Fyn and JAK2 Mediate Ras Activation by Reactive Oxygen Species
1999; Elsevier BV; Volume: 274; Issue: 30 Linguagem: Inglês
10.1074/jbc.274.30.21003
ISSN1083-351X
AutoresJun‐ichi Abe, Bradford C. Berk,
Tópico(s)Melanoma and MAPK Pathways
ResumoReactive oxygen species (ROS) activate Ras and the extracellular signal-regulated kinase (ERK) cascade. Because JAK2 is a critical mediator for Ras/Raf/ERK activation by several hormones, we examined the role of JAK2 in ROS signal events. H2O2 stimulated JAK2 activity in fibroblasts with peak at 2–5 min. To determine the specific role of Src and Fyn as mediators of JAK2 activation and its downstream events, we used fibroblasts derived from transgenic mice deficient in Src (Src−/−) or Fyn (Fyn−/−). H2O2-stimulated JAK2 activity was completely inhibited in Fyn−/− cells. Shc tyrosine phosphorylation and Ras activation by H2O2 were also significantly reduced in Fyn−/− cells, but not altered in Src−/− cells. Activation of JAK2 was restored when Fyn−/− cells were transfected with B-Fyn but not with Src. Inhibiting JAK2 activity with the specific inhibitor AG-490 prevented H2O2 stimulated Shc and Ras activation. H2O2-mediated ERK1/2 activation in Fyn−/− cells and AG-490 treated cells was completely inhibited at an early time (5 min), but not at late times (20–40 min) after stimulation. These results define a new redox-sensitive pathway for Ras activation and rapid ERK1/2 activation, which is mediated by Fyn and JAK2. Reactive oxygen species (ROS) activate Ras and the extracellular signal-regulated kinase (ERK) cascade. Because JAK2 is a critical mediator for Ras/Raf/ERK activation by several hormones, we examined the role of JAK2 in ROS signal events. H2O2 stimulated JAK2 activity in fibroblasts with peak at 2–5 min. To determine the specific role of Src and Fyn as mediators of JAK2 activation and its downstream events, we used fibroblasts derived from transgenic mice deficient in Src (Src−/−) or Fyn (Fyn−/−). H2O2-stimulated JAK2 activity was completely inhibited in Fyn−/− cells. Shc tyrosine phosphorylation and Ras activation by H2O2 were also significantly reduced in Fyn−/− cells, but not altered in Src−/− cells. Activation of JAK2 was restored when Fyn−/− cells were transfected with B-Fyn but not with Src. Inhibiting JAK2 activity with the specific inhibitor AG-490 prevented H2O2 stimulated Shc and Ras activation. H2O2-mediated ERK1/2 activation in Fyn−/− cells and AG-490 treated cells was completely inhibited at an early time (5 min), but not at late times (20–40 min) after stimulation. These results define a new redox-sensitive pathway for Ras activation and rapid ERK1/2 activation, which is mediated by Fyn and JAK2. Reduction-oxidation (redox) reactions that generate reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; RBD, Raf-binding domain; GST, glutathioneS-transferase1The abbreviations used are: ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; RBD, Raf-binding domain; GST, glutathioneS-transferaseincluding H2O2, O⨪2, and OH−, have been identified as important chemical mediators that regulate signal transduction. Because increased ROS may be a risk factor for cardiovascular events such as unstable angina, myocardial infarction, and sudden death, understanding the biological processes that generate ROS and the intracellular signals elicited by ROS will be useful to gain insights into the pathogenesis of these diseases (1Auch-Schwelk W. Bossaller C. Claus M. Graf K. Grafe M. Fleck E. J. Cardiovasc. Pharmacol. 1992; 9 (suppl.): 62-67Google Scholar, 2Stampfer M.J. Hennekens C.H. Manson J.E. Colditz G.A. Rosner B. Willett W.C. N. Engl. J. Med. 1993; 328: 1444-1449Crossref PubMed Scopus (1909) Google Scholar, 3Halliwell B. Br. J. Exp. Pathol. 1989; 70: 737-757PubMed Google Scholar, 4Abe J. Berk B.C. Trends Cardiovasc. Med. 1998; 8: 59-64Crossref PubMed Scopus (241) Google Scholar). Recently, it has been shown that ROS stimulate intracellular signal events similar to those activated by growth factors including stimulating kinases and small G proteins such as c-Src, Ras, and ERK1/2 (5Devary Y. Gottlieb R.A. Smeal T. Karin M. Cell. 1992; 71: 1081-1091Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 6Baas A.S. Berk B.C. Circ. Res. 1995; 77: 29-36Crossref PubMed Scopus (366) Google Scholar). Lander et al. (7Lander H.M. Ogiste J.S. Teng K.K. Novogrodsky A. J. Biol. Chem. 1995; 270: 21195-21198Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar) have reported that p21 Ras is a direct target of ROS and thus may be responsible for sensing redox status. In addition, Guyton et al. (8Guyton 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) have shown that H2O2-stimulated activation of ERK2 was abolished in PC12 cells by expression of dominant-negative Ras. These findings suggest that p21Ras may be an important mediator of ROS function.Previous studies have shown that c-Src is involved in signal events stimulated by ROS (5Devary Y. Gottlieb R.A. Smeal T. Karin M. Cell. 1992; 71: 1081-1091Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 9Abe J. Takahashi M. Ishida M. Lee J.-D. Berk B.C. J. Biol. Chem. 1997; 272: 20389-20394Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). It has been reported that Src family kinases and Ras are critical for ERK1/2 activation by H2O2 (10Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Tanaka M. Shiojima I. Hiroi Y. Yazaki Y. J. Clin. Invest. 1997; 100: 1813-1821Crossref PubMed Scopus (629) Google Scholar, 11Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Clermont-Goldschmidt P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1421) Google Scholar). The predominant pathway for ERK1/2 activation by angiotensin II and growth factors has also been proposed to involve Src-Ras-Raf-MEK1-ERK1/2 (12Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 13Ishida M. Ishida T. Thomas S. Berk B.C. Circ. Res. 1998; 82: 7-12Crossref PubMed Scopus (150) Google Scholar). However, it is not known how Src kinases mediate Ras activation by ROS. Several groups have suggested that JAK2 activates the Ras/Raf/ERK-signaling pathway and is required for the proliferative response initiated by many cytokines (14Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar). Han et al. (15Han Y. Leaman D.W. Watling D. Rogers N.C. Groner B. Kerr I.M. Wood W.I. Stark G.R. J. Biol. Chem. 1996; 271: 5947-5952Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) have reported that JAK2 is required to couple growth hormone receptor to pathways involving Shc. Shc is thought to function as an adapter molecule to recruit Grb2·Sos complexes to the activated receptor, which promotes formation of Ras-GTP (16Aronheim A. Engelberg D. Li N. al-Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (422) Google Scholar). In Src-transformed cells, JAK1 and JAK2 are constitutively tyrosine phosphorylated (17Campbell G.S. Yu C.L. Jove R. Carter-Su C. J. Biol. Chem. 1997; 272: 2591-2594Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) suggesting that H2O2-mediated Shc/Ras activation may be regulated by Src family kinases and JAK2.To determine the role of Src family kinases and JAK2 in H2O2-mediated Shc/Ras activation, we used specific JAK2 inhibitors and cells derived from animals deficient in c-Src and Fyn. We show here that activation of JAK2 by H2O2 is positively regulated by Fyn, but not by c-Src. Furthermore, we demonstrate that both Fyn and JAK2 are required for H2O2-mediated Shc tyrosine phosphorylation and activation of Ras. Thus, the Fyn-JAK2-Shc-Ras-signaling pathway described here may represent a new redox-sensitive mechanism.DISCUSSIONThe major findings of this study are first that H2O2 stimulates JAK2 in a Fyn-dependent and Src-independent manner, and second that JAK2 mediates phosphorylation of Shc and activation of Ras by H2O2. Redox-sensitive regulation of JAK2/Shc/Ras is thus a new function for Fyn. Data that support an essential role for Fyn and JAK2 in H2O2-mediated Shc/Ras activation include the following findings. 1) In Fyn−/− fibroblasts, there was no JAK2 tyrosine phosphorylation in response to H2O2. In contrast, in Src−/− fibroblasts, H2O2-mediated JAK2 tyrosine phosphorylation was not inhibited. 2) AG-490, a specific JAK2 inhibitor, prevented H2O2-mediated Shc tyrosine phosphorylation and Ras activation. 3) Likewise, Shc tyrosine phosphorylation and Ras activation by H2O2 were inhibited in Fyn−/− fibroblasts, but not in Src−/− fibroblasts. 4) Expression of Fyn, but not Src, in Fyn−/− cells restored the response of Shc and JAK2 to H2O2. Our results are the first to show that Fyn, but not Src, is involved specifically in oxidative stress-mediated JAK2 activation, which also regulates a Shc/Ras signaling pathway.Based on this study as well as previous work from our lab (6Baas A.S. Berk B.C. Circ. Res. 1995; 77: 29-36Crossref PubMed Scopus (366) Google Scholar, 9Abe J. Takahashi M. Ishida M. Lee J.-D. Berk B.C. J. Biol. Chem. 1997; 272: 20389-20394Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and other investigators (7Lander H.M. Ogiste J.S. Teng K.K. Novogrodsky A. J. Biol. Chem. 1995; 270: 21195-21198Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 25Rao G.N. Oncogene. 1996; 13: 713-719PubMed Google Scholar), we propose a scheme (Fig. 11) for ROS-mediated signal transduction leading to activation of Ras and ERK1/2. A novel aspect of this model is the specific role of Fyn, but not c-Src, to activate JAK2 and Ras. Previously we found that c-Src but not Fyn was required for H2O2-mediated BMK1/ERK5 activation in fibroblasts (9Abe J. Takahashi M. Ishida M. Lee J.-D. Berk B.C. J. Biol. Chem. 1997; 272: 20389-20394Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In contrast, in this study, we found that Fyn but not c-Src was required for H2O2-mediated JAK2 activation in fibroblasts. These results indicate that c-Src and Fyn have separate roles in ROS-mediated signal transduction. Campbell et al. (17Campbell G.S. Yu C.L. Jove R. Carter-Su C. J. Biol. Chem. 1997; 272: 2591-2594Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) have shown that the Janus kinases JAK1 and JAK2 (especially JAK1) are constitutively tyrosine phosphorylated in Src-transformed cells and suggested that JAK1, and possibly JAK2, are in an activated state in these cells. Furthermore, Uddin et al. (28Uddin S. Sher D.A. Alsayed Y. Pons S. Colamonici O.R. Fish E.N. White M.F. Platanias L.C. Biochem. Biophys. Res. Commun. 1997; 235: 83-88Crossref PubMed Scopus (44) Google Scholar) have shown that interferon-γ causes the SH2 domain of Fyn to interact with the activated form of interferon-γ-dependent JAK2. Future studies will be required to define the precise nature of the downstream substrates for c-Src and Fyn.In addition to activation of JAK2 by Fyn, this study demonstrates that H2O2-mediated Ras activation is partially dependent on Fyn and JAK2. Previous investigators have suggested an important relationship between two pathways used by cytokines and growth hormones to activate cells: the JAK2/STAT and the Ras/Raf/MEK/ERK pathway (14Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar). A key convergence point of these pathways may be activation of Raf. Conversion of Ras to its GTP-bound form results in the binding of Raf to Ras, and this interaction with activated Ras localizes Raf to the plasma membrane and is often the first step in Raf activation. However, other investigators have reported that tyrosine kinases, including members of the Src kinase family and JAK2 phosphorylate Raf, thereby enhancing its activity. For example, Marrero et al. (24Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) reported that angiotensin II and platelet-derived growth factor induced JAK2·Raf complex formation, Raf-1 tyrosine phosphorylation, and ERK1/2 kinase activity, which were dependent on JAK2 activity. Based on this study, we propose that JAK2 is "upstream" of the adapter protein Shc and regulates H2O2-mediated Ras activation (Fig. 11).The results for ERK1/2 activation suggest that H2O2 might activate two pathways, only one of which is dependent on Fyn and JAK2 (Fig. 11). In vascular smooth muscle cells, AG-490 inhibited the activation of ERK1/2 in response to either angiotensin II or platelet-derived growth factor (24Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Aikawa et al. (10Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Tanaka M. Shiojima I. Hiroi Y. Yazaki Y. J. Clin. Invest. 1997; 100: 1813-1821Crossref PubMed Scopus (629) Google Scholar) reported that when Csk, a negative regulator of Src tyrosine kinases was overexpressed, activation of ERK1/2 by H2O2 at 10 min was abolished. We examined the role of JAK2 and Fyn in H2O2-mediated ERK1/2 activation with AG-490 treatment and in Fyn−/− fibroblasts, and found that treatment with AG-490 or the absence of Fyn induced a temporal shift in activation of ERK1/2. However, the magnitude and duration of ERK1/2 activation were not altered. Of interest, McKenzie et al. (29McKenzie F.R. Pouyssegur J. J. Biol. Chem. 1996; 271: 13476-13483Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) have reported that pretreatment with prostaglandin E and isobutylmethylxanthine to elevate cAMP attenuated the ability of growth factors to stimulate ERK1/2, when measured 5 min after growth factor stimulation. However, similar to the effect of AG-490 and the absence of Fyn this inhibition was not apparent at late times and the magnitude of ERK1/2 activation was not decreased. Recently, York et al. (30York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar) have found that Rap1 mediates sustained ERK1/2 kinase activity induced by cAMP, so Rap1 could be a candidate to regulate this late phase activation of ERK1/2. Future studies will be required to determine the role of Rap1 in ROS-mediated signal transduction to ERK1/2.In summary, we have shown that JAK2 is activated by ROS in a Fyn-dependent manner. The fact that H2O2-mediated activation of JAK2 required Fyn, but not c-Src, suggests that these two Src family kinases serve different intracellular functions with respect to oxidative stress. In addition, the demonstration that Fyn/JAK2 regulates H2O2-mediated Shc tyrosine phosphorylation and Ras activation, suggests that Fyn/JAK2/Shc/Ras signaling pathway may involve novel intracellular mediators. Reduction-oxidation (redox) reactions that generate reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; RBD, Raf-binding domain; GST, glutathioneS-transferase1The abbreviations used are: ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; RBD, Raf-binding domain; GST, glutathioneS-transferaseincluding H2O2, O⨪2, and OH−, have been identified as important chemical mediators that regulate signal transduction. Because increased ROS may be a risk factor for cardiovascular events such as unstable angina, myocardial infarction, and sudden death, understanding the biological processes that generate ROS and the intracellular signals elicited by ROS will be useful to gain insights into the pathogenesis of these diseases (1Auch-Schwelk W. Bossaller C. Claus M. Graf K. Grafe M. Fleck E. J. Cardiovasc. Pharmacol. 1992; 9 (suppl.): 62-67Google Scholar, 2Stampfer M.J. Hennekens C.H. Manson J.E. Colditz G.A. Rosner B. Willett W.C. N. Engl. J. Med. 1993; 328: 1444-1449Crossref PubMed Scopus (1909) Google Scholar, 3Halliwell B. Br. J. Exp. Pathol. 1989; 70: 737-757PubMed Google Scholar, 4Abe J. Berk B.C. Trends Cardiovasc. Med. 1998; 8: 59-64Crossref PubMed Scopus (241) Google Scholar). Recently, it has been shown that ROS stimulate intracellular signal events similar to those activated by growth factors including stimulating kinases and small G proteins such as c-Src, Ras, and ERK1/2 (5Devary Y. Gottlieb R.A. Smeal T. Karin M. Cell. 1992; 71: 1081-1091Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 6Baas A.S. Berk B.C. Circ. Res. 1995; 77: 29-36Crossref PubMed Scopus (366) Google Scholar). Lander et al. (7Lander H.M. Ogiste J.S. Teng K.K. Novogrodsky A. J. Biol. Chem. 1995; 270: 21195-21198Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar) have reported that p21 Ras is a direct target of ROS and thus may be responsible for sensing redox status. In addition, Guyton et al. (8Guyton 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) have shown that H2O2-stimulated activation of ERK2 was abolished in PC12 cells by expression of dominant-negative Ras. These findings suggest that p21Ras may be an important mediator of ROS function. Previous studies have shown that c-Src is involved in signal events stimulated by ROS (5Devary Y. Gottlieb R.A. Smeal T. Karin M. Cell. 1992; 71: 1081-1091Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 9Abe J. Takahashi M. Ishida M. Lee J.-D. Berk B.C. J. Biol. Chem. 1997; 272: 20389-20394Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). It has been reported that Src family kinases and Ras are critical for ERK1/2 activation by H2O2 (10Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Tanaka M. Shiojima I. Hiroi Y. Yazaki Y. J. Clin. Invest. 1997; 100: 1813-1821Crossref PubMed Scopus (629) Google Scholar, 11Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Clermont-Goldschmidt P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1421) Google Scholar). The predominant pathway for ERK1/2 activation by angiotensin II and growth factors has also been proposed to involve Src-Ras-Raf-MEK1-ERK1/2 (12Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 13Ishida M. Ishida T. Thomas S. Berk B.C. Circ. Res. 1998; 82: 7-12Crossref PubMed Scopus (150) Google Scholar). However, it is not known how Src kinases mediate Ras activation by ROS. Several groups have suggested that JAK2 activates the Ras/Raf/ERK-signaling pathway and is required for the proliferative response initiated by many cytokines (14Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar). Han et al. (15Han Y. Leaman D.W. Watling D. Rogers N.C. Groner B. Kerr I.M. Wood W.I. Stark G.R. J. Biol. Chem. 1996; 271: 5947-5952Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) have reported that JAK2 is required to couple growth hormone receptor to pathways involving Shc. Shc is thought to function as an adapter molecule to recruit Grb2·Sos complexes to the activated receptor, which promotes formation of Ras-GTP (16Aronheim A. Engelberg D. Li N. al-Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (422) Google Scholar). In Src-transformed cells, JAK1 and JAK2 are constitutively tyrosine phosphorylated (17Campbell G.S. Yu C.L. Jove R. Carter-Su C. J. Biol. Chem. 1997; 272: 2591-2594Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) suggesting that H2O2-mediated Shc/Ras activation may be regulated by Src family kinases and JAK2. To determine the role of Src family kinases and JAK2 in H2O2-mediated Shc/Ras activation, we used specific JAK2 inhibitors and cells derived from animals deficient in c-Src and Fyn. We show here that activation of JAK2 by H2O2 is positively regulated by Fyn, but not by c-Src. Furthermore, we demonstrate that both Fyn and JAK2 are required for H2O2-mediated Shc tyrosine phosphorylation and activation of Ras. Thus, the Fyn-JAK2-Shc-Ras-signaling pathway described here may represent a new redox-sensitive mechanism. DISCUSSIONThe major findings of this study are first that H2O2 stimulates JAK2 in a Fyn-dependent and Src-independent manner, and second that JAK2 mediates phosphorylation of Shc and activation of Ras by H2O2. Redox-sensitive regulation of JAK2/Shc/Ras is thus a new function for Fyn. Data that support an essential role for Fyn and JAK2 in H2O2-mediated Shc/Ras activation include the following findings. 1) In Fyn−/− fibroblasts, there was no JAK2 tyrosine phosphorylation in response to H2O2. In contrast, in Src−/− fibroblasts, H2O2-mediated JAK2 tyrosine phosphorylation was not inhibited. 2) AG-490, a specific JAK2 inhibitor, prevented H2O2-mediated Shc tyrosine phosphorylation and Ras activation. 3) Likewise, Shc tyrosine phosphorylation and Ras activation by H2O2 were inhibited in Fyn−/− fibroblasts, but not in Src−/− fibroblasts. 4) Expression of Fyn, but not Src, in Fyn−/− cells restored the response of Shc and JAK2 to H2O2. Our results are the first to show that Fyn, but not Src, is involved specifically in oxidative stress-mediated JAK2 activation, which also regulates a Shc/Ras signaling pathway.Based on this study as well as previous work from our lab (6Baas A.S. Berk B.C. Circ. Res. 1995; 77: 29-36Crossref PubMed Scopus (366) Google Scholar, 9Abe J. Takahashi M. Ishida M. Lee J.-D. Berk B.C. J. Biol. Chem. 1997; 272: 20389-20394Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and other investigators (7Lander H.M. Ogiste J.S. Teng K.K. Novogrodsky A. J. Biol. Chem. 1995; 270: 21195-21198Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 25Rao G.N. Oncogene. 1996; 13: 713-719PubMed Google Scholar), we propose a scheme (Fig. 11) for ROS-mediated signal transduction leading to activation of Ras and ERK1/2. A novel aspect of this model is the specific role of Fyn, but not c-Src, to activate JAK2 and Ras. Previously we found that c-Src but not Fyn was required for H2O2-mediated BMK1/ERK5 activation in fibroblasts (9Abe J. Takahashi M. Ishida M. Lee J.-D. Berk B.C. J. Biol. Chem. 1997; 272: 20389-20394Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In contrast, in this study, we found that Fyn but not c-Src was required for H2O2-mediated JAK2 activation in fibroblasts. These results indicate that c-Src and Fyn have separate roles in ROS-mediated signal transduction. Campbell et al. (17Campbell G.S. Yu C.L. Jove R. Carter-Su C. J. Biol. Chem. 1997; 272: 2591-2594Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) have shown that the Janus kinases JAK1 and JAK2 (especially JAK1) are constitutively tyrosine phosphorylated in Src-transformed cells and suggested that JAK1, and possibly JAK2, are in an activated state in these cells. Furthermore, Uddin et al. (28Uddin S. Sher D.A. Alsayed Y. Pons S. Colamonici O.R. Fish E.N. White M.F. Platanias L.C. Biochem. Biophys. Res. Commun. 1997; 235: 83-88Crossref PubMed Scopus (44) Google Scholar) have shown that interferon-γ causes the SH2 domain of Fyn to interact with the activated form of interferon-γ-dependent JAK2. Future studies will be required to define the precise nature of the downstream substrates for c-Src and Fyn.In addition to activation of JAK2 by Fyn, this study demonstrates that H2O2-mediated Ras activation is partially dependent on Fyn and JAK2. Previous investigators have suggested an important relationship between two pathways used by cytokines and growth hormones to activate cells: the JAK2/STAT and the Ras/Raf/MEK/ERK pathway (14Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar). A key convergence point of these pathways may be activation of Raf. Conversion of Ras to its GTP-bound form results in the binding of Raf to Ras, and this interaction with activated Ras localizes Raf to the plasma membrane and is often the first step in Raf activation. However, other investigators have reported that tyrosine kinases, including members of the Src kinase family and JAK2 phosphorylate Raf, thereby enhancing its activity. For example, Marrero et al. (24Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) reported that angiotensin II and platelet-derived growth factor induced JAK2·Raf complex formation, Raf-1 tyrosine phosphorylation, and ERK1/2 kinase activity, which were dependent on JAK2 activity. Based on this study, we propose that JAK2 is "upstream" of the adapter protein Shc and regulates H2O2-mediated Ras activation (Fig. 11).The results for ERK1/2 activation suggest that H2O2 might activate two pathways, only one of which is dependent on Fyn and JAK2 (Fig. 11). In vascular smooth muscle cells, AG-490 inhibited the activation of ERK1/2 in response to either angiotensin II or platelet-derived growth factor (24Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Aikawa et al. (10Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Tanaka M. Shiojima I. Hiroi Y. Yazaki Y. J. Clin. Invest. 1997; 100: 1813-1821Crossref PubMed Scopus (629) Google Scholar) reported that when Csk, a negative regulator of Src tyrosine kinases was overexpressed, activation of ERK1/2 by H2O2 at 10 min was abolished. We examined the role of JAK2 and Fyn in H2O2-mediated ERK1/2 activation with AG-490 treatment and in Fyn−/− fibroblasts, and found that treatment with AG-490 or the absence of Fyn induced a temporal shift in activation of ERK1/2. However, the magnitude and duration of ERK1/2 activation were not altered. Of interest, McKenzie et al. (29McKenzie F.R. Pouyssegur J. J. Biol. Chem. 1996; 271: 13476-13483Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) have reported that pretreatment with prostaglandin E and isobutylmethylxanthine to elevate cAMP attenuated the ability of growth factors to stimulate ERK1/2, when measured 5 min after growth factor stimulation. However, similar to the effect of AG-490 and the absence of Fyn this inhibition was not apparent at late times and the magnitude of ERK1/2 activation was not decreased. Recently, York et al. (30York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar) have found that Rap1 mediates sustained ERK1/2 kinase activity induced by cAMP, so Rap1 could be a candidate to regulate this late phase activation of ERK1/2. Future studies will be required to determine the role of Rap1 in ROS-mediated signal transduction to ERK1/2.In summary, we have shown that JAK2 is activated by ROS in a Fyn-dependent manner. The fact that H2O2-mediated activation of JAK2 required Fyn, but not c-Src, suggests that these two Src family kinases serve different intracellular functions with respect to oxidative stress. In addition, the demonstration that Fyn/JAK2 regulates H2O2-mediated Shc tyrosine phosphorylation and Ras activation, suggests that Fyn/JAK2/Shc/Ras signaling pathway may involve novel intracellular mediators. The major findings of this study are first that H2O2 stimulates JAK2 in a Fyn-dependent and Src-independent manner, and second that JAK2 mediates phosphorylation of Shc and activation of Ras by H2O2. Redox-sensitive regulation of JAK2/Shc/Ras is thus a new function for Fyn. Data that support an essential role for Fyn and JAK2 in H2O2-mediated Shc/Ras activation include the following findings. 1) In Fyn−/− fibroblasts, there was no JAK2 tyrosine phosphorylation in response to H2O2. In contrast, in Src−/− fibroblasts, H2O2-mediated JAK2 tyrosine phosphorylation was not inhibited. 2) AG-490, a specific JAK2 inhibitor, prevented H2O2-mediated Shc tyrosine phosphorylation and Ras activation. 3) Likewise, Shc tyrosine phosphorylation and Ras activation by H2O2 were inhibited in Fyn−/− fibroblasts, but not in Src−/− fibroblasts. 4) Expression of Fyn, but not Src, in Fyn−/− cells restored the response of Shc and JAK2 to H2O2. Our results are the first to show that Fyn, but not Src, is involved specifically in oxidative stress-mediated JAK2 activation, which also regulates a Shc/Ras signaling pathway. Based on this study as well as previous work from our lab (6Baas A.S. Berk B.C. Circ. Res. 1995; 77: 29-36Crossref PubMed Scopus (366) Google Scholar, 9Abe J. Takahashi M. Ishida M. Lee J.-D. Berk B.C. J. Biol. Chem. 1997; 272: 20389-20394Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and other investigators (7Lander H.M. Ogiste J.S. Teng K.K. Novogrodsky A. J. Biol. Chem. 1995; 270: 21195-21198Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 25Rao G.N. Oncogene. 1996; 13: 713-719PubMed Google Scholar), we propose a scheme (Fig. 11) for ROS-mediated signal transduction leading to activation of Ras and ERK1/2. A novel aspect of this model is the specific role of Fyn, but not c-Src, to activate JAK2 and Ras. Previously we found that c-Src but not Fyn was required for H2O2-mediated BMK1/ERK5 activation in fibroblasts (9Abe J. Takahashi M. Ishida M. Lee J.-D. Berk B.C. J. Biol. Chem. 1997; 272: 20389-20394Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In contrast, in this study, we found that Fyn but not c-Src was required for H2O2-mediated JAK2 activation in fibroblasts. These results indicate that c-Src and Fyn have separate roles in ROS-mediated signal transduction. Campbell et al. (17Campbell G.S. Yu C.L. Jove R. Carter-Su C. J. Biol. Chem. 1997; 272: 2591-2594Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) have shown that the Janus kinases JAK1 and JAK2 (especially JAK1) are constitutively tyrosine phosphorylated in Src-transformed cells and suggested that JAK1, and possibly JAK2, are in an activated state in these cells. Furthermore, Uddin et al. (28Uddin S. Sher D.A. Alsayed Y. Pons S. Colamonici O.R. Fish E.N. White M.F. Platanias L.C. Biochem. Biophys. Res. Commun. 1997; 235: 83-88Crossref PubMed Scopus (44) Google Scholar) have shown that interferon-γ causes the SH2 domain of Fyn to interact with the activated form of interferon-γ-dependent JAK2. Future studies will be required to define the precise nature of the downstream substrates for c-Src and Fyn. In addition to activation of JAK2 by Fyn, this study demonstrates that H2O2-mediated Ras activation is partially dependent on Fyn and JAK2. Previous investigators have suggested an important relationship between two pathways used by cytokines and growth hormones to activate cells: the JAK2/STAT and the Ras/Raf/MEK/ERK pathway (14Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar). A key convergence point of these pathways may be activation of Raf. Conversion of Ras to its GTP-bound form results in the binding of Raf to Ras, and this interaction with activated Ras localizes Raf to the plasma membrane and is often the first step in Raf activation. However, other investigators have reported that tyrosine kinases, including members of the Src kinase family and JAK2 phosphorylate Raf, thereby enhancing its activity. For example, Marrero et al. (24Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) reported that angiotensin II and platelet-derived growth factor induced JAK2·Raf complex formation, Raf-1 tyrosine phosphorylation, and ERK1/2 kinase activity, which were dependent on JAK2 activity. Based on this study, we propose that JAK2 is "upstream" of the adapter protein Shc and regulates H2O2-mediated Ras activation (Fig. 11). The results for ERK1/2 activation suggest that H2O2 might activate two pathways, only one of which is dependent on Fyn and JAK2 (Fig. 11). In vascular smooth muscle cells, AG-490 inhibited the activation of ERK1/2 in response to either angiotensin II or platelet-derived growth factor (24Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Aikawa et al. (10Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Tanaka M. Shiojima I. Hiroi Y. Yazaki Y. J. Clin. Invest. 1997; 100: 1813-1821Crossref PubMed Scopus (629) Google Scholar) reported that when Csk, a negative regulator of Src tyrosine kinases was overexpressed, activation of ERK1/2 by H2O2 at 10 min was abolished. We examined the role of JAK2 and Fyn in H2O2-mediated ERK1/2 activation with AG-490 treatment and in Fyn−/− fibroblasts, and found that treatment with AG-490 or the absence of Fyn induced a temporal shift in activation of ERK1/2. However, the magnitude and duration of ERK1/2 activation were not altered. Of interest, McKenzie et al. (29McKenzie F.R. Pouyssegur J. J. Biol. Chem. 1996; 271: 13476-13483Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) have reported that pretreatment with prostaglandin E and isobutylmethylxanthine to elevate cAMP attenuated the ability of growth factors to stimulate ERK1/2, when measured 5 min after growth factor stimulation. However, similar to the effect of AG-490 and the absence of Fyn this inhibition was not apparent at late times and the magnitude of ERK1/2 activation was not decreased. Recently, York et al. (30York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar) have found that Rap1 mediates sustained ERK1/2 kinase activity induced by cAMP, so Rap1 could be a candidate to regulate this late phase activation of ERK1/2. Future studies will be required to determine the role of Rap1 in ROS-mediated signal transduction to ERK1/2. In summary, we have shown that JAK2 is activated by ROS in a Fyn-dependent manner. The fact that H2O2-mediated activation of JAK2 required Fyn, but not c-Src, suggests that these two Src family kinases serve different intracellular functions with respect to oxidative stress. In addition, the demonstration that Fyn/JAK2 regulates H2O2-mediated Shc tyrosine phosphorylation and Ras activation, suggests that Fyn/JAK2/Shc/Ras signaling pathway may involve novel intracellular mediators. We thank Dr. D. Shalloway for providing the GST-RBD construct and Drs. H. Umemori and Tadashi Yamamoto for providing B-Fyn and Src cDNA construct. We also thank Drs. C. Yan, H. Ueba, M. Okuda, B. Gallis, and G. Daum for their invaluable assistance and critical reading of this manuscript.
Referência(s)