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• W2160770127 abstract "Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that transduce signals from the cell membrane to the nucleus upon activation by tyrosine phosphorylation. Several protein-tyrosine kinases can induce phosphorylation of STATs in cells, including Janus kinase (JAK) and Src family kinases. One STAT family member, Stat3, is constitutively activated in Src-transformed NIH3T3 cells and is required for cell transformation. However, it is not entirely clear whether Src kinase can phosphorylate Stat3 directly or through another pathway, such as JAK family kinases. To address this question, we investigated the phosphorylation of STATs in baculovirus-infected Sf-9 insect cells in the presence of Src. Our results show that Src can tyrosine-phosphorylate Stat1 and Stat3 but not Stat5 in this system. The phosphorylated Stat1 and Stat3 proteins are functionally activated, as measured by their abilities to specifically bind DNA oligonucleotide probes. In addition, the JAK family member Jak1 efficiently phosphorylates Stat1 but not Stat3 in Sf-9 cells. By contrast, we observe that AG490, a JAK family-selective inhibitor, and dominant negative Jak1 protein can significantly inhibit Stat3-induced DNA binding activity as well as Stat3-mediated gene activation in NIH3T3 cells. Furthermore, wild-type or kinase-inactive platelet-derived growth factor receptor enhances Stat3 activation by v-Src, consistent with the receptor serving a scaffolding function for recruitment and activation of Stat3. Our results demonstrate that Src kinase is capable of activating STATs in Sf-9 insect cells without expression of JAK family members; however, Jak1 and platelet-derived growth factor receptor are required for maximal Stat3 activation by Src kinase in mammalian cells. Based on these findings, we propose a model in which Jak1 serves to recruit Stat3 to a receptor complex with Src kinase, which in turn directly phosphorylates and activates Stat3 in Src-transformed fibroblasts. Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that transduce signals from the cell membrane to the nucleus upon activation by tyrosine phosphorylation. Several protein-tyrosine kinases can induce phosphorylation of STATs in cells, including Janus kinase (JAK) and Src family kinases. One STAT family member, Stat3, is constitutively activated in Src-transformed NIH3T3 cells and is required for cell transformation. However, it is not entirely clear whether Src kinase can phosphorylate Stat3 directly or through another pathway, such as JAK family kinases. To address this question, we investigated the phosphorylation of STATs in baculovirus-infected Sf-9 insect cells in the presence of Src. Our results show that Src can tyrosine-phosphorylate Stat1 and Stat3 but not Stat5 in this system. The phosphorylated Stat1 and Stat3 proteins are functionally activated, as measured by their abilities to specifically bind DNA oligonucleotide probes. In addition, the JAK family member Jak1 efficiently phosphorylates Stat1 but not Stat3 in Sf-9 cells. By contrast, we observe that AG490, a JAK family-selective inhibitor, and dominant negative Jak1 protein can significantly inhibit Stat3-induced DNA binding activity as well as Stat3-mediated gene activation in NIH3T3 cells. Furthermore, wild-type or kinase-inactive platelet-derived growth factor receptor enhances Stat3 activation by v-Src, consistent with the receptor serving a scaffolding function for recruitment and activation of Stat3. Our results demonstrate that Src kinase is capable of activating STATs in Sf-9 insect cells without expression of JAK family members; however, Jak1 and platelet-derived growth factor receptor are required for maximal Stat3 activation by Src kinase in mammalian cells. Based on these findings, we propose a model in which Jak1 serves to recruit Stat3 to a receptor complex with Src kinase, which in turn directly phosphorylates and activates Stat3 in Src-transformed fibroblasts. signal transducers and activators of transcription Janus kinase platelet-derived growth factor PDGF receptor phosphate-buffered saline electrophoretic mobility shift assay Signal transducers and activators of transcription (STATs)1 are a family of latent cytoplasmic transcription factors that are activated in response to various extracellular polypeptide ligands, including cytokines and growth factors (1Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar[email protected] , 2Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar). Upon cytokine stimulation, cytokine receptors dimerize and thereby activate receptor-associated tyrosine kinases of the Janus kinase (JAK) family (3Leonard W.J. O'Shea J.J. Annu. Rev. Immunol. 1998; 16: 293-322Crossref PubMed Scopus (1460) Google Scholar). The activated JAKs induce STAT activation by a two-step mechanism. First, JAKs phosphorylate receptor tyrosine residues, which in turn become docking sites for the recruitment of cytoplasmic STAT proteins. Second, the recruited STAT proteins are directly phosphorylated by the receptor-associated JAKs. Activated STATs then dimerize and translocate to the nucleus, where they bind to specific promoter sequences of target genes and induce transcription (4Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3346) Google Scholar). This signaling mechanism is often referred to as the JAK-STAT pathway (2Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar, 3Leonard W.J. O'Shea J.J. Annu. Rev. Immunol. 1998; 16: 293-322Crossref PubMed Scopus (1460) Google Scholar).Seven mammalian STAT family members have been identified and characterized, and they share similar structural features as well as activation mechanism (1Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar, 4Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3346) Google Scholar). The different STATs are involved in mediating a variety of biological functions in diverse cell types. For example, Stat1 is critical for interferon functions as well as innate immunity (5Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar, 6Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1383) Google Scholar), while Stat3 is required for IL-6 signaling in hematopoietic cells as well as anti-apoptosis (7Fukada T. Hibi M. Yamanaka Y. Takahashi-Tezuka M. Fujitani Y. Yamaguchi T. Nakajima K. Hirano T. Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 8Catlett-Falcone R. Landowski T.H. Oshiro M.M. Turkson J. Levitzki A. Savino R. Ciliberto G. Moscinski L. Fernandez-Luna J.L. Nunez G. Dalton W.S. Jove R. Immunity. 1999; 10: 105-115Abstract Full Text Full Text PDF PubMed Scopus (1441) Google Scholar, 9Hirano T. Nakajima K. Hibi M. Cytokine Growth Factor Rev. 1997; 8: 241-252Crossref PubMed Scopus (330) Google Scholar). Targeted disruption of the mouse Stat3 gene is embryonic lethal (10Takeda K. Noguchi K. Shi W. Tanaka T. Matsumoto M. Yoshida N. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3801-3804Crossref PubMed Scopus (1089) Google Scholar), which demonstrates the importance of Stat3 in the development of mouse embryos. In addition, Stat5 has been shown to be important in lactation and hematopoiesis (11Gouilleux F. Moritz D. Humar M. Moriggl R. Berchtold S. Groner B. Endocrinology. 1995; 136: 5700-5708Crossref PubMed Scopus (67) Google Scholar, 12Frank D.A. Mol. Med. 1999; 5: 432-456Crossref PubMed Google Scholar, 13Akira S. Stem Cells. 1999; 17: 138-146Crossref PubMed Scopus (270) Google Scholar).STAT activation has also been observed to be induced by epidermal growth factor and PDGF receptors with intrinsic tyrosine kinase activities (14Fu X.Y. Zhang J.J. Cell. 1993; 74: 1135-1145Abstract Full Text PDF PubMed Scopus (272) Google Scholar, 15Ruff-Jamison S. Chen K. Cohen S. Science. 1993; 261: 1733-1736Crossref PubMed Scopus (240) Google Scholar, 16Zhong Z. Wen Z. Darnell J.E. Science. 1994; 264: 95-98Crossref PubMed Scopus (1692) Google Scholar, 17Leaman D.W. Leung S. Li X. Stark G.R. FASEB J. 1996; 10: 1578-1588Crossref PubMed Scopus (271) Google Scholar). While receptor tyrosine kinases may directly phosphorylate STATs, some reports suggest that JAKs are involved in PDGF-induced STAT activation. For example, the JAK family kinases, Jak1, Jak2, and Tyk2, are activated in cells stimulated with PDGF (18Vignais M.L. Sadowski H.B. Watling D. Rogers N.C. Gilman M. Mol. Cell. Biol. 1996; 16: 1759-1769Crossref PubMed Scopus (218) Google Scholar). Furthermore, recent studies suggest that Stat3 activation by PDGF receptor is mediated by JAK kinases but that Stat1 activation is not (19Vignais M.L. Gilman M. Mol. Cell. Biol. 1999; 19: 3727-3735Crossref PubMed Scopus (53) Google Scholar). PDGF can also activate the nonreceptor tyrosine kinase c-Src (20Gould K.L. Hunter T. Mol. Cell. Biol. 1988; 8: 3345-3356Crossref PubMed Scopus (171) Google Scholar,21Courtneidge S.A. Fumagalli S. Koegl M. Superti-Furga G. Twamley-Stein G.M. Dev. Suppl. 1993; : 57-64PubMed Google Scholar), and it has been suggested that c-Src activates Stat1 and Stat3 in PDGF-stimulated murine fibroblast cells (22Cirri P. Chiarugi P. Marra F. Raugei G. Camici G. Manao G. Ramponi G. Biochem. Biophys. Res. Commun. 1997; 239: 493-497Crossref PubMed Scopus (56) Google Scholar). These findings indicate that STAT activation by polypeptide hormones involves nonreceptor tyrosine kinases in addition to the intrinsic tyrosine kinase of their receptors.We and others have observed the constitutive activation of Stat3 in v-Src-transformed cells (23Yu C.L. Meyer D.J. Campbell G.S. Larner A.C. Carter-Su C. Schwartz J. Jove R. Science. 1995; 269: 81-83Crossref PubMed Scopus (818) Google Scholar, 24Cao X. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Crossref PubMed Scopus (338) Google Scholar, 25Chaturvedi P. Sharma S. Reddy E.P. Mol. Cell. Biol. 1997; 17: 3295-3304Crossref PubMed Scopus (99) Google Scholar). Further studies demonstrated that this Stat3 activation results in gene activation and is essential for v-Src transformation (26Turkson J. Bowman T. Garcia R. Caldenhoven E. De Groot R.P. Jove R. Mol. Cell. Biol. 1998; 18: 2545-2552Crossref PubMed Scopus (590) Google Scholar, 27Bromberg J.F. Horvath C.M. Besser D. Lathem W.W. Darnell Jr., J.E. Mol. Cell. Biol. 1998; 18: 2553-2558Crossref PubMed Scopus (568) Google Scholar). Although direct activation of Stat3 by Src has been suggested (24Cao X. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Crossref PubMed Scopus (338) Google Scholar, 28Chaturvedi P. Reddy M.V. Reddy E.P. Oncogene. 1998; 16: 1749-1758Crossref PubMed Scopus (125) Google Scholar), the mechanism of Stat3 activation is not entirely clear (29Garcia R. Jove R. J. Biomed. Sci. 1998; 5: 79-85Crossref PubMed Scopus (183) Google Scholar) and may employ different mechanisms that are dependent on the cell type. In v-Src-transformed mouse fibroblasts, Jak1 and, to a much lesser extent, Jak2 are also constitutively activated (30Campbell 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). To investigate the mechanism of Stat3 activation by Src, we examined STAT activation by Src and Jak1 expressed from recombinant baculoviruses in Sf-9 insect cells as well as the role of Jak1 in Stat3 activation in mouse fibroblasts transformed by v-Src.Here we report that Stat1 and Stat3 are tyrosine-phosphorylated in Sf-9 cells by activated Src in the absence of other mammalian tyrosine kinases. The phosphorylated STAT proteins bind to specific DNA sequences in gel shift assays, indicating that this phosphorylation induces functional activation of the STAT proteins. Furthermore, Jak1 enhances activation of Stat1 but not Stat3 when co-expressed with Src in Sf-9 cells, and the phosphorylation level of Jak1 is also increased with the expression of Src. By contrast, in NIH3T3 cells, Jak1 activity is required for maximal Stat3-mediated gene induction. In addition, activation of Stat3 by Src in mammalian cells is enhanced by the PDGF receptor independently of receptor kinase activity, consistent with a scaffolding function for the receptor. Our results indicate that, although Src can directly activate Stat3 in insect cells, Jak1 plays an important role in the activation of Stat3 in Src-transformed mouse fibroblasts. These findings support a model in which Src and Jak1 cooperate together with the PDGF receptor and possibly other receptors to activate Stat3 in the context of oncogenesis.DISCUSSIONWhile constitutive activation of Stat3 signaling has previously been shown to be required for cell transformation by the oncogenic Src tyrosine kinase (26Turkson J. Bowman T. Garcia R. Caldenhoven E. De Groot R.P. Jove R. Mol. Cell. Biol. 1998; 18: 2545-2552Crossref PubMed Scopus (590) Google Scholar, 27Bromberg J.F. Horvath C.M. Besser D. Lathem W.W. Darnell Jr., J.E. Mol. Cell. Biol. 1998; 18: 2553-2558Crossref PubMed Scopus (568) Google Scholar), the mechanism of Stat3 activation by Src was not entirely clear. Our results presented here demonstrate that maximal activation of Stat3 requires Jak1 and PDGF-R in v-Src-transformed NIH3T3 cells, indicating that the mechanism of Stat3 activation induced by oncogenic Src is more complex than a simple interaction between Stat3 and Src. However, in Sf-9 insect cells, Src is much more efficient than Jak1 at phosphorylating Stat3, arguing that Jak1 is not acting as an intermediary kinase between Src and Stat3. Furthermore, the role of PDGF-R in Stat3 activation by Src in NIH3T3 cells does not require the receptor's intrinsic tyrosine kinase activity. Based on our findings, we propose that the oncogenic Src kinase activates Jak1 kinase, which in turn phosphorylates tyrosine sites on PDGF-R and possibly other receptors that provide docking sites for Stat3 (Fig.11). In this model, activation of Jak1 is required for the recruitment of Stat3 proteins into a receptor complex with Src kinase, which then directly phosphorylates Stat3 at Tyr-705. Our model is consistent with the earlier findings that Stat3 is co-immunoprecipitated with Src (24Cao X. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Crossref PubMed Scopus (338) Google Scholar, 25Chaturvedi P. Sharma S. Reddy E.P. Mol. Cell. Biol. 1997; 17: 3295-3304Crossref PubMed Scopus (99) Google Scholar) and that Jak1 is constitutively activated in Src transformed cells (30Campbell 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).Our data (Fig. 1) demonstrate that Src can efficiently activate Stat1 and Stat3, but not Stat5A, in Sf-9 cells in the absence of other mammalian kinases, suggesting that Stat1 and Stat3 are immediate substrates of Src. Although we cannot quantitatively compare the phosphorylation levels of Stat1 and Stat3 with each other, since different antibodies are used for each protein, we can determine the phosphorylation status of these STAT proteins (Fig. 3). Either Stat5A is not a substrate of Src or else an additional component is required for its activation. This observation is consistent with our previous findings that c-Fes activates Stat3 but not Stat5A in Sf-9 cells (39Nelson K.L. Rogers J.A. Bowman T.L. Jove R. Smithgall T.E. J. Biol. Chem. 1998; 273: 7072-7077Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and that v-Src does not activate Stat5 in NIH3T3 cells (43Garcia R., Yu, C.L. Hudnall A. Catlett R. Nelson K.L. Smithgall T. Fujita D.J. Ethier S.P. Jove R. Cell Growth Differ. 1997; 8: 1267-1276PubMed Google Scholar). Furthermore, the observation that baculovirus-expressed Bcr-Abl can activate Stat5A rules out the possibility that Stat5A expressed in insect cells is resistant to activation (39Nelson K.L. Rogers J.A. Bowman T.L. Jove R. Smithgall T.E. J. Biol. Chem. 1998; 273: 7072-7077Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Although we could not exclude the possible involvement of an insect equivalent of mammalian JAK kinase, the endogenous insect JAK counterpart would probably be expressed at very low levels compared with baculovirus-overexpressed STAT and Src proteins. We did not observe a rate-limiting step in STAT activation by Src in insect cells, suggesting that low levels of endogenous insect kinases are not involved in STAT activation by overexpressed Src. Moreover, co-expression of Jak1 did not significantly increase Stat3 activation by Src in Sf-9 cells, indicating that JAK family kinases are not a factor in Stat3 activation by Src in insect cells.Several lines of evidence support our model shown in Fig. 11. First, we show in insect cells that Src is able to activate Stat1 and Stat3 selectively, consistent with Src being the immediate upstream kinase for phosphorylation of Stat1 and Stat3. Second, Jak1 is unable to phosphorylate Stat3 efficiently, which is in striking contrast to the result that it phosphorylates Stat1 equally as well as Src does. This finding suggests that Jak1 is unlikely to be the kinase for Stat3 activation acting downstream of Src kinase in v-Src-transformed cells. Third, the JAK-selective inhibitor, AG490, and the Src-selective inhibitor, PD180970, significantly inhibit Stat3 DNA binding activity in v-Src-transformed NIH3T3 cells, which indicates a requirement for both Src and JAKs in Stat3 activation. Fourth, Jak1 is hyperphosphorylated in the presence of active Src in insect cells and NIH3T3 fibroblasts. In addition, inhibition of Src kinase activity by PD180970 abolishes Jak1 tyrosine phosphorylation in v-Src-transformed NIH3T3 cells. Since the tyrosine phosphorylation level of Jak1 has been found to correlate with its kinase activity (30Campbell 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), these results suggest that Jak1 is directly activated by Src in v-Src transformed NIH3T3 cells. Fifth, both AG490 and dominant negative Jak1 inhibit Stat3-mediated gene regulation, further establishing a requirement of Jak1 for Stat3 activation by Src in NIH3T3 cells. Sixth, the lack of Stat1 activation in v-Src-transformed fibroblast cells may be due to the inaccessibility (possibly resulting from the lack of Stat1-specific docking sites) to the kinases, which is consistent with the notion that membrane-bound receptors contribute to the specificity of STAT signaling (4Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3346) Google Scholar). Previous studies (24Cao X. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Crossref PubMed Scopus (338) Google Scholar) have shown that Stat1 can be activated by interferon-γ stimulation of v-Src-transformed NIH3T3 cells, indicating that the normal Jak1-Stat1 pathway is intact in v-Src-transformed fibroblasts. These findings point to different mechanisms for activation of Stat1 and Stat3. One plausible reason why Stat1 signaling is down-regulated in v-Src-transformed cells may be that Stat1 is involved in growth-inhibitory and proapoptosis functions (51Bromberg J.F. Horvath C.M. Wen Z. Schreiber R.D. Darnell J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7673-7678Crossref PubMed Scopus (440) Google Scholar, 52Chin Y.E. Kitagawa M. Kuida K. Flavell R.A. Fu X.Y. Mol. Cell. Biol. 1997; 17: 5328-5337Crossref PubMed Scopus (464) Google Scholar).In myeloid cells stimulated with interleukin-3, c-Src but not JAKs is required for activation of Stat3 (28Chaturvedi P. Reddy M.V. Reddy E.P. Oncogene. 1998; 16: 1749-1758Crossref PubMed Scopus (125) Google Scholar), consistent with a direct role for c-Src in Stat3 activation. Normal c-Src has been shown to be activated in epidermal growth factor- and PDGF-stimulated cells and to interact with epidermal growth factor and PDGF receptors (17Leaman D.W. Leung S. Li X. Stark G.R. FASEB J. 1996; 10: 1578-1588Crossref PubMed Scopus (271) Google Scholar, 21Courtneidge S.A. Fumagalli S. Koegl M. Superti-Furga G. Twamley-Stein G.M. Dev. Suppl. 1993; : 57-64PubMed Google Scholar, 29Garcia R. Jove R. J. Biomed. Sci. 1998; 5: 79-85Crossref PubMed Scopus (183) Google Scholar,53Wang Y. Wharton W. Garcia R. Kraker A. Jove R. Pledger W.J. Oncogene. 2000; 19: 2075-2085Crossref PubMed Scopus (99) Google Scholar). Furthermore, one recent study (54Olayioye M.A. Beuvink I. Horsch K. Daly J.M. Hynes N.E. J. Biol. Chem. 1999; 274: 17209-17218Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) reported that the activation of STAT proteins induced by epidermal growth factor receptor is mediated by c-Src. Using a cell-free system, another recent study provided evidence for differences between Stat1 and Stat3 activation by PDGF (19Vignais M.L. Gilman M. Mol. Cell. Biol. 1999; 19: 3727-3735Crossref PubMed Scopus (53) Google Scholar). In particular, JAK kinases are indispensable for Stat3 activation induced by PDGF but not for Stat1 activation; however, these studies did not address the involvement of c-Src in activation of Stat1 and Stat3 in response to PDGF stimulation (19Vignais M.L. Gilman M. Mol. Cell. Biol. 1999; 19: 3727-3735Crossref PubMed Scopus (53) Google Scholar). Other studies have also suggested that c-Src activates Stat1 and Stat3 in PDGF-stimulated NIH3T3 cells (22Cirri P. Chiarugi P. Marra F. Raugei G. Camici G. Manao G. Ramponi G. Biochem. Biophys. Res. Commun. 1997; 239: 493-497Crossref PubMed Scopus (56) Google Scholar, 53Wang Y. Wharton W. Garcia R. Kraker A. Jove R. Pledger W.J. Oncogene. 2000; 19: 2075-2085Crossref PubMed Scopus (99) Google Scholar), and a multiprotein complex containing PDGF-R, c-Src, and STAT proteins has been detected (53Wang Y. Wharton W. Garcia R. Kraker A. Jove R. Pledger W.J. Oncogene. 2000; 19: 2075-2085Crossref PubMed Scopus (99) Google Scholar). Moreover, there is evidence that Src and JAK family kinases are both required for PDGF-mediated Stat3 signaling in normal NIH3T3 cells (Ref. 53Wang Y. Wharton W. Garcia R. Kraker A. Jove R. Pledger W.J. Oncogene. 2000; 19: 2075-2085Crossref PubMed Scopus (99) Google Scholar; our unpublished results). Therefore, cooperation among Src, JAKs, and PDGF-R may be required for Stat3 activation in normal growth factor signaling events. We have shown that the expression of PDGF-R is essential for maximal Stat3 activation induced by v-Src in mammalian cells (Fig. 10). Importantly, the intrinsic tyrosine kinase activity of PDGF-R is not required for Stat3 activation by v-Src, consistent with the receptor's proposed role as a scaffolding complex for recruitment of Stat3 into close proximity of Src. However, our findings do not exclude the possibility that other receptors may also provide this scaffold function for recruitment and activation of Stat3 by v-Src.The requirement of JAK kinases for STAT activation has been previously observed in other oncogenic signaling events (55Catlett-Falcone R. Dalton W.S. Jove R. Curr. Opin. Oncol. 1999; 11: 490-496Crossref PubMed Scopus (163) Google Scholar). The inhibitor of JAK family kinases, AG490, blocks IL-6-dependent Stat3 activation in human multiple myeloma tumor cells (8Catlett-Falcone R. Landowski T.H. Oshiro M.M. Turkson J. Levitzki A. Savino R. Ciliberto G. Moscinski L. Fernandez-Luna J.L. Nunez G. Dalton W.S. Jove R. Immunity. 1999; 10: 105-115Abstract Full Text Full Text PDF PubMed Scopus (1441) Google Scholar). In human mycosis fungoides tumor cell lines, Jak3 and Tyk2 are in a complex with Stat3 and are required for Stat3 activation as well as for cell growth (56Nielsen M. Kaltoft K. Nordahl M. Ropke C. Geisler C. Mustelin T. Dobson P. Svejgaard A. Odum N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6764-6769Crossref PubMed Scopus (210) Google Scholar). In the case of v-Abl-transformed cells, Jak1 is required for proliferation in BAF/3 cells (57Danial N.N. Losman J.A. Lu T. Yip N. Krishnan K. Krolewski J. Goff S.P. Wang J.Y. Rothman P.B. Mol. Cell. Biol. 1998; 18: 6795-6804Crossref PubMed Scopus (61) Google Scholar). Direct interaction of Jak1 and v-Abl has been observed, and this interaction is essential for STAT activation (57Danial N.N. Losman J.A. Lu T. Yip N. Krishnan K. Krolewski J. Goff S.P. Wang J.Y. Rothman P.B. Mol. Cell. Biol. 1998; 18: 6795-6804Crossref PubMed Scopus (61) Google Scholar). In v-Src-transformed NIH3T3 cells, Jak1, but not Jak2 or Tyk2, is important in Stat3 activation by v-Src, since dominant negative Jak2 and Tyk2 did not affect Stat3-mediated gene activation by v-Src. 2Y. Zhang, J. Turkson, C. Carter-Su, T. Smithgall, A. Levitzki, A. Kraker, J. J. Krolewski, P. Medveczky, and R. Jove, unpublished results. By contrast, JAK kinases are neither activated nor required for Bcr-Abl-induced STAT activation (12Frank D.A. Mol. Med. 1999; 5: 432-456Crossref PubMed Google Scholar), and the activated Lck kinase can directly phosphorylate Stat3 (58Lund T.C. Coleman C. Horvath E. Sefton B.M. Jove R. Medveczky M.M. Medveczky P.G. Cell. Signal. 1999; 11: 789-796Crossref PubMed Scopus (38) Google Scholar). These findings suggest that the requirement of JAK family kinases is dependent on the specific cell type as well as the particular oncogenic signals involved.Consistent with the results presented here, recent studies demonstrate that both Src and JAK tyrosine kinases are required for constitutive Stat3 activation in human breast cancer cell lines. 3R. Garcia, J. Sun, T. L. Bowman, G. Niu, Y. Zhang, S. Minton, C. A. Muro-Cacho, N. N. Ku, R. Falcone, C. Cox, A. Kraker, A. Levitzki, S. Parsons, S. M. Sebti, and R. Jove, submitted for publication. Inhibition of Src or JAKs by PD180970 or AG490, respectively, results in inactivation of Stat3 DNA binding activity and growth inhibition of these breast cancer cells. Thus, the cooperation between Src and JAK tyrosine kinases is important for the constitutive Stat3 activation in various cell types, including human tumor cell lines. Our findings provide evidence for a novel mechanism of Stat3 activation that requires cooperation of Src and Jak1 kinase in v-Src-transformed mouse fibroblasts. In this model, Jak1 has a critical role in recruiting Stat3 to a receptor complex with Src kinase, which in turn directly phosphorylates Stat3. This model may be relevant not only to oncogenic signaling by tyrosine kinases but also to normal growth factor receptor signaling. Signal transducers and activators of transcription (STATs)1 are a family of latent cytoplasmic transcription factors that are activated in response to various extracellular polypeptide ligands, including cytokines and growth factors (1Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar[email protected] , 2Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar). Upon cytokine stimulation, cytokine receptors dimerize and thereby activate receptor-associated tyrosine kinases of the Janus kinase (JAK) family (3Leonard W.J. O'Shea J.J. Annu. Rev. Immunol. 1998; 16: 293-322Crossref PubMed Scopus (1460) Google Scholar). The activated JAKs induce STAT activation by a two-step mechanism. First, JAKs phosphorylate receptor tyrosine residues, which in turn become docking sites for the recruitment of cytoplasmic STAT proteins. Second, the recruited STAT proteins are directly phosphorylated by the receptor-associated JAKs. Activated STATs then dimerize and translocate to the nucleus, where they bind to specific promoter sequences of target genes and induce transcription (4Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3346) Google Scholar). This signaling mechanism is often referred to as the JAK-STAT pathway (2Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar, 3Leonard W.J. O'Shea J.J. Annu. Rev. Immunol. 1998; 16: 293-322Crossref PubMed Scopus (1460) Google Scholar). Seven mammalian STAT family members have been identified and characterized, and they share similar structural features as well as activation mechanism (1Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar, 4Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3346) Google Scholar). The different STATs are involved in mediating a variety of biological functions in diverse cell types. For example, Stat1 is critical for interferon functions as well as innate immunity (5Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar, 6Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1383) Google Scholar), while Stat3 is required for IL-6 signaling in hematopoietic cells as well as anti-apoptosis (7Fukada T. Hibi M. Yamanaka Y. Takahashi-Tezuka M. Fujitani Y. Yamaguchi T. Nakajima K. Hirano T. Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 8Catlett-Falcone R. Landowski T.H. Oshiro M.M. Turkson J. Levitzki A. Savino R. Ciliberto G. Moscinski L. Fernandez-Luna J.L. Nunez G. Dalton W.S. Jove R. Immuni" @default.
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