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• W2059442882 abstract "Recently we reported that simultaneous treatment of NIH 3T3 cells with the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H2O2) resulted in synergistic activation of Raf-1 kinase (Lee, M., Petrovics, G., and Anderson, W. B. (2003) Biochem. Biophys. Res. Commun. 311, 1026–1033). In this study we have demonstrated that PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, greatly potentiated the ability of PMA and/or H2O2 to activate Raf-1 kinase, whereas it blocked the tyrosine phosphorylation of Raf-1. Unlike PMA/H2O2 treatment, which showed transient activation, PP2-mediated Raf-1 activation was sustained and continued to increase through 4 h of treatment. Transient transfection studies with a dominant-negative mutant of Ras (N19Ras) indicated that this PP2-induced activation of Raf-1 was Ras-independent. Moreover, PP2 showed no effect on platelet-derived growth factor-induced Raf-1 activation. Interestingly, mutation of the reported Raf-1 Src family tyrosine kinase phosphorylation site by conversion of tyrosines 340 and 341 to phenylalanine (YY340/341FF Raf) had limited effect on the ability of PP2 to induce significant stimulation of Raf-1 kinase activity. Taken together, our results suggest that a tyrosine phosphorylation event is involved in the negative feedback regulation of Raf-1. Inhibition of a Src family tyrosine kinase by PP2 appears to alleviate this tyrosine kinase-mediated inhibition of Raf-1 and allow activating modification(s) of Raf-1 to proceed. This PP2 effect resulted in significant and sustained Ras-independent activation of Raf-1 by PMA and H2O2. Recently we reported that simultaneous treatment of NIH 3T3 cells with the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H2O2) resulted in synergistic activation of Raf-1 kinase (Lee, M., Petrovics, G., and Anderson, W. B. (2003) Biochem. Biophys. Res. Commun. 311, 1026–1033). In this study we have demonstrated that PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, greatly potentiated the ability of PMA and/or H2O2 to activate Raf-1 kinase, whereas it blocked the tyrosine phosphorylation of Raf-1. Unlike PMA/H2O2 treatment, which showed transient activation, PP2-mediated Raf-1 activation was sustained and continued to increase through 4 h of treatment. Transient transfection studies with a dominant-negative mutant of Ras (N19Ras) indicated that this PP2-induced activation of Raf-1 was Ras-independent. Moreover, PP2 showed no effect on platelet-derived growth factor-induced Raf-1 activation. Interestingly, mutation of the reported Raf-1 Src family tyrosine kinase phosphorylation site by conversion of tyrosines 340 and 341 to phenylalanine (YY340/341FF Raf) had limited effect on the ability of PP2 to induce significant stimulation of Raf-1 kinase activity. Taken together, our results suggest that a tyrosine phosphorylation event is involved in the negative feedback regulation of Raf-1. Inhibition of a Src family tyrosine kinase by PP2 appears to alleviate this tyrosine kinase-mediated inhibition of Raf-1 and allow activating modification(s) of Raf-1 to proceed. This PP2 effect resulted in significant and sustained Ras-independent activation of Raf-1 by PMA and H2O2. Raf-1 is a cytoplasmic serine/threonine protein kinase that plays an important role in the transmission of signals initiated at the plasma membrane to modulate transcriptional activation and mitogenesis (1Morrison D.K. Cutler R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (534) Google Scholar, 2Hagemann C. Rapp U.R. Exp. Cell Res. 1999; 253: 34-46Crossref PubMed Scopus (219) Google Scholar). Raf-1 activation is mediated by an interaction with Ras-GTP, which recruits Raf-1 to the plasma membrane and induces a conformation change that relieves an inhibition imposed by the N terminus on the catalytic domain (3Marshall M. Mol. Reprod. Dev. 1995; 42: 493-499Crossref PubMed Scopus (85) Google Scholar). This is, however, insufficient to activate Raf-1, and additional phosphorylations on tyrosine and serine/threonine residues are required for activation (4Cai H. Smola U. Wixler V. Eisenmann-Tappe I. Diaz-Meco M.T. Moscat J. Rapp U. Cooper G.M. Mol. Cell. Biol. 1997; 17: 732-741Crossref PubMed Scopus (263) Google Scholar, 5Jelinek T. Dent P. Sturgill T.W. Weber M.J. Mol. Cell. Biol. 1996; 16: 1027-1034Crossref PubMed Scopus (123) Google Scholar, 6Morrison D.K. Mol. Reprod. Dev. 1995; 42: 507-514Crossref PubMed Scopus (62) Google Scholar, 7Dhillon A.S. Kolch W. Arch. Biochem. Biophys. 2002; 404: 3-9Crossref PubMed Scopus (155) Google Scholar). In addition, dimerization and interaction with other proteins also play important roles in the regulation of Raf-1 activity (8Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (386) Google Scholar, 9Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1204) Google Scholar).Protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PDGF, platelet-derived growth factor; PMA, phorbol myristate acetate; PP2, 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; RKIP, Raf kinase inhibitor protein; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; wt, wild type.1The abbreviations used are: PKC, protein kinase C; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PDGF, platelet-derived growth factor; PMA, phorbol myristate acetate; PP2, 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; RKIP, Raf kinase inhibitor protein; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; wt, wild type. involvement in the regulation of the Ras/Raf/MAP kinase pathway has been demonstrated in a variety of systems ranging from yeast to higher eukaryotes (10Liao D.-F. Monia B. Dean N. Berk B.C. J. Biol. Chem. 1997; 272: 6146-6150Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 11Marshall M.S. FASEB J. 1995; 9: 1311-1318Crossref PubMed Scopus (271) Google Scholar, 12Morrison P. Saltiel A.R. Rosner M.R. J. Biol. Chem. 1996; 271: 12891-12896Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The PKC family is composed of at least 11 closely related isozymes that are classified into three major groups. The conventional PKCs (PKCα, -β1, -β2, and -γ) are Ca2+-dependent and are regulated by diacylglycerol and phorbol 12-myristate 13-acetate (PMA). The novel PKCs (PKCδ, -ϵ, -ι, and -θ) are Ca2+-independent but exhibit similar response to diacylglycerol and PMA. In contrast, the atypical aPKCs (PKCζ and -λ) are insensitive to Ca2+, diacylglycerol, and PMA (13Parekh D.B. Ziegler W. Parker P.J. EMBO J. 2000; 19: 496-503Crossref PubMed Scopus (508) Google Scholar). Both conventional PKCα and novel PKCϵ have been shown to activate Raf-1 in vitro (4Cai H. Smola U. Wixler V. Eisenmann-Tappe I. Diaz-Meco M.T. Moscat J. Rapp U. Cooper G.M. Mol. Cell. Biol. 1997; 17: 732-741Crossref PubMed Scopus (263) Google Scholar). Other studies also have reported that PKCϵ either directly or indirectly is involved in the activation of Raf-1 (14Cacace A.M. Ueffing M. Philipp A. Han E.K.H. Kolch W. Weinstein I.B. Oncogene. 1996; 13: 2517-2526PubMed Google Scholar, 15Ueffing M. Lovric J. Philipp A. Mischak H. Kolch W. Oncogene. 1997; 15: 2921-2927Crossref PubMed Scopus (71) Google Scholar, 16Perletti G.P. Concari P. Brusaferri S. Marras E. Piccinini F. Tashjian A.H.J. Oncogene. 1998; 16: 3345-3348Crossref PubMed Scopus (68) Google Scholar). In addition, evidence has been presented that implicates both aPKCλ and aPKCζ in the regulation of Ras/Raf/MAP kinase pathway (10Liao D.-F. Monia B. Dean N. Berk B.C. J. Biol. Chem. 1997; 272: 6146-6150Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 17Berra E. Diaz-Meco M.T. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (252) Google Scholar, 18Bjorkoy G. Perander M. Overvatn A. Johansen T. J. Biol. Chem. 1997; 272: 11557-11565Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar).In recent years it has become increasingly evident that the cellular generation of reactive oxygen species, including H2O2, plays an important role in cellular signal transduction and modulation of transmembrane signaling pathways (19Rhee S.G. Exp. Mol. Med. 1999; 31: 53-59Crossref PubMed Scopus (561) Google Scholar, 20Finkel T. FEBS Lett. 2000; 476: 52-59Crossref PubMed Scopus (489) Google Scholar, 21Allen R.G. Tresini M. Free Radic. Biol. Med. 2000; 28: 463-499Crossref PubMed Scopus (1062) Google Scholar, 22Finkel T. Curr. Opin. Cell Biol. 2003; 15: 247-254Crossref PubMed Scopus (1206) Google Scholar). The oxygen radicals generated appear to act as second messengers to regulate transmembrane signaling and to modulate cellular functions such as cell proliferation, differentiation, and apoptosis. A number of cell types produce H2O2 in response to a variety of growth factors, including platelet-derived growth factor (23Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2300) Google Scholar) and epidermal growth factor (24Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar). Furthermore, the PDGF-induced activation of the ERK/MAP kinase mitogenic cascade was found to require the generation of H2O2 (25Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1001) Google Scholar). Exposure of cells to H2O2 has been shown to increase the level of cellular tyrosine phosphorylation (26Barrett W.C. DeGnore J.P. Keng Y.F. Zhang Z.Y. Yim M.B. Chock P.B. J. Biol. Chem. 1999; 274: 34543-34546Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 27Konishi H. Yamauchi E. Taniguchi H. Yamamoto T. Matsuzaki H. Takemura Y. Ohmae K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6587-6592Crossref PubMed Scopus (217) Google Scholar, 28Rao G.N. Oncogene. 1996; 13: 713-719PubMed Google Scholar) and to stimulate the phosphorylation of MAP kinase (29Abe M.K. Kartha S. Karpova A.Y. Li J. Liu P.T. Kuno W.-L. Hershenson M.B. Am. J. Respir. Cell Mol. Biol. 1998; 18: 562-569Crossref PubMed Scopus (109) Google Scholar, 30Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1138) Google Scholar, 31Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1421) Google Scholar). Moreover, we found that a 33-kDa C-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to stimulation of NIH 3T3 cells with H2O2 (32Ferrier A.F. Lee M. Anderson W.B. Benvenuto G. Morrison D.K. Lowy D.R. DeClue J.E. J. Biol. Chem. 1997; 272: 2136-2142Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), whereas Abe et al. (29Abe M.K. Kartha S. Karpova A.Y. Li J. Liu P.T. Kuno W.-L. Hershenson M.B. Am. J. Respir. Cell Mol. Biol. 1998; 18: 562-569Crossref PubMed Scopus (109) Google Scholar) reported that treatment of bovine tracheal myocytes with H2O2 led to the activation of Raf-1 kinase. In related studies, treatment of cells with ionizing radiation was found to increase the level of membrane-bound, tyrosine-phosphorylated Raf-1 and stimulate Raf-1 kinase activity (29Abe M.K. Kartha S. Karpova A.Y. Li J. Liu P.T. Kuno W.-L. Hershenson M.B. Am. J. Respir. Cell Mol. Biol. 1998; 18: 562-569Crossref PubMed Scopus (109) Google Scholar, 33Kasid U. Suy S. Dent P. Ray S. Whiteside T.L. Sturgill T.W. Nature. 1996; 382: 813-816Crossref PubMed Scopus (154) Google Scholar).Maximal activation of Raf-1 has been shown to require phosphorylation events (4Cai H. Smola U. Wixler V. Eisenmann-Tappe I. Diaz-Meco M.T. Moscat J. Rapp U. Cooper G.M. Mol. Cell. Biol. 1997; 17: 732-741Crossref PubMed Scopus (263) Google Scholar, 5Jelinek T. Dent P. Sturgill T.W. Weber M.J. Mol. Cell. Biol. 1996; 16: 1027-1034Crossref PubMed Scopus (123) Google Scholar, 6Morrison D.K. Mol. Reprod. Dev. 1995; 42: 507-514Crossref PubMed Scopus (62) Google Scholar, 7Dhillon A.S. Kolch W. Arch. Biochem. Biophys. 2002; 404: 3-9Crossref PubMed Scopus (155) Google Scholar). In particular, phosphorylation of serine 338 and tyrosine 341 are required for Ras-dependent activation of Raf-1 (34Diaz B. Barnard D. Filson A. MacDonald S. King A. Marshall M. Mol. Cell. Biol. 1997; 17: 4509-4516Crossref PubMed Scopus (163) Google Scholar, 35Mason C.S. Springer C.J. Cooper R.G. Superti-Furga G. Marshall C.J. Marias R. EMBO J. 1999; 18: 2137-2148Crossref PubMed Scopus (364) Google Scholar, 36Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (398) Google Scholar). Phosphorylation of tyrosine 341 has been reported to be carried out by Src family tyrosine kinases (35Mason C.S. Springer C.J. Cooper R.G. Superti-Furga G. Marshall C.J. Marias R. EMBO J. 1999; 18: 2137-2148Crossref PubMed Scopus (364) Google Scholar, 37Marais R. Light Y. Paterson H.F. Marshall C.J. EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (520) Google Scholar, 38Fabian J.R. Daar I.O. Morrison D.K. Mol. Cell. Biol. 1993; 13: 7170-7179Crossref PubMed Scopus (300) Google Scholar). The Src family of non-receptor tyrosine kinases plays an important role in the regulation of cell adhesion, growth, and differentiation through the activation of multiple intracellular signaling pathways (39Bjorge J.D. Jakymiw A. Fujita D.J. Oncogene. 2000; 19: 5620-5635Crossref PubMed Scopus (333) Google Scholar, 40Schwartzberg P.L. Oncogene. 1998; 17: 1463-1468Crossref PubMed Scopus (132) Google Scholar). Src kinase normally is maintained in an inactive state but can be transiently activated during cellular events such as mitosis or constitutively activated by abnormal events such as mutation (39Bjorge J.D. Jakymiw A. Fujita D.J. Oncogene. 2000; 19: 5620-5635Crossref PubMed Scopus (333) Google Scholar). The activation of Src occurs as a result of disruption of negative regulatory processes that normally suppress Src tyrosine kinase activity (40Schwartzberg P.L. Oncogene. 1998; 17: 1463-1468Crossref PubMed Scopus (132) Google Scholar).Recently we determined that exposure of NIH 3T3 cells to H2O2 also resulted in stimulation of Raf-1 kinase activity (41Lee M. Petrovics G. Anderson W.B. Biochem. Biophys. Res. Commun. 2003; 311: 1026-1033Crossref PubMed Scopus (6) Google Scholar). Interestingly, treatment of cells with H2O2 in combination with PMA to stimulate PKCϵ resulted in synergistic activation of Raf-1 (41Lee M. Petrovics G. Anderson W.B. Biochem. Biophys. Res. Commun. 2003; 311: 1026-1033Crossref PubMed Scopus (6) Google Scholar). Here we have employed the tyrosine kinase inhibitor PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) to determine the possible involvement of tyrosine phosphorylation in the modulation of H2O2/PMA stimulation of Raf-1 kinase activity. PP2 is a potent inhibitor of Src family tyrosine kinases but only weakly inhibits ZAP-70 and JAK2 (42Hanke J.H. Gardner J.P. Dow R.L. Changelianb P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1780) Google Scholar). Interestingly, treatment of cells with PP2 to inhibit Src family kinase was found to enhance PMA/H2O2-mediated activation of Raf-1. These results indicate that the generation of H2O2 along with the activation of PKCϵ and inhibition of Src family tyrosine kinase all play an important role in the Ras-independent, sustained activation of Raf-1 kinase. Furthermore, the results presented here suggest that tyrosine phosphorylation events may be involved in both the stimulation and down-regulation of Raf-1 kinase.EXPERIMENTAL PROCEDURESMaterials—Rabbit polyclonal anti-Raf (C-12) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), whereas monoclonal mouse anti-Raf and anti-phosphotyrosine RC20H were from BD Biosciences Pharmingen. Protein A-agarose was from Roche Applied Science. Dulbecco's modified Eagle's medium, fetal calf serum, and penicillin-streptomycin were purchased from Invitrogen. Reagents for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. [γ-32P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. PP2, GF 109203X, Gö 6976, and wortmannin were purchased from Calbiochem. Monoclonal anti-FLAG M2, PMA, and H2O2 were obtained from Sigma. PMA and PP2 were dissolved in Me2SO and freshly diluted for each experiment.Plasmid DNA—The pcDNA vector encoding a dominant-negative mutant of Ras (N17Ras) was kindly provided by Dr. Toren Finkel (NIH, Bethesda, MD) with Dr. Silvio Gutkind's (NIH, Bethesda, MD) permission. NIH 3T3/BXB Raf cells overexpressing N-terminally truncated and constitutively activated Raf-1 were produced by cloning into the pMTH vector (43Olah Z. Ferrier A. Lehel C. Anderson W.B. Biochem. Biophys. Res. Commun. 1995; 214: 340-347Crossref PubMed Scopus (10) Google Scholar). The pcDNA-FLAG-Raf construct, obtained from Dr. Jeffrey E. DeClue (NIH, Bethesda, MD) with Dr. Richard Kolesnick's (Memorial Sloan-Kettering Cancer Center, New York, New York) permission (44Zhang Y. Yao B. Delikat S. Bayoumy S. Lin X.H. Basu S. McGinley M. Chan-Hui P.Y. Lichenstein H. Kolesnick R. Cell. 1997; 89: 63-72Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar), was used as the template to generate FLAG-tagged Raf-1 point mutants using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The presence of point mutations was verified by DNA sequence analysis.Mammalian Cell Culture and Transient Transfection—Parental and v-Ha-ras-transformed NIH 3T3 cells were maintained at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin-streptomycin, and glutamine. Where indicated the cells were transiently transfected with the pcDNA vector encoding the indicated FLAG-tagged Raf-1 point mutants or encoding dominant-negative Ras (N17Ras) by the electroporation method as specified by the manufacturer (BTX). After 48 h the transfected cells were serum-deprived overnight before treatment with PMA, H2O2, and PP2 as indicated.Preparation of Cell Lysates—The indicated treatments of cells were carried out at 37 °C in serum-free medium as described in the figure legends. After treatment whole cell lysates were prepared as follows. Cells were washed twice with ice-cold phosphate-buffered saline and harvested by scraping the cells into lysis buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 10 μg/ml leupeptin, 20 mm β-glycerophosphate, and 2 mm sodium fluoride). Cell lysates were clarified by centrifugation at 15,000 × g for 10 min at 4 °C, and lysate protein concentrations were determined with a BCA protein assay reagent kit as described by the manufacturer (Pierce).Immunoprecipitation and Immunoblot Analysis—Immunoprecipitation was performed on the whole cell lysates using either polyclonal anti-Raf or monoclonal anti-FLAG M2 and protein A-agarose beads. After incubation for 2 h at 4 °C, immunoprecipitates were washed twice with ice-cold lysis buffer. For immunoblotting, immunoprecipitates were denatured in Laemmli sample buffer and resolved by either 7.5 or 12% SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose, and immunoblot analysis was performed using the antibody described in the figure legends. Endogenous Raf-1 kinase and the oncogenically active BXB Raf fragment (the C-terminal fragment starting with the amino acid 302 of c-Raf-1) were detected using a 1:1000 dilution of anti-Raf antibody (BD Biosciences Pharmingen). Immune complexes on nitrocellulose were detected by enzyme-linked chemiluminescence (Amersham Biosciences). Fluorescent images were captured using a CCD camera in a ChemiDoc system (Bio-Rad) and saved to an IBM-PC computer. Bands were quantified using Quantity One analysis software, Version 4.4 (Bio-Rad).In Vitro Raf-1 Kinase Activity Assay—Raf-1 proteins were specifically immunoprecipitated from lysates of NIH 3T3 cells, washed three times with lysis buffer and once with kinase buffer (20 mm Tris, pH 7.4, 20 mm NaCl, 1 mm dithiothreitol, 10 mm MgCl2). Raf-1 kinase activity was measured by phosphorylation of recombinant MEK (Santa Cruz Biotechnology) as previously described (32Ferrier A.F. Lee M. Anderson W.B. Benvenuto G. Morrison D.K. Lowy D.R. DeClue J.E. J. Biol. Chem. 1997; 272: 2136-2142Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The washed immunoprecipitates were incubated in 40 μl of kinase buffer containing 10 mm ATP, 1 μg of the recombinant MEK, and 5 μCi of [γ-32P]ATP at 30 °C for 30 min in the presence of MEK inhibitor, PD 98059, which was used to inhibit the autokinase activity of MEK-1. Assays were terminated by the addition of gel-loading buffer. The samples were resolved by 7.5% SDS-PAGE, and phosphorylated MEK protein bands were visualized by autoradiography.RESULTSThe Src-family Tyrosine Kinase Inhibitor PP2 Markedly Enhanced PMA/H2O2-mediated Activation of Raf-1 Kinase—Our previous studies showed that simultaneous treatment of cells with H2O2 and PMA resulted in synergistic activation of Raf-1 kinase (41Lee M. Petrovics G. Anderson W.B. Biochem. Biophys. Res. Commun. 2003; 311: 1026-1033Crossref PubMed Scopus (6) Google Scholar). Since tyrosine phosphorylation has been shown to be an important event in Raf-1 activation (6Morrison D.K. Mol. Reprod. Dev. 1995; 42: 507-514Crossref PubMed Scopus (62) Google Scholar, 7Dhillon A.S. Kolch W. Arch. Biochem. Biophys. 2002; 404: 3-9Crossref PubMed Scopus (155) Google Scholar), and because one mechanism by which reactive oxygen species has been reported to modulate signal transduction pathway is via alteration in protein tyrosine phosphorylation (26Barrett W.C. DeGnore J.P. Keng Y.F. Zhang Z.Y. Yim M.B. Chock P.B. J. Biol. Chem. 1999; 274: 34543-34546Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 45Natarajan V. Scribner W.M. al-Hassani M. Vepa S. Environ. Health Perspect. 1998; 106: 1205-1212Crossref PubMed Scopus (57) Google Scholar), several tyrosine kinase inhibitors were tested to determine their relative effects on Raf-1 kinase activation by the combination of PMA and H2O2. In these studies v-Ha-ras-transformed cells were used because these cells were found to be more susceptible to PMA and H2O2 activation than parental NIH 3T3 cells. In addition, NIH 3T3 cells expressing truncated BXB Raf also were used in these experiments. No apparent inhibition of PMA/H2O2-induced Raf-1 activation was observed with the tyrosine kinase inhibitors tested with the exception of AG490, an inhibitor of epidermal growth factor receptor tyrosine (Fig. 1A). Unexpectedly, PP2, an inhibitor of Src-family tyrosine kinases (42Hanke J.H. Gardner J.P. Dow R.L. Changelianb P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1780) Google Scholar), significantly increased PMA/H2O2-mediated activation of Raf-1 kinase (Fig. 1A). Importantly, PP2 showed an even greater stimulatory effect of PMA and H2O2 activation of constitutively active BXB Raf (the N-terminal deletion mutant of Raf-1) (Fig. 1B). The kinetics of Raf-1 activation after exposure of v-Ha-ras-transformed cells to PP2 are shown in Fig. 1C. It was found that promotion of PMA/H2O2-induced activation of Raf-1 was optimal within 30 min of PP2 treatment, and this level of activation was sustained through at least 4 h of exposure to PP2. The ability of PP2 to enhance PMA/H2O2 activation of Raf-1 decreased after 12 h of treatment and approached control levels of Raf-1 kinase activity by 24 h of exposure to PP2 (inset, Fig. 1C).PP2 Blocked PMA and H2O2-induced Tyrosine Phosphorylation of Raf-1 and BXB Raf—Next, studies were carried out to determine whether PP2 treatment inhibited the tyrosine phosphorylation of Raf-1 noted with exposure of v-Ha-ras-transformed (Fig. 2A) and BXB Raf-overexpressing (Fig. 2B) NIH 3T3 cells to PMA and H2O2. In this experiment both Raf-1 and the BXB Raf fragment were immunoprecipitated using anti-Raf antibody, and the presence of phosphotyrosine was detected by immunoblotting with anti-phosphotyrosine antibody. As shown, the presence of PP2 blocked the slight increase in Raf-1 (Fig. 2A) and BXB Raf (Fig. 2B) tyrosine phosphorylation induced by PMA and H2O2. There was no change in the levels of either Raf-1 or BXB Raf protein under these conditions, as determined by reprobing the stripped immunoblots with anti-Raf antibody.Fig. 2Treatment of cells with PP2 inhibited the tyrosine phosphorylation (P-tyr) of Raf-1 and BXB Raf induced by exposure to PMA and H2O2. Panel A, v-Ha-ras-transformed NIH 3T3 cells serum-deprived for 24 h were pretreated in the presence and absence of PP2 as indicated for 30 min before stimulation with 100 nm PMA and/or 1 mm H2O2 for 5 min. IP, immunoprecipitation. Ab, antibody. Panel B, BXB Raf-overexpressing NIH 3T3 cells serum-deprived for 24 h were pretreated with and without 10 μm PP2 for 30 min before stimulation with the combination of 100 nm PMA and 1 mm H2O2 for 5 min. In panels A and B, Raf-1 and BXB Raf proteins were immunoprecipitated from cell lysates with anti-Raf antibody, and the immunoprecipitates were resolved by 7.5% SDS-PAGE. The presence of phosphotyrosine was detected by immunoblotting with anti-phosphotyrosine antibody RC20H. The same blots were stripped and then reprobed with anti-Raf-1 antibody to determine the amount of Raf-1 and BXB Raf protein present in each lane. The results are representative of three independent experiments.View Large Image Figure ViewerDownload (PPT)The Stimulatory Effect of PP2 Is Ras-independent—The phorbol ester tumor promoter PMA serves as a potent, non-physiological activator of PKC by acting as a mimic of diacylglycerol, an endogenous activator of PKC. Thus, studies were carried out with the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) to determine whether OAG can replace PMA to induce synergistic activation of Raf-1 in combination with H2O2. Although not as effective as PMA, simultaneous treatment of cells with OAG along with H2O2 did result in transient activation of Raf-1 (Fig. 3A). Raf-1 activation by OAG/H2O2 reached its peak within 5–10 min after treatment and was found to be markedly enhanced by pretreatment of cells with PP2. Conversely, PP2 showed no effect on PDGF-induced Raf-1 kinase activation, which is Ras-dependent (Fig. 3B). Thus, experiments were carried out to determine whether Ras is required for PP2 stimulation of Raf-1 activation induced by PMA/H2O2. Inhibition of Ras function was achieved by transient transfection with a vector encoding dominant-negative Ras (RasN17). As shown in Fig. 3C, expression of dominant-negative RasN17 to block Ras function did not inhibit the PMA/H2O2-induced activation of Raf-1 noted in the presence of PP2. However, in experiments carried out to establish that expression of RasN17 in these cells effectively blocked Ras function, as determined by inhibition of PDGF-induced ERK phosphorylation, it was found that the elevated levels of ERK phosphorylation noted with v-Ha-ras-transformed cells made it difficult to detect consistent, significant stimulation of ERK phosphorylation induced by PDGF alone. Thus, parental NIH 3T3 cells were used to establish that expression of RasN17 did block Ras function, as determined by inhibition of PDGF-induced stimulation of ERK phosphorylation, but had a much less pronounced effect on PMA/H2O2-induced ERK activation (Fig. 3C, inset). Importantly, transient transfection of RasN17 into NIH 3T3 cells did not significantly block PMA/H2O2/PP2-induced activation of Raf-1. These results established that PMA/H2O2-induced activation of Raf-1 was Ras-independent. Of added interest was the finding that the presence of PP2 did not enhance PMA/H2O2-induced ERK activation with v-Ha-ras-transformed cells, as determined by increased ERK phosphorylation, despite the noted hyperactivation of Raf-1 under these conditions (Fig. 3D). These results suggested that PMA/H2O2/PP2-mediated activation of Raf-1 did not appear to potentiate signaling through the Ras/MEK/ERK pathway.Fig. 3PP2 potentiation of Raf-1 activity is Ras-independent and specific to the activation induced by PMA or OAG and H O. Panel A, PP2 also enhanced Raf-1 kinase activation by the combination of the diacylglycerol analogue OAG and H2O2. Subconfluent v-Ha-ras-transformed cells were serum-deprived for 24 h and then exposed to 10 μm PP2 where indicated for 30 min before treatment with the combination of 50 μm OAG and 1 mm H2O2 for the times indicated. Panel B, PP2 does not enhance the ability of PDGF to stimulate Raf-1 kinase. Subconfluent NIH 3T3 cells were serum-deprived for 24 h along with exposure to the indicated tyrosine kinase inhibitors for the following time periods. The serum-deprived cells were treated with 60 μm genistein for the final 60 min, with 10 μm PP2 for the final 30 min, with 50 μm AG1296 (PDGFR kinase inhibitor) for the final 4 h, and with 50 μm AG1478 (epidermal growth factor receptor kinase inhibitor) for the final 30 min before exposure to 20 ng/ml PDG" @default.
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• W2059442882 date "2004-11-01" @default.
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• W2059442882 title "Src Tyrosine Kinase Inhibitor PP2 Markedly Enhances Ras-independent Activation of Raf-1 Protein Kinase by Phorbol Myristate Acetate and H2O2" @default.
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• W2059442882 doi "https://doi.org/10.1074/jbc.m403132200" @default.
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