine and threonine residues on its substrate proteins, leading to subsequent activation/inactivation signals in the cell (6Lincoln T.M. Cornwell T.L. Komalavilas P. Boerth N. Methods Enzymol. 1996; 269: 149-166Crossref PubMed Google Scholar). Activation of the Ras-binding protein, Raf-1, by serine and threonine phosphorylation is a primary step for initiation of the MAP kinase cascade. Activation of this cascade has been shown to be a prerequisite for growth factor-induced proliferation in many systems (7Nishida E. Gotoh Y. Trends Pharmacol. Sci. 1993; 18: 128-131Scopus (964) Google Scholar). The MAP kinase cascade is initiated by the binding of Ras to Raf-1, facilitating phosphorylation and activation of Raf-1 by an adjacent ligand-dependent protein kinase (8Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (886) Google Scholar). Activated Raf-1 phosphorylates and activates MAP kinase kinase, or MEK, which in turn phosphorylates and stimulates MAP kinase, or ERK. Activation of ERK stimulates downstream signaling, including activation of ternary control factor, c-Fos, and Elk-1. These events promote transcription and activation of the AP-1 promoter, which in turn initiates DNA synthesis and proliferation (9Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1397) Google Scholar). The present study was designed to characterize the transduction pathways through which VEGF stimulates endothelial proliferation. Recent reports have shown that NO is an intermediary in this pathway and is necessary for both proliferation and ERK1/2 activation (5Parenti A. Morbidelli L. Cui X.-L. Douglas J.G. Hood J.D. Granger H.J. Ledda F. Ziche M. J. Biol. Chem. 1998; 273: 4220-4226Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). In order to better understand the mechanisms underlying this pathway we assessed the role of PKG activation in stimulation of human umbilical vein endothelial cell (HUVEC) proliferation and Raf-1 kinase activation in the VEGF signal cascade. Unless otherwise noted all supplies were purchased from Sigma. HUVECs were purchased from Clonetics. The cells were cultured at 37 °C on gelatin-coated plates in the basal nutrient medium, MCDB-131, supplemented with 5% fetal bovine serum (Hyclone), 20 units/ml heparin, 1 mm sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin. HUVECs were passaged by trypsinization in Dulbecco's phosphate-buffered saline containing 0.25% trypsin and 0.02% EDTA. The cells used in this study were from passages 2 and 4. Endothelial cell proliferation was monitored as described previously (10Connolly D.T. Knight M.B. Harakas N.K. Wittwer A.J. Feder J. Anal. Biochem. 1986; 152: 136-140Crossref PubMed Scopus (182) Google Scholar). Briefly, HUVECs (2 × 104/well) were incubated with described growth factors and pharmacological agents in MCDB-131 containing 0.1% bovine serum albumin and 0.2% fetal bovine serum in 96-well plates. After a 3-day incubation, cellular acid phosphatase was measured using a Abacus kit (CLONTECH) according to manufacturer's instructions. Endothelial cell proliferation is quantified as units of optical density measured at 405 nm. These values were standardized by comparison to measurements made by cell counting with a hemocytometer under each pharmacological environment. After a 10-min treatment with VEGF (Peprotech), confluent 60-mm dishes of treated (or untreated control) HUVECs were rinsed with Dulbecco's phosphate-buffered saline and scraped into 0.5 ml of TME lysis buffer (10 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm EDTA, 25 mm NaF, 1 μg/ml aprotinin, 0.5 mg/ml Pefabloc™, 1 μg/ml pepstatin, 1 μg/ml leupeptin). Cell lysates were prepared by subjecting samples to two to three freeze-thaw cycles, sonication for 3 s, shearing with a 21-gauge needle, and centrifugation for 15 min at 15,000 × g. Protein concentration in the supernatant was quantified using the bicinchoninic acid protocol (Pierce) with bovine serum albumin as a standard. The lysates were subjected to overnight immunoprecipitation at 4 °C with either anti-Raf-1 (Santa Cruz Biotechnology) or anti-PKG (Upstate Biotechnology, Inc.) polyclonal antibodies. Immune complexes were recovered by addition of protein A-agarose (Santa Cruz), incubation overnight at 4 °C, and centrifugation. The beads were washed once with TME buffer, twice with TTBS (20 mm Tris, pH 7.5, 150 mm NaCl, and 0.1% Tween), and once more with TME buffer. Immunoprecipitated proteins were removed by boiling in polyacrylamide gel electrophoresis sample buffer and separated by SDS-polyacrylamide gel electrophoresis (7.5% acrylamide, 1.5-mm thick slab gel). Proteins then were transferred to a nitrocellulose membrane (Bio-Rad) by electroblotting. The membrane was blocked for 12 h at 4 °C in TTBS containing SuperblockTM (Pierce), and incubated with antibodies against either H-Ras, Raf-1, pan-PKC (Santa Cruz), or PKG for 12 h at 4 °C. After washing, the membrane was incubated with 0.1 μg/ml donkey anti-mouse immunoglobulin G antibody conjugated to horseradish peroxidase; peroxidase activity was visualized using the SuperSignalTM enhanced chemiluminescence substrate system (Pierce). Raf-1 protein kinase activity was determined in vitro using the synthetic peptide, syntide 2 (Santa Cruz), as a substrate. The phosphotransferase assay was performed by incubating Raf-1 immunoprecipitates along with the exogenous substrate for 20 min at 30 °C in 40 μl of reaction buffer (25 mm HEPES, pH 7.4, 25 mmβ-glycerophosphate, 1 mm dithiothreitol, 10 mm MnCl2, 100 μm ATP, and 10 μCi of [γ-32P]ATP. The assay was terminated by spotting 20 μl of the reaction mix onto pieces of P81 phosphocellulose paper. The filters were washed four times for 15 min in 0.85% phosphoric acid. The syntide 2-associated 32P radioactivity bound to the filters was quantitated by scintillation counting. Reconstitution experiments were performed using recombinant catalytically active PKG (Upstate Biotechnology). In these experiments phosphotransfer activity was visualized using MEK-1-glutathioneS-transferase (Upstate Biotechnology) as a substrate, followed by size fractionation on 9% SDS-polyacrylamide gel electrophoresis, gel drying, and autoradiography. Data is expressed as mean ± S.E. Comparisons of data from different groups were made by analysis of variance followed by a Fisher's test of least significant differences. For comparison of two variables where paired data were available, paired t tests were used. Probability values <0.05 were considered significant. The goal of this study was to elucidate the mechanisms of VEGF-induced proliferation in human endothelial cells. Recent reports have shown that in bovine coronary venular endothelial cells blocking NO production withl-NAME interfered with VEGF-stimulated proliferation and ERK 1/2 activation (4Morbidelli L. Chang C.-H. Douglas J.G. Granger H.J. Ledda F. Ziche M. Am. J. Physiol. 1996; 270: H411-H415Crossref PubMed Google Scholar, 5Parenti A. Morbidelli L. Cui X.-L. Douglas J.G. Hood J.D. Granger H.J. Ledda F. Ziche M. J. Biol. Chem. 1998; 273: 4220-4226Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Activation of the MAP kinase system has been shown to be a requirement for growth factor-stimulated mitogenesis in many systems (7Nishida E. Gotoh Y. Trends Pharmacol. Sci. 1993; 18: 128-131Scopus (964) Google Scholar). To ascertain whether this held true in human endothelial cells stimulated with VEGF, HUVECs were serum-starved for 24 h, pretreated with the appropriate blockers for 30 min, and assayed for proliferation and Raf-1 activation. PD 98059, which inhibits MAP kinase kinase (MEK) activity, effectively blocked VEGF-mediated proliferation as did the NOS inhibitor,l-NAME, indicating that activation of both the NO generating system and the MAP kinase cascade are necessary for the proliferative response of VEGF in HUVECs (Fig. 1 A). Furthermore,l-NAME also prevented VEGF-stimulated Raf-1 kinase activity, indicating that activation of the NO pathway is necessary for stimulation of this upstream effector of the MAP kinase pathway (Fig. 1 B). The classical downstream target of the NO pathway is protein kinase G. To determine if this protein kinase played a role in VEGF-mediated mitogenesis, blockers of either the regulatory (Rp-8-pCPT-cGMPs) or catalytic (KT5823) domain were used to pretreat HUVECs in proliferation and Raf-1 kinase assays. cAMP-dependent protein kinase (PKA) can be cross-activated by cGMP. To test the possibility that PKA was playing a role in this system, some cells were pretreated with the PKA inhibitor, Rp-8-pCPT-cAMPs, and assayed. VEGF activates phospholipase C (11Brock T.A. Dvorak H.F. Senger D.R. Am. J. Pathol. 1991; 138: 213-221PubMed Google Scholar) and consequently protein kinase C (12O'Brian C.A. Ward N.E. Cancer Metastasis Rev. 1989; 8: 199-214Crossref PubMed Scopus (108) Google Scholar), a reported activator of Raf-1 kinase (13Kolch W. Heidecker G. Kochs G. Humme R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1130) Google Scholar); therefore, we also tested the effects of bisindolylmaleimide, a protein kinase C blocker, on VEGF-stimulated proliferation and Raf-1 kinase activation. Neither bisindolylmaleimide nor Rp-8-pCPT-cAMPs interfered with VEGF-induced mitogenesis, while PKG blockers prevented VEGF-induced proliferation (Fig. 2 A). Furthermore, whereas PKG blockade prevented Raf-1 activation, neither down-regulation of PKC by 24-h pretreatment with phorbol esters or bisindolylmaleimide I nor PKA inhibition with Rp-8-pCPT-cAMPs significantly curbed the Raf-1 kinase activation elicited by VEGF (Fig. 2 B). This indicates that PKG, the downstream effector of NO, stimulates Raf-1 kinase and proliferation in HUVECs. Activation of the MAP kinase cascade by growth factors has been shown to involve Raf-1 interaction with membrane-bound Ras (14Egan S.E. Weinberg R.A. Nature. 1993; 365: 781-783Crossref PubMed Scopus (526) Google Scholar). It has also been shown that, while this association is necessary, it is not sufficient to activate Raf-1 in the absence of an associated serine-threonine protein kinase (8Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (886) Google Scholar). To determine if VEGF-stimulation induced a similar interaction of Raf-1 with Ras and Raf-1 with PKG or PKC, we immunoprecipitated Raf-1 from control and treated HUVECs and used Western blot analysis to determine which proteins were associated with it. Raf electrophoretic mobility was noticeably retarded ("band shift") by VEGF treatment compared with control cells (Fig. 3 A). The retardation of Raf mobility has been previously reported to correlate with increased phosphorylation of Raf (15Molloy C.J. Taylor D.S. Weber H. J. Biol. Chem. 1993; 268: 7338-7345Abstract Full Text PDF PubMed Google Scholar). The band shift was ameliorated by pretreatment of cells with l-NAME or Rp-8-pCPT-cGMPs, but not by Rp-8-pCPT-cAMPs. Treatment with 8-pCPT-cGMPs also caused a band shift, but the population of the immunoprecipitated Raf that showed electrophoretic retardation was not as complete as that witnessed with VEGF treatment. While treatment with VEGF for 5 min increased Raf-1 association with Ras (Fig. 3 B) and PKG (Fig. 3 C), no PKC association could be detected (Fig. 3 D). The efficiency of the PKC antibody was confirmed by treatment in medium containing 1% serum (Fig. 3 D). These observations were further corroborated by the findings that PKG immunoprecipitates contained Raf and Ras in VEGF-treated cells, but not in control untreated cells (Fig. 3 E). Interestingly, although pretreatment with NOS or PKG inhibitors blocked Raf-1 activation, as visualized in this assay as a Raf-1 bandshift (Fig. 3 A), pretreatment with NOS or PKG inhibitors did not completely repress Raf-1 association with PKG or Ras. This seems to indicate that while NOS activity is necessary for PKG activation it is not necessary for Raf-1 protein localization. Together these results suggest that PKG may serve as the Ras-associated activator of Raf-1 in the VEGF-activated MAP kinase cascade. While both the necessity of PKG for VEGF-stimulated activation of Raf-1, and the PKG association with Raf-1 in the VEGF-treated cells, strongly infer that PKG is the activator of Raf-1, the mechanism is not clear. One possibility is that PKG directly phosphorylates Raf-1 leading to its activation. The capacity of PKG to phosphorylate and activate Raf-1 has not been previously reported. To test this possibility, Raf-1 immunoprecipitated from HUVECs, recombinant catalytically active PKG, and catalytically inactive MEK-1-glutathione S-transferase (Raf-1 substrate) were incubated in kinase buffer with [γ-32P]ATP, then fractionated on a polyacrylamide gel, and visualized by autoradiography. Combining PKG, Raf-1, and MEK resulted in the appearance of a Raf-1 and MEK phosphorylation band, while neither Raf-1 alone nor PKG alone were capable of phosphorylating MEK (Fig. 4 A). This indicates that the PKG → Raf-1 → MEK cascade is feasible. In order to ascertain if PKG activation was sufficient for stimulation of HUVEC proliferation and Raf-1 activity, cells were serum-starved for 24 h, treated with the PKG activator 8-pCPT-cGMPs, and assayed. PKG activation resulted in both an increase in Raf-1 kinase activity and endothelial cell proliferation (Fig. 4, B and C). VEGF is an endothelial specific mitogen released by a variety of cell types. The rate of VEGF production and release into the extracellular fluid is increased in hypoxia, thus providing a rational explanation for the dependence of angiogenesis on tissuepO2 (16Shima D.T. Adamis A.P. Ferrara N. Yeo K.-T. Folkman J. D'Amore P.A. Mol. Med. 1995; 2: 64-71Google Scholar, 17Banai S. Shweiki D. Pinson A. Chandra M. Lazarovici G. Keshet E. Cardiovasc. Res. 1994; 28: 1176-1179Crossref PubMed Scopus (412) Google Scholar). VEGF isoforms are ligands for two receptor tyrosine kinases, flt1 and flk1/KDR (18deVries C. Escobedo J.A. Ueno H. Houck K.A. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1896) Google Scholar). The lower affinity flk1/KDR receptor is responsible for activation of several stages of angiogenesis, including detachment, migration, proliferation, and tube formation (19Seethram L. Gotoh N. Maru Y. Neufield G. Yamaguchi S. Shibuya M. Oncogene. 1995; 10: 135-147PubMed Google Scholar). VEGF was first identified as vascular permeability factor, reflecting the increased microvascular permeability associated with endothelial cell detachment processes (1Thomas K.A. J. Biol. Chem. 1996; 271: 603-606Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 2Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar, 20Connolly D.T. Olander J.V. Heuvelman D. Nelson R. Monsell R. Siegel N. Haymore B.L. Leingruber R. Feder J. J. Biol. Chem. 1989; 264: 20017-20024Abstract Full Text PDF PubMed Google Scholar). Ligation of flk1/KDR also leads to activation of phospholipase Cγ (11Brock T.A. Dvorak H.F. Senger D.R. Am. J. Pathol. 1991; 138: 213-221PubMed Google Scholar) and the formation of diacylglycerol and inositol triphosphate; the former promotes PKC activity and the latter elicits the release of calcium from the endoplasmic reticulum (21De Jonge H.W. Van Heugten H.A. Lamers J.M. J. Mol. Cell. Cardiol. 1995; 27: 93-106Abstract Full Text PDF PubMed Scopus (62) Google Scholar). The elevated calcium stimulates a constitutive form of nitric oxide synthase (ecNOS) found in endothelial cells and initiates production of nitric oxide and citrulline from the substrate l-arginine (22Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). Nitric oxide, a key messenger in the phospholipase C/ecNOS/soluble guanylate cyclase/PKG signaling pathway, stimulates endothelial proliferation at low to moderate concentrations (e.g. 10 μm or less) (23Ziche M. Morbidelli L. Masini E. Granger H.J. Geppetti P. Ledda F. Bochem. Biophys. Res. Commun. 1993; 192: 1198-1203Crossref PubMed Scopus (116) Google Scholar, 24Ziche M. Morbidelli L. Masini E. Amerini W. Granger H.J. Maggi C.A. Geppetti P. ledda F. J. Clin. Invest. 1994; 94: 2036-2044Crossref PubMed Scopus (774) Google Scholar). VEGF has previously been shown to stimulate NO production acutely and chronically in HUVECs (25van der Zee R. Murohara T. Luo Z. Zollmann F. Passeri J. Lekutat C. Isner J.M. Circulation. 1997; 95: 1030-1037Crossref PubMed Scopus (372) Google Scholar, 26Hood J.D. Meininger C.J. Ziche M. Granger H.J. Am. J. Physiol. 1998; 274: H1054-H1058PubMed Google Scholar). Furthermore, the increased rate of endothelial cell division elicited by VEGF is NO-dependent, as evidenced by the ability of inhibitors of ecNOS, soluble guanylate cyclase, and PKG to prevent mitogenesis (4Morbidelli L. Chang C.-H. Douglas J.G. Granger H.J. Ledda F. Ziche M. Am. J. 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PKC has been reported to activate Raf in several systems (30Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1161) Google Scholar, 31Ueda Y. Hirai S. Osada S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar), and VEGF phosphorylation of phospholipase Cγ could lead to PKC activation (32Quest A.F. Enzyme Protein. 1996; 49: 231-261Crossref PubMed Scopus (65) Google Scholar). However, PKC blockade by inhibitors of the conventional and novel PKC isozymes that might be activated by phospholipase Cγ (33Hofmann J. FASEB J. 1997; 11: 649-669Crossref PubMed Scopus (335) Google Scholar, 34Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. 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Recently we found that VEGF-induced NO stimulation of the MAP kinase system is dependent on cGMP production (5Parenti A. Morbidelli L. Cui X.-L. Douglas J.G. Hood J.D. Granger H.J. Ledda F. Ziche M. J. Biol. Chem. 1998; 273: 4220-4226Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Several recent studies have found that at high concentrations cGMP can "crossover" and activate PKA (38Cornwell T.L. Arnold E.A. Boerth N.J. Lincoln T.M. Am. J. Physiol. 1994; 267: C1405-C1413Crossref PubMed Google Scholar, 39Forte L.R. Thorne P.K. Eber S.L. Krause W.J. Freeman R. Francis S.H. Corbin J.D. Am. J. Physiol. 1992; 263: C607-C615Crossref PubMed Google Scholar), whose regulatory site is very similar to that of PKG (40Lincoln T.M. Cornwell T.L. FASEB J. 1993; 7: 328-338Crossref PubMed Scopus (543) Google Scholar). Because PKA is a reported inhibitor of Raf-1 (41Yamazaki T. Komuro I. Zou Y. Kudoh S. Mizuno T. Hiroi Y. Shiojima I. Takano H. Kinugawa K. Kohmoto O. Takahashi T. Yazaki Y. J. Mol. Cell. Cardiol. 1997; 29: 2491-2501Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 42Hafner S. Adler H.S. Mischak H. Janosch P. Heidecker G. Wolfman A. Pippig S. Lohse M. Ueffing M. Kolch W. Mol. Cell. Biol. 1994; 14: 6696-6703Crossref PubMed Scopus (292) Google Scholar), it is doubtful that transactivation of PKA by cGMP has a significant effect on our observations of PKG-induced Raf-1 activation. Nevertheless, in order to ensure that our observations were not due to PKA transactivation, we carefully used PKG agonist and antagonist doses in ranges that do not affect PKA activity and in some experiments ran trials with the highly selective inhibitor of PKA, Rp-8-pCPT-cAMPs. The PKA antagonist had no effect on VEGF-stimulated Raf-1 activation or proliferation, indicating that cross-talk with the PKA system was not playing a significant role in the VEGF system. Unlike cAMP, which acts almost exclusively through protein kinase A activation, cGMP has several intracellular target proteins: PKG, cGMP-gated ion channels, and cGMP-stimulated or cGMP-inhibited phosphodiesterases (43Lohmann S.M. Vaandrager A.B. Smolenski A. Walter U. De Jonge H.R. Trends Biochem Sci. 1997; 22: 307-312Abstract Full Text PDF PubMed Scopus (352) Google Scholar). In the present study, inhibition of PKG activity by blocking either the regulatory site with Rp-8-pCPT-cGMPs or the catalytic domain with KT5823 throttled VEGF-induced Raf activation and HUVEC proliferation. On the other hand, stimulation of PKG in the absence of VEGF led to an increase in endothelial cell Raf-1 kinase activity and proliferation. Thus, our findings are the first to demonstrate a key role for protein kinase G in activation of the MAP kinase cascade. The dependence of VEGF-induced HUVEC proliferation on the NO signaling pathway is explained by the phosphorylation of Raf-1 by activated PKG. This correlates well with other recent studies that have found that PKG activators stimulate gene expression of c-Fos, c-Jun, and transcription from AP-1 responsive promoters (44Gudi T. Huvar I. Meinecke M. Lohmann S.M. Boss G.R. Pilz R.B. J. Biol. Chem. 1996; 271: 4597-4600Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 45Pilz R.B. Suhasini M. Idriss S. Meinkoth J.L. Boss G.R. FASEB J. 1995; 9: 552-558Crossref PubMed Scopus (178) Google Scholar). An important commonalty of these gene products is that they are all downstream of Raf-1 activation and upstream of MAP kinase-stimulated proliferation (46Seger, R., and Krebs, E. G. FASEB J. 9, 726–735Google Scholar). In the present study, reconstitution of the converging components of the NO and Raf-1/MEK pathways strongly supports the idea that PKG modulates the MAP kinase cascade by phosphorylation and activation of the Raf-1 protein. The activation of Raf-1 in the reconstituted system is evidenced by its ability to phosphorylate a catalytically inactive form of MEK. Addition of catalytically active PKG led to phosphorylation of Raf-1 and subsequent phosphorylation of the catalytically inactive MEK. Removal of either Raf-1 or PKG prevented the MEK phosphorylation. Coprecipitation of Raf-1 and PKG in HUVECs also supports the central concept that PKG activates the MAP kinase cascade via phosphorylation of Raf-1. However, the association of PKG with Raf-1 and Raf-1 with Ras appears to be independent of PKG activation. This suggests that complexing of PKG, Ras, and Raf-1 is through a NO-independent mechanism, perhaps dependent on Ras.
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