Bisindolylmaleimide I Suppresses Fibroblast Growth Factor-mediated Activation of Erk MAP Kinase in Chondrocytes by Preventing Shp2 Association with the Frs2 and Gab1 Adaptor Proteins
2006; Elsevier BV; Volume: 282; Issue: 5 Linguagem: Inglês
10.1074/jbc.m606144200
ISSN1083-351X
AutoresPavel Krejčı́, Bernard Masri, Lisa Salazar, Claire Farrington‐Rock, Hervé Prats, Leslie M. Thompson, William R. Wilcox,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoFibroblast growth factors (FGFs) inhibit chondrocyte proliferation via the Erk MAP kinase pathway. Here, we explored the role of protein kinase C in FGF signaling in chondrocytes. Erk activity in FGF2-treated RCS (rat chondrosarcoma) chondrocytes or human primary chondrocytes was abolished by the protein kinase C inhibitor bisindolylmaleimide I (Bis I). Bis I inhibited FGF2-induced activation of MEK, Raf-1, and Ras members of Erk signaling module but not the FGF2-induced tyrosine phosphorylation of Frs2 or the kinase activity of FGFR3, demonstrating that it targets the Erk cascade immediately upstream of Ras. Indeed, Bis I abolished the FGF2-mediated association of Shp2 tyrosine phosphatase with Frs2 and Gab1 adaptor proteins necessary for proper Ras activation. We also determined which PKC isoform is involved in FGF2-mediated activation of Erk. When both conventional and novel PKCs expressed by RCS chondrocytes (PKCα, -γ, -δ, and -ϵ) were down-regulated by phorbol ester, cells remained responsive to FGF2 with Erk activation, and this activation was sensitive to Bis I. Moreover, treatment with PKCλ/ζ pseudosubstrate lead to significant reduction of FGF2-mediated activation of Erk, suggesting involvement of an atypical PKC. Fibroblast growth factors (FGFs) inhibit chondrocyte proliferation via the Erk MAP kinase pathway. Here, we explored the role of protein kinase C in FGF signaling in chondrocytes. Erk activity in FGF2-treated RCS (rat chondrosarcoma) chondrocytes or human primary chondrocytes was abolished by the protein kinase C inhibitor bisindolylmaleimide I (Bis I). Bis I inhibited FGF2-induced activation of MEK, Raf-1, and Ras members of Erk signaling module but not the FGF2-induced tyrosine phosphorylation of Frs2 or the kinase activity of FGFR3, demonstrating that it targets the Erk cascade immediately upstream of Ras. Indeed, Bis I abolished the FGF2-mediated association of Shp2 tyrosine phosphatase with Frs2 and Gab1 adaptor proteins necessary for proper Ras activation. We also determined which PKC isoform is involved in FGF2-mediated activation of Erk. When both conventional and novel PKCs expressed by RCS chondrocytes (PKCα, -γ, -δ, and -ϵ) were down-regulated by phorbol ester, cells remained responsive to FGF2 with Erk activation, and this activation was sensitive to Bis I. Moreover, treatment with PKCλ/ζ pseudosubstrate lead to significant reduction of FGF2-mediated activation of Erk, suggesting involvement of an atypical PKC. Activating mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human dwarfisms characterized by diminished long bone growth (1Passos-Bueno M.R. Wilcox W.R. Jabs E.W. Sertie A.L. Alonso L.G. Kitoh H. Hum. Mutat. 1999; 14: 115-125Crossref PubMed Scopus (250) Google Scholar). In cartilage, FGFR3 alters chondrocyte proliferation and differentiation by up-regulation of cell cycle inhibitors and stimulation of cartilage matrix degradation (2Chen L. Adar R. Yang X. Monsonego E.O. Li C. Hauschka P.V. Yayon A. Deng C. J. Clin. Investig. 1999; 104: 1517-1525Crossref PubMed Scopus (214) Google Scholar, 3Li C. Chen L. Iwata T. Kitagawa M. Fu X.Y. Deng C.X. Hum. Mol. Genet. 1999; 8: 35-44Crossref PubMed Scopus (194) Google Scholar, 4Legeai-Mallet L. Benoist-Lasselin C. Munnich A. Bonaventure J. Bone. 2004; 34: 26-36Crossref PubMed Scopus (84) Google Scholar, 5Krejci P. Masri B. Fontaine V. Mekikian P.B. Weis M. Prats H. Wilcox W.R. J. Cell Sci. 2005; 118: 5089-5100Crossref PubMed Scopus (123) Google Scholar). The anti-proliferative action of FGF 2The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; RCS, rat chondrosarcoma; Bis I, bisindolylmaleimide I; PKC, protein kinase C; aPKC, atypical PKC; MAP, mitogen-activated protein; MAPKAP, MAP kinase-activated protein; MEK, MAP kinase/extracellular signal-regulated kinase kinase; PMA, phorbol 12-myristate 13-acetate; WB, Western immunoblotting; GFP, green fluorescent protein; GTPγS, guanosine 5′-3-O-(thio)triphosphate. signaling in cartilage contrasts with the usual mitogenic response of cells to FGF stimulus (6Boilly B. Vercoutter-Edouart A.S. Hondermarck H. Nurcombe V. Le Bourhis X. Cytokine Growth Factor Rev. 2000; 11: 295-302Crossref PubMed Scopus (229) Google Scholar), but the molecular basis of this paradox remains unclear. 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A. 2004; 101: 609-614Crossref PubMed Scopus (101) Google Scholar). The requirement of PKC for sustained Ras/Erk signaling prompted us, in this study, to investigate the role of PKC in FGF signaling in chondrocytes. Cell Culture, Western Immunoblotting (WB), and Immunoprecipitation—FGFR2 and FGFR3-expressing (32Aikawa T. Segre G.V. Lee K. J. Biol. Chem. 2001; 276: 29347-29352Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) 3P. Krejci and W. R. Wilcox, unpublished data. rat chondrosarcoma (RCS) chondrocytes and Chinese hamster ovary cells were propagated in Dulbecco's modified Eagle's media or Opti-MEM media (Invitrogen) containing 10% fetal bovine serum (Atlanta Biological, Nordcross, GA) and antibiotics. To obtain human chondrocytes, cartilage was dissected from the ends of long bones of 20–28 week-of-gestation fetus and cleared of the soft tissues. Chondrocytes were isolated by a 24-h treatment with 0.1% bacterial collagenase (Invitrogen) and grown in monolayer in Dulbecco's modified Eagle's media. Cells were lysed in immunoprecipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA, 25 mm NaF, 0.1 mm dithiothreitol, 1 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, 1 mm phenylmethylsulfonyl fluoride, 8 mm β-glycerolphosphate, 10 mm Na3VO4, 1 μg/ml aprotinin). Lysates were resolved by SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, and visualized by luminescence (Amersham Biosciences). The following antibodies were used: actin, Erk2, FGFR1–4, Frs2, Grb2, MEK1, and PKCη, -μ, -θ, and -ζ (Santa Cruz Biotechnology, Santa Cruz, CA); Grb2, PKCα, -β, -γ, -δ, -ϵ, and -λ, Raf-1, Shc, and Shp2 (BD Transduction Laboratories); P-ElkS383, Erk1/2, P-Erk1/2T202/Y204, P-MEKS217/221, P-Raf-1S338, and P-FGFRY653/654 (Cell Signaling, Beverly, MA); 4G10, Gab1, Ras, and Rap1 (Upstate Biotechnology, Lake Placid, NY); PKCζ (Calbiochem); FLAG (Sigma); and Shb (Abcam, Cambridge, MA). For immunoprecipitation, 2 mg of total protein was incubated with Raf-1, Frs2, Gab1, PKCλ, PKCζ, Raf-1, Shc, Shb (2 μg), or FLAG antibody (5 μg) for 2 h at 4°C. Immunocomplexes were isolated using A/G agarose (Santa Cruz Biotechnology). To quantify the WB signal, the integrated optical density of a given band was determined using Scion Image software (Scion Corp., Frederick, MA). Signal Transduction Studies—Cells were serum-starved for 12 h before treatment with 10 ng/ml FGF2 (R&D Systems, Minneapolis, MN) for 30 min in the presence of heparin (1 μg/ml; Invitrogen). When Bis I, Gö6983, Gö6976, GSK3β inhibitor I (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), H89, Raf1 inhibitor I (5-iodo-3-((3,5-dibromo-4-hydroxyphenyl) methylene)-2-indolinone), Ro-31-8220, SU5402 (Calbiochem), U0126, or rapamycin (Cell Signaling) were used, cells were treated for 30 min prior to FGF2 treatment. When PMA (200 nm; Sigma) was used, cells were treated for 5 min or 12 h, respectively. The phosphorylation status of Raf-1, Erk, and MEK was detected by WB using the antibodies described above. Erk activity was determined using a kinase assay kit (Cell Signaling). Briefly, Erk was immunoprecipitated from 200 μg of total protein and incubated with recombinant Elk-1 in the presence of ATP. Phosphorylation of Elk-1 at Ser-383 was determined by WB. For the Raf-1 kinase assay, Raf-1 immunocomplexes were washed with Raf-1 kinase buffer (25 mm Tris, pH 7.5, 5 mm β-glycerolphosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, 10 mm MgCl2), and the kinase reaction was performed for 30 min at 30 °C in the presence of 20 μm ATP and 500 ng of recombinant MEK1 (Santa Cruz Biotechnology) in 40 μl of kinase buffer. MEK1 phosphorylation was determined by WB. For the FGFR3 kinase assay using recombinant FGFR3 intracellular domain, the Frs2 immunocomplexes were washed with FGFR3 kinase buffer (60 mm HEPES-NaOH, pH 7.5, 3 mm MgCl2, 3 mm MnCl2, 3 μm Na3VO4, 1.2 mm dithiothreitol), and the kinase reaction was performed for 30 min at 30 °C in the presence of 2.5 μg of polyethylene glycol, 10 μm ATP, and 300 ng of recombinant FGFR3 intracellular domain (Glu-322–Thr-806; Cell Signaling) in 50 μl of kinase buffer. For the FGFR3 kinase assay using full-length FGFR3, Chinese hamster ovary cells were transfected with vectors carrying C-terminally FLAG-tagged human wild-type or K650E-FGFR3. Ninety-six hours later, FGFR3 was immunoprecipitated with FLAG antibody. Immunocomplexes were washed with FGFR3 kinase buffer, and the kinase reaction was performed for 30 min at 30 °C in the presence of 10 μm ATP and 150 ng of recombinant Frs2 (Abnova, Taipei City, Taiwan) in 40 μl of kinase buffer. Ras and Rap1 activation was determined using a Ras or the Rap-1 activation assay (Upstate Biotechnology). Briefly, active GTPase was purified from cell lysates using the agarose-bound glutathione S-transferase fusion protein containing the Ras-binding domain of Raf-1 or Rap1-binding domain of Ral GDS and detected by WB. Myristoylated atypical PKC pseudosubstrate (N-myristoyl-SIYRRGARRWR KL) was obtained from Biomol (Plymouth, PA). Cells were treated with pseudosubstrate for 30 min, treated with 10 ng/ml FGF2 for 30 min, and analyzed for active Erk by WB. Vectors, Cell Transfection, and Cell Sorting—The pRK7-FGFR3 vector was made by cloning the full-length human wild-type or K650E-FGFR3 cDNA into the HindIII site of pRK7. The FLAG-tagged FGFR3 constructs were prepared by PCR amplification of pRK7-FGFR3 segment between the MluI site and the 3′ end using primers that added the FLAG tag and a BamHI site immediately 3′ of the stop codon (5′-CTGGAGTCCAACGCGTCCATGAGCTC-3′ and 5′-GTTGGGGATCCAGTGGCCCTTCACTTATCGTCGTCATCCTTGTAATCCATCGTCCGCGAGCCCCCACTGC-3′). The PCR product was recloned into the pRK7 vector at the MluI and BamHI sites. Cells were transfected with FuGENE 6 (Roche Diagnostics, Penzberg, Germany) according to the manufacturer's protocol. For cell sorting, cells were co-transfected with a GFP-expressing vector (pCCEY) and pRK7-FGFR3 vector in a 1:3 ratio. Twenty-four hours later, the GFP-positive cells were isolated using a FACStar+ cell sorter (BD Biosciences) and plated. Bis I Inhibits FGF2-mediated Activation of Erk MAP Kinase in Chondrocytes—To determine whether PKC is involved in FGF signaling in chondrocytes, we asked whether the FGF2-mediated activation of Erk MAP kinase was sensitive to the chemical inhibition of PKC. We treated RCS chondrocytes with FGF2 alone or together with three different PKC inhibitors (Gö6983, Bis I, Gö6976) and determined their effect on the FGF2-mediated Erk activity. Fig. 1A shows that Bis I almost entirely abolished Erk activation at concentrations higher than 5 μm in contrast to Gö6983 or Gö6976 that only partially modulated the Erk activation. Since sustained Erk activation underlies the growth inhibitory effect of FGF signaling in chondrocytes (8Raucci A. Laplantine E. Mansukhani A. Basilico C. J. Biol. Chem. 2004; 279: 1747-1756Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 9Krejci P. Bryja V. Pachernik J. Hampl A. Pogue R. Mekikian P. Wilcox W.R. Exp. Cell Res. 2004; 297: 152-164Crossref PubMed Scopus (63) Google Scholar), we determined the effect of Bis I on long term FGF2-mediated Erk activation. Fig. 1B demonstrates that Bis I inhibits both short term and long term FGF2-mediated Erk activation in RCS cells. Similar data were obtained with human primary chondrocytes (Fig. 1C). The effect of Bis I on Erk activity is not direct since no inhibition of Erk activity was detected by kinase assay in the presence of up to 50 μm Bis I (Fig. 1D). Bis I Inhibits FGF2-mediated Activation of the Erk Pathway Upstream of Ras—Fig. 1 demonstrates that Bis I inhibits activation of Erk by FGF signaling upstream of Erk. We asked at which level Bis I targets the Erk module. FGF2 treatment led to activatory phosphorylation of both MEK and Raf-1, and this phosphorylation was abolished by Bis I (Fig. 2, A and B). In the following experiment, Raf-1 was immunoprecipitated from cells treated with FGF2 alone or in combination with Bis I and probed for its kinase activity using recombinant MEK1 as a substrate. Raf-1 kinase activity was inhibited by Bis I, thus confirming that Bis I targets the Erk module either at the level or upstream of Raf-1 (Fig. 2C). Next, we tested whether Bis I targets Raf-1 directly. Active Raf-1 was immunoprecipitated from FGF2-treated cells and subjected to a kinase assay with Bis I added into the kinase reaction. No significant Raf-1 inhibition was detected in the presence of up to 50 μm Bis I (Fig. 2D), suggesting that Bis I inhibits the Erk module upstream of Raf-1. It is well established that FGF receptors (FGFR) activate the Erk module via Frs2-mediated recruitment of Grb2-Sos complexes that in turn activate Ras (33Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar). Recently, an alternative mode was described in nerve growth factor-treated PC12 cells whereby Frs2 recruits Crk-C3G complexes and activates Erk through Rap1 GTPase and B-Raf kinase (34Kao S. Jaiswal R.K. Kolch W. Landreth G.E. J. Biol. Chem. 2001; 276: 18169-18177Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). We therefore determined whether FGF signaling in RCS cells utilizes Ras or Rap1 for Erk activation. Although FGF2 triggered significant and prolonged Ras activity, a weak, if any, activation of Rap1 was detected (Fig. 3A). FGF2-mediated activation of Ras was abolished by Bis I (Fig. 3B). Bis I Does Not Inhibit FGFR3—Fig. 3B demonstrates that Bis I inhibits FGF2-mediated activation of the Erk pathway upstream of Ras. We asked whether Bis I inhibits FGFR3 kinase itself. First, we determined the FGF2- and Bis I-induced changes in the tyrosine phosphorylation status of Frs2, which reflects FGFR3 activity. Fig. 4A shows that FGF2 treatment led to an increase of Frs2 tyrosine phosphorylation that was not inhibited by Bis I. However, a significant electrophoretic mobility shift of Frs2 was induced by FGF2 that was eliminated by Bis I (Fig. 4B). This shift appears to be caused by Erk-mediated phosphorylation since it was nearly eliminated by the MEK inhibitor U0126. The tyrosine phosphorylation status of Frs2 (Fig. 4A) represents an indirect method of monitoring the FGFR3 activity. Since we could only detect weak Frs2 tyrosine phosphorylation and Bis I is structurally similar to staurosporine (both staurosporine and its derivatives such as PKC412 inhibit FGFRs (35Chen J. Deangelo D.J. Kutok J.L. Williams I.R. Lee B.H. Wadleigh M. Duclos N. Cohen S. Adelsperger J. Okabe R. Coburn A. Galinsky I. Huntly B. Cohen P.S. Meyer T. Fabbro D. Roesel J. Banerji L. 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We therefore probed the Bis I inhibitory activity toward FGFR3 by a kinase assay utilizing the recombinant, kinase active intracellular domain of FGFR3 and Frs2, immunoprecipitated from RCS cells, as a substrate. Fig. 4C shows that in this system, Bis I did not significantly inhibit the Frs2 tyrosine phosphorylation induced by recombinant FGFR3. This contrasted with the FGFR-specific inhibitor SU5402 that completely inhibited Frs2 tyrosine phosphorylation. Next, we tested the ability of Bis I to inhibit the activity of full-length FGFR3. C-terminally FLAG-tagged wild type or constitutively active FGFR3 mutant (K650E) was transfected into Chinese hamster ovary cells, immunoprecipitated, and subjected to a kinase assay with recombinant Frs2 as a substrate, and Bis I was added into the kinase reaction. Fig. 4D shows that Bis I did not inhibit FGFR3 activity at concentrations up to 20 μm, although partial inhibition was apparent at 50 μm. Bis I Prevents FGF2-induced Shp2 Association with Frs2 and Gab1 Adaptor Proteins—Fig. 4 shows that FGFR3 is not directly inhibited by Bis I concentrations sufficient to abolish FGF2-mediated activation of Erk pathway in cells, suggesting that Bis I inhibits the Erk pathway immediately upstream of Ras (Figs. 1, 3, and 4). To activate Ras, FGFRs phosphorylate several adaptor proteins such as Frs2, Gab1, Shc, and Shb, which recruit Ras guanine nucleotide exchange factor Sos, complexed with Grb2 or Shp2-Grb2, to the site of FGFR activation (33Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar, 36Klint P. Kanda S. Claesson-Welsh L. J. Biol. Chem. 1995; 270: 23337-23344Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 37Ong S.H. Hadari Y.R. Gotoh N. Guy G.R. Schlessinger J. Lax I. Proc. Natl. Acad. Sci. U. S. 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Atypical PKC Pseudosubstrate Inhibits FGF2-mediated Activation of the Erk Pathway—Although considered to be PKC-selective, Bis I may inhibit other kinases such as cAMP-dependent protein kinase, GSK-3, p70S6K, and MAPKAP-1β (39Hers I. Tavare J.M. Denton R.M. FEBS Lett. 1999; 460: 433-436Crossref PubMed Scopus (136) Google Scholar, 40Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 41Alessi D. FEBS Lett. 1997; 402: 121-123Crossref PubMed Scopus (198) Google Scholar). We therefore used inhibitors specific to cAMP-dependent protein kinase (H89; 1–10 μm), GSK-3 (GSK-3β inhibitor I; 1–10 μm), p70S6K (rapamycin; 1–20 nm), and MAPKAP-1β (Ro-31-8220; 1–10 μm) to evaluate their role in FGF2-mediated Erk activation. None of the compounds inhibited FGF2-mediated Erk activity (not shown), suggesting that Bis I abolishes FGF2-mediated Erk activation by inhibiting PKC. Next, we determined which PKC isoform is involved in FGF2-mediated Erk activation. At the protein level, RCS cells expressed PKCα, -γ, -δ, -ϵ, -λ, and -ζ (Fig. 6A). We first asked whether phorbol ester-responsive PKCs are involved in Erk activation by FGF2. Brief PMA treatment led to potent Erk activation that was completely inhibited by 5 μm of Gö6983 or Bis I, demonstrating the ability of PMA-responsive PKCs to activate Erk (Fig. 6B) (14Adams P.D. Parker P.J. FEBS Lett. 1991; 290: 77-82Crossref PubMed Scopus (47) Google Scholar). Chronic PMA treatment led to down-regulation of PKCα, -γ, -δ, and -ϵ (42Szallasi Z. Kosa K. Smith C.B. Dlugosz A.A. Williams E.K. Yuspa S.H. Blumberg P.M. Mol. Pharmacol. 1995; 47: 258-265PubMed Google Scholar), and their absence was functionally confirmed by brief PMA treatment, which failed to activate Erk (Fig. 6, A and B). When cells with down-regulated PKCα, -γ, -δ, and -ϵ were treated with FGF2, they responded with the usual levels of Erk activation that remained sensitive to Bis I, suggesting that a PMA-unresponsive aPKC (PKCλ and/or PKCζ) is involved in FGF2-mediated Erk activation in RCS cells (Fig. 6, A and C). To challenge this hypothesis, we determined the effect of cell-permeable, aPKC-specific pseudosubstrate peptide (SIYRRGARRWRKL) on FGF2-mediated Erk activation. The pseudosubstrate suppresses PKC catalytic activity by blocking the substrate-binding site and thus can be used as an isoform-specific PKC inhibitor (43Zhou G. Seibenhener M.L. Wooten M.W. J. Biol. Chem. 1997; 272: 31130-31137Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Treatment with PKCλ/ζ pseudosubstrate inhibited FGF2-mediated Erk activation in RCS cells (Fig. 6D). Although it is well established that FGF signaling inhibits chondrocyte growth, the molecular mechanism of this effect is not clearly defined. Considering the usual mitogenic outcome of FGF signaling, the effect of the FGF signal in chondrocytes appears to be unique. It was recently shown that the Ras/Erk pathway is a candidate for FGF-mediated growth arrest, and in contrast to most other cell types, the FGF stimulus elicits a remarkably prolonged Erk activation in chondrocytes (8Raucci A. Laplantine E. Mansukhani A. Basilico C. J. Biol. Chem. 2004; 279: 1747-1756Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 9Krejci P. Bryja V. Pachernik J. Hampl A. Pogue R. Mekikian P. Wilcox W.R. Exp. Cell Res. 2004; 297: 152-164Crossref PubMed Scopus (63) Google Scholar). This correlates with the known cellular responses to Erk, where transient activation appears to be crucial for mitogenic signaling of growth factors, but sustained activity frequently leads to growth arrest (44Roovers K. Assoian R.K. BioEssays. 2000; 22: 818-826Crossref PubMed Scopus (425) Google Scholar). Therefore, a part of the unique chondrocyte response to FGF may lie in their ability to maintain sustained Erk activity following the FGF stimulus. The molecular mechanism of this feature is unknown. The role of PKC in sustained or in constitutive activation of Erk in growth factor and oncogenic Ras signaling (26Bhalla U.S. Ram P.T. Iyengar R. Science. 2002; 297: 1018-1023Crossref PubMed Scopus (528) Google Scholar, 27Überall F. Kampfer S. Doppler W. Grunicke H.H. Cell. Signal. 1994; 6: 285-297Crossref PubMed Scopus (10) Google Scholar, 28Kampfer S. Hellbert K. Villunger A. Doppler W. Baier G. Grunicke H.H. Überall F. EMBO J. 1998; 14: 4046-4055Crossref Scopus (62) Google Scholar, 29Kampfer S. Windegger M. Hochholdinger F. Schwaiger W. Pestell R.G. Baier G. Grunicke H.H. Überall F. J. Biol. Chem. 2001; 276: 42834-42842Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) prompted us to explore the role of PKC in FGF signaling in chondrocytes. We found that the PKC inhibitor Bis I abolishes FGF2-mediated activation of Erk in chondrocytes. Both short term and long term Erk activity were equally inhibited by Bis I with somewhat higher effectiveness than either FGFR-specific or MEK-specific inhibitors, suggesting a critical role for Bis I-inhibited molecule in both the initiation and the maintenance of Erk activation following FGF2 treatment (Figs. 1 and 4). Bis I inhibited FGF2-mediated MEK activity, demonstrating that it targets the Erk pathway upstream of MEK. It is well established that FGFRs activate the Erk pathway through phosphorylation of the ad
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