Activation of STAT3 by the Src Family Kinase Hck Requires a Functional SH3 Domain
2002; Elsevier BV; Volume: 277; Issue: 47 Linguagem: Inglês
10.1074/jbc.m204255200
ISSN1083-351X
AutoresSteven J. Schreiner, Anthony P. Schiavone, Thomas E. Smithgall,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoSTAT3 is a member of a family of transcription factors with Src homology 2 (SH2) domains that are activated by tyrosine phosphorylation in response to a wide variety of cytokines and growth factors. In this study, we investigated the mechanism of STAT3 activation by the Src family of nonreceptor tyrosine kinases, which have been linked to STAT activation in both normal and transformed cell types. Using Sf-9 insect cells, we demonstrate direct STAT3 tyrosine phosphorylation and stimulation of DNA binding activity by five members of the Src kinase family (Src, Hck, Lyn, Fyn, and Fgr). We also observed stable STAT3·Src family kinase complex formation in this system. Recombinant Src family kinase SH3 domains were sufficient for interaction with STAT3, suggesting a mechanistic basis for the Src kinase-STAT3 interaction. To test the contribution of Src family kinase SH3 domains to the recruitment and activation of STAT3 in vivo, we used Rat-2 fibroblasts expressing activated mutants of the myeloid Src family member Hck. Transformation of fibroblasts by an activated Hck mutant lacking the negative regulatory tail tyrosine residue (Hck-YF) induced strong DNA binding activity of endogenous STAT3. Inactivation of Hck SH3 function by Ala replacement of a conserved Trp residue (W93A mutant) completely abolished STAT3 activation by Hck-YF and reduced transforming activity by 50% without affecting Hck kinase activity. Finally, overexpression of STAT3 in Rat-2 cells transiently stimulated Hck and c-Src kinase activity in the absence of extracellular signals, an effect that was dependent upon a putative SH3 binding motif in STAT3. These results support a model in which Src family kinases recruit STAT3 through an SH3-dependent mechanism, resulting in transient kinase activation and STAT3 phosphorylation. STAT3 is a member of a family of transcription factors with Src homology 2 (SH2) domains that are activated by tyrosine phosphorylation in response to a wide variety of cytokines and growth factors. In this study, we investigated the mechanism of STAT3 activation by the Src family of nonreceptor tyrosine kinases, which have been linked to STAT activation in both normal and transformed cell types. Using Sf-9 insect cells, we demonstrate direct STAT3 tyrosine phosphorylation and stimulation of DNA binding activity by five members of the Src kinase family (Src, Hck, Lyn, Fyn, and Fgr). We also observed stable STAT3·Src family kinase complex formation in this system. Recombinant Src family kinase SH3 domains were sufficient for interaction with STAT3, suggesting a mechanistic basis for the Src kinase-STAT3 interaction. To test the contribution of Src family kinase SH3 domains to the recruitment and activation of STAT3 in vivo, we used Rat-2 fibroblasts expressing activated mutants of the myeloid Src family member Hck. Transformation of fibroblasts by an activated Hck mutant lacking the negative regulatory tail tyrosine residue (Hck-YF) induced strong DNA binding activity of endogenous STAT3. Inactivation of Hck SH3 function by Ala replacement of a conserved Trp residue (W93A mutant) completely abolished STAT3 activation by Hck-YF and reduced transforming activity by 50% without affecting Hck kinase activity. Finally, overexpression of STAT3 in Rat-2 cells transiently stimulated Hck and c-Src kinase activity in the absence of extracellular signals, an effect that was dependent upon a putative SH3 binding motif in STAT3. These results support a model in which Src family kinases recruit STAT3 through an SH3-dependent mechanism, resulting in transient kinase activation and STAT3 phosphorylation. The Src family of protein-tyrosine kinases regulates a diverse array of cellular processes in both normal and transformed cells, including proliferation, survival, differentiation, adhesion, and motility (1Lowell C.A. Soriano P. Genes Dev. 1996; 10: 1845-1857Google Scholar, 2Corey S.J. Anderson S.M. Blood. 1999; 93: 1-14Google Scholar, 3Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Google Scholar, 4Abram C.L. Courtneidge S.A. Exp. Cell Res. 2000; 254: 1-13Google Scholar, 5Parsons J.T. Parsons S.J. Curr. Opin. Cell Biol. 1997; 9: 187-192Google Scholar). Of the eight members of the Src kinase family expressed in mammalian cells, several are ubiquitously expressed (Src, Yes, Fyn), whereas the others are restricted to specific lineages of cells including macrophages (Hck, Fgr) and lymphocytes (Lyn, Lck, Blk). Tissue-specific expression and constitutive activation in multiple tumor types have led to renewed interest in Src family kinases as targets for anti-cancer drug design (6Biscardi J.S. Tice D.A. Parsons S.J. Adv. Cancer Res. 1999; 76: 61-119Google Scholar, 7Bjorge J.D. Jakymiw A. Fujita D.J. Oncogene. 2000; 19: 5620-5635Google Scholar, 8Irby R.B. Yeatman T.J. Oncogene. 2000; 19: 5636-5642Google Scholar, 9Susva M. Missbach M. Green J. Trends Pharmacol. Sci. 2000; 21: 489-495Google Scholar). Src kinases share the same overall structural architecture and regulation (10Sicheri F. Kuriyan J. Curr. Opin. Struct. Biol. 1997; 7: 777-785Google Scholar, 11Williams J.C. Wierenga R.K. Saraste M. Trends Biochem. Sci. 1998; 23: 179-184Google Scholar). N-terminal sequences are unique to each family member and provide targeting signals for myristoylation and in some cases palmitoylation, which target Src kinases to the plasma membrane. Conserved structural features include SH3, 1The abbreviations used are: SH3 and SH2 domains, Src homology 3 and 2 domains, respectively; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; GST, glutathioneS-transferase; HIV-1, human immunodeficiency virus type 1; KE, kinase-defective; RIPA, radioimmune precipitation assay; SIE, sis-inducible element; STAT, signal transducers and activators of transcription; STAT3YF, STAT3 mutant lacking the conserved tyrosine phosphorylation site at Tyr-705. SH2, and kinase domains, followed by a short C-terminal tail region containing a conserved tyrosine phosphorylation site. Recent structural analyses of c-Src and Hck demonstrate that the SH2 and SH3 domains contribute to the negative regulation of kinase activity through intramolecular interactions (12Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Google Scholar, 13Schindler T. Sicheri F. Pico A. Gazit A. Levitzki A. Kuriyan J. Mol. Cell. 1999; 3: 639-648Google Scholar, 14Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Google Scholar, 15Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Google Scholar, 16Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Google Scholar). The SH2 domain binds to the negative regulatory tail, an interaction that is dependent upon prior phosphorylation by the Src tail kinase, Csk (17Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Scopus (510) Google Scholar). The importance of tail-SH2 interaction to negative regulation is illustrated by the constitutive tyrosine kinase and transforming activities of the v-Src oncogene, which lacks C-terminal tail residues including the conserved tyrosine. The crystal structures also revealed intramolecular contacts between the SH3 domain and a polyproline type II helix formed by the linker connecting the SH2 domain with the N-terminal lobe of the kinase domain. Point mutations of linker prolines as well as some SH3 mutations can also release kinase activity and induce a transformed phenotype in fibroblasts, providing evidence for an essential role of this SH3-mediated interaction to overall regulation of kinase activity (18Briggs S.D. Smithgall T.E. J. Biol. Chem. 1999; 274: 26579-26583Google Scholar, 19Gonfloni S. Williams J.C. Hattula K. Weijland A. Wierenga R.K. Superti-Furga G. EMBO J. 1997; 16: 7261-7271Google Scholar). Together, these SH2 and SH3-mediated interactions work through an allosteric mechanism to push the two lobes of the kinase domain together, thus preventing substrate binding and stabilizing the inactive form of the kinase domain. The crystal structures of the down-regulated forms of Src family kinases suggest that physiological protein-protein interactions through these domains may induce transient kinase activation, leading to substrate phosphorylation. In this report, we investigated the SH3-dependent interaction of the STAT3 transcription factor with Src family kinases in vitro and in vivo. STATs are latent cytoplasmic transcription factors that become activated by tyrosine phosphorylation, which induces STAT dimerization, nuclear translocation, and transcriptional regulation (20Ihle J.N. Curr. Opin. Cell Biol. 2001; 13: 211-217Google Scholar, 21Horvath C.M. Trends Biochem. Sci. 2000; 25: 496-502Google Scholar). A growing body of evidence strongly supports the idea that STATs are substrates for Src family kinases, particularly STAT3 (22Reddy E.P. Korapati A. Chaturvedi P. Rane S. Oncogene. 2000; 19: 2532-2547Google Scholar). For example, transformation of rodent fibroblasts with constitutively activated Src kinases results in strong activation of STAT3, and activation appears to be essential for transformation (23Turkson J. Bowman T.L. Garcia R. Caldenhoven E. de Groot R.P. Jove R. Mol. Cell. Biol. 1998; 18: 2545-2552Google Scholar, 24Yu C.-L. Meyer D.J. Campbell G.S. Larner A.C. Carter-Su C. Schwartz J. Jove R. Science. 1995; 269: 81-83Google Scholar, 25Cao X.M. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Google Scholar, 26Bromberg J.F. Horvath C.M. Besser D. Lathem W.W. Darnell Jr., J.E. Mol. Cell. Biol. 1998; 18: 2553-2558Google Scholar). Here we show that STAT3 is a direct substrate for all members of the Src kinase family tested and that the SH3 domains of Src kinases are sufficient for STAT3 binding in vitro. Disruption of the SH3 domain of an activated Hck mutant causes a complete loss of STAT3 activation in fibroblasts, indicating a necessary role for Src family kinase SH3 domains in the recognition of STAT3 by Src kinases in vivo. In addition, we show that interaction with STAT3 induces a transient increase in Src kinase activity. Together these results suggest a model in which STAT3 is recruited to Src family kinases via an SH3-dependent mechanism, leading to transient kinase activation and STAT3 tyrosine phosphorylation. SH3-dependent substrate recruitment is sufficient to induce Src activation without dephosphorylation of the negative regulatory tail, allowing Src family kinases to return to the inactive state after release of phosphorylated STAT3. Sf-9 cells (Invitrogen) were cultured in Grace's complete insect cell medium containing 10% fetal bovine serum and 50 μg/ml gentamycin. Recombinant STAT3, STAT3YF (STAT3 mutant lacking the conserved tyrosine phosphorylation site at Tyr-705), Hck, c-Src, Lyn, Fyn, and Fgr baculoviruses were generated as described previously (27Briggs S.D. Lerner E.C. Smithgall T.E. Biochemistry. 2000; 39: 489-495Google Scholar, 28Nelson K. Rogers J.A. Bowman T.L. Jove R. Smithgall T.E. J. Biol. Chem. 1998; 273: 7072-7077Google Scholar). STAT3 proline residues 331 and 334 were replaced by alanines to create the STAT3–2PA mutant using the Gene Editor oligonucleotide-directed mutagenesis kit (Promega). The resulting mutant cDNA was subcloned into the baculovirus transfer vector pVL1392 (Pharmingen). STAT3 was subcloned into the pVL-GST transfer vector (29Rogers J.A. Read R.D. Li J. Peters K.L. Smithgall T.E. J. Biol. Chem. 1996; 271: 17519-17525Google Scholar) to generate the GST-STAT3 baculovirus. The STAT3–2PA and GST-STAT3 baculoviruses were produced using BaculoGold DNA and the manufacturer's protocol (Pharmingen). For protein expression, 2.5 × 106 Sf-9 cells were infected with high titer viral stocks for 1 h at room temperature and lysed 48 h later in 1 ml of Hck lysis buffer containing protease and phosphatase inhibitors (50 mm Tris-HCl, pH 7.4, 50 mm NaCl, 1 mm EDTA, 10 mmMgCl2, 1% Triton X-100, 0.5 mmphenylmethylsulfonyl fluoride, 20 mm NaF, 1 mmNa3VO4,). Lysates were clarified by centrifugation before further analysis. The antibodies used in this study include anti-Hck (C-30; Santa Cruz Biotechnology), anti-Src phosphospecific (Src pY418; BIOSOURCE International), anti-phosphotyrosine (PY99; Santa Cruz), anti-STAT3 (C-20; Santa Cruz), anti-STAT3 phosphospecific (STAT3 Tyr-705; Santa Cruz), anti-cyclin D1 (Ab-3; Calbiochem), anti-actin (mAB1501; Chemicon) and anti-GST (B-14; Santa Cruz). For immunoprecipitation, clarified cell lysates were incubated with 1 μg of primary antibody and 20 μl of protein G-Sepharose (50% slurry, Amersham Biosciences) for 2 h at 4 °C. The immunoprecipitates were collected by centrifugation and washed twice with cold radioimmune precipitation assay (RIPA) buffer (18Briggs S.D. Smithgall T.E. J. Biol. Chem. 1999; 274: 26579-26583Google Scholar, 27Briggs S.D. Lerner E.C. Smithgall T.E. Biochemistry. 2000; 39: 489-495Google Scholar). For immunoblotting, clarified lysates and immunoprecipitates were heated in SDS-PAGE sample buffer and resolved on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. After incubation with primary antibody, immunoreactive proteins were detected with an appropriate secondary antibody-alkaline phosphatase conjugate and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate colorimetric substrate (Sigma) or CDP-Star chemiluminescent alkaline phosphatase substrate (PerkinElmer Life Sciences). For GSH-agarose precipitation experiments, GSH-agarose beads (20 μl of a 50% w/v slurry; Sigma) were used to precipitate the GST-STAT3·Src kinase protein complexes; beads were washed with RIPA buffer, and associated Src kinases were visualized by immunoblotting. For GST-SH3 fusion protein expression, PCR fragments encoding the SH3 domains of Hck, c-Src, Lyn, Fyn, and Hck-W93A were subcloned into the bacterial expression vector pGEX-2T (AmershamBiosciences). GST and GST-SH3 fusion proteins were expressed inEscherichia coli and immobilized on GSH-agarose beads as described elsewhere (30Briggs S.D. Bryant S.S. Jove R. Sanderson S.D. Smithgall T.E. J. Biol. Chem. 1995; 270: 14718-14724Google Scholar, 31Peters K.L. Smithgall T.E. Cell. Signal. 1999; 11: 507-514Google Scholar). Clarified lysates from Sf-9 cells expressing recombinant STAT3 were incubated with 10 μg of each GST-SH3 fusion protein or GST alone for 2 h at 4 °C. STAT3·SH3 complexes were precipitated by centrifugation, washed extensively in RIPA buffer, and eluted by heating in SDS-PAGE sample buffer. The samples were resolved by SDS-PAGE, and STAT3 was detected by immunoblotting. Thesis-inducible element (SIE) double-stranded oligonucleotide probe for STAT3 binding was 32P labeled as described elsewhere (24Yu C.-L. Meyer D.J. Campbell G.S. Larner A.C. Carter-Su C. Schwartz J. Jove R. Science. 1995; 269: 81-83Google Scholar, 28Nelson K. Rogers J.A. Bowman T.L. Jove R. Smithgall T.E. J. Biol. Chem. 1998; 273: 7072-7077Google Scholar). The STAT3 EMSA was performed on equal amounts of nuclear protein extracts (1–5 μg of protein) from Rat-2 fibroblasts prepared as described by Skorski et al. (32Skorski T. Nieborowska-Skorska M. Wlodarski P. Wasik M. Trotta R. Kanakaraj P. Salomoni P. Antonyak M. Martinez R. Majewski M. Wong A. Perussia B. Calabretta B. Blood. 1998; 91: 406-418Google Scholar). Binding reactions (20 μl) contained 40,000 cpm of SIE probe in 10 mm HEPES, pH 7.9, 25 mm KCl, 0.5 mmdithiothreitol, 0.5 mm EDTA, 1 μg of poly(dI·dC), and 5 μg of bovine serum albumin. Control reactions for binding specificity contained a 100-fold molar excess of unlabeled SIE probe. Binding reactions were incubated at 30 °C for 30 min and quenched on ice. STAT3·SIE complexes were resolved on 5% nondenaturing polyacrylamide gels, and radiolabeled bands were visualized by autoradiography. Retroviral expression vectors for wild-type Hck, the activated tail mutant (Hck-YF), and kinase-defective Hck (Hck-KE) have been described elsewhere (18Briggs S.D. Smithgall T.E. J. Biol. Chem. 1999; 274: 26579-26583Google Scholar, 33Lerner E.C. Smithgall T.E. Nat. Struct. Biol. 2002; 9: 365-369Google Scholar, 34Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Google Scholar). The SH3-inactivating mutation W93A was introduced into full-length Hck using the Gene Editor oligonucleotide-directed mutagenesis kit (Promega). This mutation was combined with Hck-YF by restriction fragment swapping to create Hck-YFW. These Hck clones as well as the coding sequences for GFP, STAT3, and STAT3–2PA were subcloned into the retroviral expression vector pSRαMSVtkneo (35Muller A.J. Young J.C. Pendergast A.M. Pondel M. Landau R.N. Littman D.R. Witte O.N. Mol. Cell. Biol. 1991; 11: 1785-1792Google Scholar). These vectors were used to generate high titer retroviral stocks in 293T cells by cotransfection with an ecotropic packaging vector (36Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Google Scholar). Rat-2 fibroblasts (ATCC) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 50 μg/ml gentamycin. Retroviral stocks were supplemented with Polybrene (hexadimethrine bromide, Sigma) to 4 μg/ml and added to fibroblasts in six-well plates (2 × 105 cells/well). The plates were centrifuged at 1,000 × g for 3 h at room temperature to enhance infection efficiency. For the focus-forming assay, 2 × 104 infected cells were plated in 60-mm culture dishes in in the presence of 800 μg/ml G418. Transformed foci were visualized 10–14 days later by Wright-Giemsa staining. For the soft agar assay, 1 × 104 infected cells were plated in 35-mm culture dishes in Dulbecco's modified Eagle's medium containing 0.3% Seaplaque agarose (FMC). Colonies were stained with iodonitrotetrazolium violet formazan (Sigma) and counted using a BioRad imaging densitometer and colony counting software (37Briggs S.D. Scholtz B. Jacque J.M. Swingler S. Stevenson M. Smithgall T.E. J. Biol. Chem. 2001; 276: 25605-25611Google Scholar). Rat-2 fibroblasts stably expressing GFP, STAT3, or STAT3–2PA were infected with c-Src or Hck retroviruses as described above. Kinase activity was assessed 48 h later using the in vitro kinase assay described elsewhere (18Briggs S.D. Smithgall T.E. J. Biol. Chem. 1999; 274: 26579-26583Google Scholar, 27Briggs S.D. Lerner E.C. Smithgall T.E. Biochemistry. 2000; 39: 489-495Google Scholar). This assay employs a 50-kDa GST fusion protein containing residues 331–443 Sam-68 as substrate (38Taylor S.J. Shalloway D. Nature. 1994; 368: 867-871Google Scholar, 39Fumagalli S. Totty N.F. Hsuan J.J. Courtneidge S.A. Nature. 1994; 368: 871-874Google Scholar) (Santa Cruz Biotechnology) and [γ-32P]ATP (PerkinElmer Life Sciences). Recent studies have implicated Src family kinases in STAT3 activation, both in normal and transformed cell types (22Reddy E.P. Korapati A. Chaturvedi P. Rane S. Oncogene. 2000; 19: 2532-2547Google Scholar, 40Bowman T. Garcia R. Turkson J. Jove R. Oncogene. 2000; 19: 2474-2488Google Scholar). To investigate whether STAT3 is a direct substrate for the Src kinase family, c-Src, Hck, Lyn, Fyn, and Fgr were coexpressed with STAT3 in Sf-9 insect cells using recombinant baculovirus vectors. Sf-9 cells provide a useful system to study direct tyrosine kinase-STAT3 interaction because they lack homologs of mammalian tyrosine kinases involved in STAT3 activation (28Nelson K. Rogers J.A. Bowman T.L. Jove R. Smithgall T.E. J. Biol. Chem. 1998; 273: 7072-7077Google Scholar, 41Zhang Y. Turkson J. Carter-Su C. Smithgall T. Levitzki A. Kraker A. Krolewski J.J. Medveczky P. Jove R. J. Biol. Chem. 2000; 275: 24935-24944Google Scholar). In addition, Src family kinases are constitutively active in Sf-9 cells because of the absence of Csk. STAT3 was immunoprecipitated from coinfected cell lysates and immunoblotted with anti-phosphotyrosine antibodies. As shown in Fig.1, STAT3 was strongly phosphorylated by all of the Src family members tested. Immunoblotting of a duplicate membrane with phosphospecific antibodies that recognize the tyrosine phosphorylation site responsible for STAT3 dimerization and activation (Tyr-705) indicates that Src kinases phosphorylate STAT3 on Tyr-705. To confirm this result, a STAT3 mutant lacking this tyrosine residue (STAT3YF) was coexpressed with Src family kinases and analyzed in the same manner. Anti-phosphotyrosine immunoblotting shows that Tyr-705 mutagenesis substantially reduced or eliminated STAT3 phosphorylation by Src family kinases. Interestingly, c-Src and Hck induced low level phosphorylation of STAT3YF, suggesting that alternative phosphorylation sites may exist for these two Src family members. Control immunoblots show equal amounts of STAT3 in each lane. In addition, a control immunoblot of cell lysates with an antibody specific for the autophosphorylated Src activation loop reveals approximately equal levels of activated Src family members in each cell lysate. We next investigated whether tyrosine phosphorylation of STAT3 by Src family kinases correlated with STAT3 DNA binding activity. Sf-9 insect cells were infected with recombinant Src family kinase baculoviruses in combination with the STAT3 or STAT3YF viruses. EMSAs were then performed using clarified cell lysates and a radiolabeled STAT3-specific DNA probe (SIE) to check for the presence of STAT3 DNA binding activity. As shown in Fig. 2, coexpression of Src family kinases with STAT3 induced a strong gel shift, whereas coexpression of the kinases with STAT3YF did not. Overexpression of STAT3 alone (Fig. 2) or the kinases alone (data not shown) did not result in a shifted band. STAT3 EMSA performed in the presence of a 100-fold molar excess of unlabeled probe resulted in complete inhibition of STAT3·DNA complex formation, indicating the specificity of the DNA binding activity for the probe (data not shown). Although all of the Src kinases induced STAT3 DNA binding activity, Fyn and Fgr produced consistently lower levels of STAT3·DNA binding compared with the other kinases tested. However, this effect does not appear to be caused by differences in STAT3 protein levels or tyrosine phosphorylation (Fig. 1) and may instead relate to less efficient release of STAT3 from these kinases after tyrosine phosphorylation. These results indicate that STAT3 is a common substrate for the Src kinase family and show that phosphorylation and activation occur via a direct interaction. Previous reports have shown that Src can be coimmunoprecipitated with STAT3 from fibroblasts and other cell types, suggesting that these proteins interact in vivo (25Cao X.M. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Google Scholar, 42Chaturvedi P. Reddy M.V.R. Reddy E.P. Oncogene. 1998; 16: 1749-1758Google Scholar). To determine whether this interaction is dependent upon other mammalian proteins, Src kinases were coexpressed with a GST-STAT3 fusion protein in Sf-9 insect cells. GST-STAT3 was precipitated from Sf-9 cell lysates with GSH-agarose beads, and STAT3-bound Src kinases were detected by immunoblotting. As shown in Fig. 3, all of the Src kinases tested in this assay readily formed complexes with GST-STAT3, whereas no interaction was observed with GST alone. These results indicate that Src family kinases have the potential to bind directly to STAT3in vivo. Control blots show that GST and GST-STAT3 were expressed at the same level in the infected cell lysates and that each culture contained the same amount of each active Src family member. We also investigated the dependence of the interaction on tyrosine phosphorylation using a kinase-defective mutant of Hck. This mutant also formed a stable complex with GST-STAT3, indicating that recruitment of STAT3 is independent of kinase function (data not shown). We next investigated the mechanism of Src family kinase-STAT3 interaction. Inspection of the amino acid sequence of STAT3 revealed an extended PXXP motif identical in spacing to one found in HIV-1 Nef, a well characterized binding protein for the Hck and Lyn SH3 domains (43Saksela K. Cheng G. Baltimore D. EMBO J. 1995; 14: 484-491Google Scholar) (Fig. 4 A). To determine the binding activity of Src family kinase SH3 domains toward STAT3, the Hck, c-Src, Lyn, and Fyn SH3 domains were expressed in bacteria as GST fusion proteins, immobilized on GSH-agarose beads, and used in binding reactions with clarified lysates from Sf-9 cells expressing recombinant STAT3. As shown in Fig. 4 B, all four recombinant SH3 domains were sufficient for STAT3 binding. To determine whether STAT3-SH3 binding involved a typical interaction with the hydrophobic surface of SH3, we replaced Trp-93 in the Hck SH3 domain with Ala, a mutation previously shown to destroy Hck SH3 function (W93A mutant) (44Hassaine G. Courcoul M. Bessou G. Barthalay Y. Picard C. Olive D. Collette Y. Vigne R. Decroly E. J. Biol. Chem. 2001; 276: 16885-16893Google Scholar). Fig. 4 C shows that the W93A mutation substantially reduces binding of the Hck SH3 domain to STAT3 compared with the wild-type SH3 protein. We next investigated the role of Src family kinase SH3 domains in the recruitment and activation of STAT3 in mammalian cells. For these experiments, we employed Rat-2 fibroblasts, which are readily transformed by activated mutants of Hck (18Briggs S.D. Smithgall T.E. J. Biol. Chem. 1999; 274: 26579-26583Google Scholar, 33Lerner E.C. Smithgall T.E. Nat. Struct. Biol. 2002; 9: 365-369Google Scholar, 34Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Google Scholar). We first investigated whether fibroblast transformation by activated Hck correlates with constitutive activation of endogenous STAT3, as shown previously for Src (24Yu C.-L. Meyer D.J. Campbell G.S. Larner A.C. Carter-Su C. Schwartz J. Jove R. Science. 1995; 269: 81-83Google Scholar, 25Cao X.M. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Google Scholar). Fibroblasts were infected with recombinant retroviruses carrying either an activated mutant of Hck in which the negative regulatory tail tyrosine has been replaced by phenylalanine (Hck-YF), or with wild-type Hck as a negative control. Wild-type Hck is phosphorylated on the tail tyrosine by Csk, resulting in suppression of its kinase and transforming activities in this cell type (33Lerner E.C. Smithgall T.E. Nat. Struct. Biol. 2002; 9: 365-369Google Scholar). Nuclear extracts were prepared from these cells and analyzed for active STAT3 by EMSA using the same SIE probe described above for the Sf-9 cell experiments. As shown in Fig.5 A, Rat-2 fibroblasts expressing Hck-YF showed strong STAT3·SIE complex formation, consistent with the transformed phenotype. In contrast, STAT3 DNA binding activity was not observed in Rat-2 cells expressing wild-type Hck. These results suggest that STAT3 is an endogenous substrate for Hck in mammalian cells. To address whether the SH3 domain of Hck is required for STAT3 activation in fibroblasts, the Hck-YF mutant was combined with the W93A SH3 mutation to create the double mutant, Hck-YFW. In Fig.4 C the W93A mutation was shown to block binding of the Hck SH3 domain to STAT3. The Hck-YFW mutant was expressed in Rat-2 fibroblasts, and STAT3 DNA binding activity was assessed by EMSA. As shown in Fig. 5 A, the SH3 mutation completely blocked the ability of Hck-YF to activate endogenous STAT3. As a control, the W93A mutation was also introduced into wild-type Hck, and the effect of this SH3 mutation on endogenous STAT3 activation was assessed by gel shift assay. Fig. 5 A shows that this mutant was also unable to activate endogenous STAT3, despite release of kinase activity as a result of the mutation (see below). These results provide evidence for an SH3-dependent mechanism of STAT3 recognition by Src family kinases in vivo. To determine whether the loss of STAT3 activation correlates with reduced expression of a STAT3 target gene, we examined cyclin D1 expression in the same panel of Rat-2 fibroblasts. Previous studies have shown that activation of STAT3 is sufficient to induce cyclin D1 expression in this cell type (45Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell Jr., J.E. Cell. 1999; 98: 295-303Google Scholar). Rat-2 fibroblasts expressing each of the Hck constructs were serum starved, and cyclin D1 levels were determined by immunoblotting. As shown in Fig. 5 B, Rat-2 fibroblasts expressing the constitutively active Hck-YF mutant show an elevated level of cyclin D1 protein relative to cells expressing wild-type or kinase-defective Hck. However, introduction of the W93A SH3 mutation reduced the induction of cyclin D1 expression by Hck-YF, consistent with the inability of this mutant to activate STAT3. Similarly, introduction of the W93A mutation into wild-type Hck has very little effect on cyclin D1 expression, consistent with the lack of STAT3 activation by this Hck mutant. Results shown in Fig. 5 demonstrate that mutation of the Hck SH3 domain causes a loss of STAT3 activation by Hck-YF in Rat-2 cells. To establish that this SH3 mutation affects STAT3 recognition and not kinase function, we investigated the kinase activity of this mutant by immunoblot analysis. As shown in Fig.6, Hck-YF and the corresponding SH3 mutant (Hck-YFW) were autophosphorylated to the same extentin vivo, as determined by immunoblotting with phosphospecific antibodies for the phosphorylated activation loop. We also examined tyrosine phosphorylation of p40, an endogenous Hck substrate reported previously (18Briggs S.D. Smithgall T.E. J. Biol. Chem. 1999; 274: 26579-26583Google Scholar, 27Briggs S.D. Lerner E.C. Smithgall T.E. Biochemistry. 2000; 39: 489-495Google Scholar, 33Lerner E.C. Smithgall T.E. Nat. Struct. Biol. 2002; 9: 365-369Google Scholar, 34Briggs S.D. Sharkey M. Stevenson M. Smithgall
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