Identification of the signal transduction pathways used by PRL is essential for understanding the role of PRL receptors in growth and differentiation processes. Early cellular mediators of PRL receptor activation include tyrosine kinases of the Janus kinase (JAK) and SRC families, with rapid nuclear signaling via tyrosine phosphorylated signal transducers and activators of transcription. In the present study we provide the first demonstration of PRL-induced activation of Ras, an oncogenic protein that supports an alternative signaling route from the membrane to the nucleus. PRL stimulated Ras in rat Nb2-SP lymphoma cells, as detected by a 2.0-fold increase in the GTP-bound state of the molecule (P < 0.01). This activation was associated with marked tyrosine phosphorylation and increased membrane association of the 52-kilodalton form of SHC. Moreover, PRL induced binding of SHC to growth factor receptor bound 2 and the guanine-nucleotide exchange factor son of sevenless, a common method used by growth factor receptors to activate Ras. In contrast, no apparent regulation by PRL of Ras via VAV or p120 Ras-guanosine triphosphatase-activating protein was detected, based upon an absence of PRL-inducible tyrosine phosphorylation of these proteins. Collectively, these results provide a molecular bridge between activation of PRL receptor-associated tyrosine kinases and subsequent stimulation of the serine/threonine kinase Raf-1, an established Ras target that was recently shown to be activated by PRL in Nb2 cells. We conclude that PRL is able to activate Ras via recruitment of the signaling proteins SHC, growth factor receptor bound 2, and son of sevenless in Nb2 cells. Moreover, PRL induced tyrosine phosphorylation of SHC in two of three PRL-responsive human breast cancer cell lines, suggesting that SHC-mediated Ras activation is a commonly used signaling strategy by PRL.
The haematopoietic protein, p95vav, has been shown to be a tyrosine kinase substrate and to have tyrosine kinase-modulated guanine-nucleotide-releasing-factor activity. This implies a function in the control of ras or ras-like proteins. Because ras activation has been shown to be a downstream event following stimulation of the interleukin-2 (IL-2) receptor, we investigated the possibility that vav was involved in IL-2 signal transduction pathways, using human T cells as a model. We found rapid tyrosine phosphorylation of vav in response to IL-2 within 1 min, with maximum increase of phosphorylation of 5-fold occurring by 5 min after treatment in normal human T cells. IL-2 stimulation of the human T-cell line YT and a subclone of the YT cell line (YTlck-) that does not express message for the src-family kinase p56lck also results in a rapid rate of tyrosine phosphorylation of vav of more than 5-fold by 5 min. These results suggest that vav may play an important role in IL-2-stimulated signal transduction and that there is not a strict requirement for the tyrosine kinase p56lck.
Identification of the signal transduction pathways used by PRL is essential for understanding the role of PRL receptors in growth and differentiation processes. Early cellular mediators of PRL receptor activation include tyrosine kinases of the Janus kinase (JAK) and SRC families, with rapid nuclear signaling via tyrosine phosphorylated signal transducers and activators of transcription. In the present study we provide the first demonstration of PRL-induced activation of Ras, an oncogenic protein that supports an alternative signaling route from the membrane to the nucleus. PRL stimulated Ras in rat Nb2-SP lymphoma cells, as detected by a 2.0-fold increase in the GTP-bound state of the molecule (P < 0.01). This activation was associated with marked tyrosine phosphorylation and increased membrane association of the 52-kilodalton form of SHC. Moreover, PRL induced binding of SHC to growth factor receptor bound 2 and the guanine-nucleotide exchange factor son of sevenless, a common method used by growth factor receptors to activate Ras. In contrast, no apparent regulation by PRL of Ras via VAV or p120 Ras-guanosine triphosphatase-activating protein was detected, based upon an absence of PRL-inducible tyrosine phosphorylation of these proteins. Collectively, these results provide a molecular bridge between activation of PRL receptor-associated tyrosine kinases and subsequent stimulation of the serine/threonine kinase Raf-1, an established Ras target that was recently shown to be activated by PRL in Nb2 cells. We conclude that PRL is able to activate Ras via recruitment of the signaling proteins SHC, growth factor receptor bound 2, and son of sevenless in Nb2 cells. Moreover, PRL induced tyrosine phosphorylation of SHC in two of three PRL-responsive human breast cancer cell lines, suggesting that SHC-mediated Ras activation is a commonly used signaling strategy by PRL.
The activation of Janus kinases (JAKs) is crucial for propagation of the proliferative response initiated by many cytokines. The proliferation of various cell lines, particularly those of hematopoietic origin, is also modulated by mediators of oxidative stress such as nitric oxide and thiol redox reagents. Herein we demonstrate that nitric oxide and other thiol oxidants can inhibit the autokinase activity of rat JAK2 in vitro , presumably through oxidation of crucial dithiols to disulfides within JAK2. The reduced form of JAK2 is the most active form, and the oxidized JAK2 form is inactive. Nitric oxide pretreatment of quiescent Ba/F3 cells also inhibits the interleukin 3-triggered in vivo activation of JAK2, a phenomenon that correlates with inhibited proliferation. Furthermore, we observed that the autokinase activity of JAK3 responds in a similar fashion to thiol redox reagents in vitro and to nitric oxide donors in vivo . We suggest that the thiol redox regulation of JAKs may partially explain the generally immunosuppressive effects of nitric oxide and of other thiol oxidants.
Many cytokines, hormones, and growth factors activate Janus kinases to tyrosine phosphorylate select members of the Stat transcription factors. For full transcriptional activation, Stat1 and Stat3 also require phosphorylation of a conserved serine residue within a mitogen-activated protein kinase phosphorylation consensus site. On the other hand, two recently identified and highly homologous Stat5a and Stat5b proteins lack this putative mitogen-activated protein kinase phosphorylation site. The present study set out to establish whether Stat5a and Stat5b are under the control of an interleukin-2 (IL2)-activated Stat5 serine kinase. We now report that IL2 stimulated marked phosphorylation of serine and tyrosine residues of both Stat5a and Stat5b in human T lymphocytes and in several IL2-responsive lymphocytic cell lines. No Stat5a/b phosphothreonine was detected. Phosphoamino acid analysis also revealed that Stat5a/b phosphotyrosine levels were maximized within 1–5 min of IL2 stimulation, whereas serine phosphorylation kinetics were slower. Interestingly, IL2-induced serine phosphorylation of Stat5a differed quantitatively and temporally from that of Stat5b with Stat5a serine phosphorylation leveling off after 10 min and the more pronounced Stat5b response continuing to rise for at least 60 min of IL2 stimulation. Furthermore, we identified two discrete domains of IL2 receptor β (IL2Rβ) that could independently restore the ability of a truncated IL2Rβ mutant to mediate Stat5a/b phosphorylation and DNA binding to the γ-activated site of the β-casein gene promoter. These observations demonstrated that there is no strict requirement for one particular IL2Rβ region for Stat5 phosphorylation. Finally, we established that the IL2-activated Stat5a/b serine kinase is insensitive to several selective inhibitors of known IL2-stimulated kinases including MEK1/MEK2 (PD98059), mTOR (rapamycin), and phosphatidylinositol 3-kinase (wortmannin) as determined by phosphoamino acid and DNA binding analysis, thus suggesting that a yet-to-be-identified serine kinase mediates Stat5a/b activation. Many cytokines, hormones, and growth factors activate Janus kinases to tyrosine phosphorylate select members of the Stat transcription factors. For full transcriptional activation, Stat1 and Stat3 also require phosphorylation of a conserved serine residue within a mitogen-activated protein kinase phosphorylation consensus site. On the other hand, two recently identified and highly homologous Stat5a and Stat5b proteins lack this putative mitogen-activated protein kinase phosphorylation site. The present study set out to establish whether Stat5a and Stat5b are under the control of an interleukin-2 (IL2)-activated Stat5 serine kinase. We now report that IL2 stimulated marked phosphorylation of serine and tyrosine residues of both Stat5a and Stat5b in human T lymphocytes and in several IL2-responsive lymphocytic cell lines. No Stat5a/b phosphothreonine was detected. Phosphoamino acid analysis also revealed that Stat5a/b phosphotyrosine levels were maximized within 1–5 min of IL2 stimulation, whereas serine phosphorylation kinetics were slower. Interestingly, IL2-induced serine phosphorylation of Stat5a differed quantitatively and temporally from that of Stat5b with Stat5a serine phosphorylation leveling off after 10 min and the more pronounced Stat5b response continuing to rise for at least 60 min of IL2 stimulation. Furthermore, we identified two discrete domains of IL2 receptor β (IL2Rβ) that could independently restore the ability of a truncated IL2Rβ mutant to mediate Stat5a/b phosphorylation and DNA binding to the γ-activated site of the β-casein gene promoter. These observations demonstrated that there is no strict requirement for one particular IL2Rβ region for Stat5 phosphorylation. Finally, we established that the IL2-activated Stat5a/b serine kinase is insensitive to several selective inhibitors of known IL2-stimulated kinases including MEK1/MEK2 (PD98059), mTOR (rapamycin), and phosphatidylinositol 3-kinase (wortmannin) as determined by phosphoamino acid and DNA binding analysis, thus suggesting that a yet-to-be-identified serine kinase mediates Stat5a/b activation. Interleukin-2 (IL2) 1The abbreviations used are: IL2, interleukin-2; IL2R, IL2 receptor; Stat, signal transducers and activators of transcription; MAPK, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; JAK, Janus kinase; MEK, MAPK kinase; SH, Src homology. is a key regulator of normal immune function and acts on a variety of lymphoid cell types including T lymphocytes, B lymphocytes, and natural killer cells (1Smith K.A. Science. 1988; 240: 1169-1176Crossref PubMed Scopus (1906) Google Scholar). IL2-induced effects are mediated by heterodimerization of two related transmembrane proteins of the hematopoietin receptor family that are designated IL2 receptor β- and γ-chains (IL2Rβ and ILR2γ) (2Kondo M. Takeshita T. Ishi N. Nakamura M. Watanabe S. Arai K-i. Sugamura K. Science. 1993; 262: 1874-1877Crossref PubMed Scopus (735) Google Scholar, 3Noguchi M. Nakamura Y. Russell S.M. Ziegler S.F. Tsang M. Cao X. Leonard W. Science. 1993; 262: 1877-1880Crossref PubMed Scopus (787) Google Scholar, 4Russell S.M. Keegan A.D. Harada N. Nakamura Y. Noguchi M. Leland P. Friedmann M.C. Miyajima A. Puri R.K. Paul W.E. Leonard W.L. Science. 1993; 262: 1880-1883Crossref PubMed Scopus (743) Google Scholar). In addition to this pair of essential receptor subunits, a third non-conforming protein with a short cytoplasmic domain (TAC or IL2Rα) represents an accessory receptor subunit that can serve as a positive affinity modulator through its regulated expression (5Nikaido T. Schimizu A. Ishida N. Sabe H. Teshigawara K. Maeda M. Uchiyama T. Yodoi J. Honjo T. Nature. 1984; 311: 631-635Crossref PubMed Scopus (396) Google Scholar, 6Cosman D. Cerratti D.P. Larson A. Park L. March C. Dower S. Gillis S. Urdal D. Nature. 1984; 312: 768-771Crossref PubMed Scopus (198) Google Scholar, 7Leonard W.J. Depper J.M. Crabtree G.R. Rudikoff S.J. Pumphrey J. Robb R.J. Kronke M. Svetlik P.B. Peffer N.J. Waldman T.A. Greene W.C. Nature. 1984; 311: 626-631Crossref PubMed Scopus (607) Google Scholar). IL2-induced dimerization of IL2Rβ and IL2Rγ results in stimulation of the receptor-associated Janus kinases (JAK) JAK1 and/or JAK3 through intermolecular transphosphorylation (8Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedman M. Berg M. Witthuhn B.A. Goldman A.S. Schmalstieg F.C. Ihle J.N. O'Shea J.J. Leonard W.J. Science. 1994; 266: 1042-1045Crossref PubMed Scopus (592) Google Scholar, 9Kirken R.A. Rui H. Malabarba M.G. Howard O.M.Z. Kawamura M. O'Shea J.J. Farrar W.L. Cytokine. 1995; 7: 689-700Crossref PubMed Scopus (79) Google Scholar). Activation of JAKs initiates intracellular signaling cascades that include Src tyrosine kinases, phosphatases, serine-threonine kinases, and a family of transcription factors known as signal transducers and activators of transcription (Stats). Stats act in concert with other transcription factors to control cell growth and differentiation (10Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar,11Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (676) Google Scholar). At present, seven members of the Stat transcription factor family have been identified (10Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar, 12Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1657) Google Scholar). Stat proteins are characterized by a central DNA-binding motif, a COOH-terminal transactivation domain, a Src homology (SH) domain 2, and an SH3-like domain (12Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1657) Google Scholar). Current models hold that newly phosphorylated tyrosine residues within activated receptor complexes direct the recruitment of Stats from the cytoplasm via their SH2 domains (13Fu X.Y. Cell. 1992; 70: 323-335Abstract Full Text PDF PubMed Scopus (304) Google Scholar). Subsequently, JAK enzymes catalyze Stat tyrosine phosphorylation, which facilitates dimerization and disengagement of Stats from the receptor complex. Serine phosphorylation of Stat1α and Stat3 was also recently reported to be critical for interferon-induced nuclear translocalization and maximal transcriptional activation (14Wen Z. Zhong Z. Darnell J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1755) Google Scholar). Serine-threonine kinases of the mitogen-activated protein kinase (MAPK) family were suggested to perform this function (14Wen Z. Zhong Z. Darnell J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1755) Google Scholar, 15Zhang X. Belnis J. Li H.C. Schindler C. Chen-Kiang S. Science. 1995; 267: 1990-1994Crossref PubMed Scopus (525) Google Scholar). In support of this proposal, mutation of the serine residue of a conserved MAPK consensus phosphorylation site (X-Pro-X-Ser-Pro) (corresponding to Ser727 of human Stat1α) to alanine abolished interferon-γ- and interferon-α-induced serine phosphorylation of Stat1α and Stat3, respectively (15Zhang X. Belnis J. Li H.C. Schindler C. Chen-Kiang S. Science. 1995; 267: 1990-1994Crossref PubMed Scopus (525) Google Scholar). Moreover, the MAPK ERK2 reportedly binds to the α-chain of the interferon-α/β receptor and coprecipitates with Stat1α in an interferon-β-inducible manner (16David M. Petricoin III, E. Benjamin C. Pine R. Weber M.J. Larner A.C. Science. 1995; 269: 1721-1723Crossref PubMed Scopus (529) Google Scholar). However, the involvement and significance of MAPKs as general Stat serine kinases are still controversial (17Ihle J.N. Bioessays. 1996; 18: 95-98Crossref PubMed Scopus (73) Google Scholar). In contrast to Stat1 and Stat3, two more recently identified and highly homologous Stat5 proteins do not contain this conserved putative MAPK phosphorylation site (18Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (301) Google Scholar, 19Hou J. Schindler U. Henzel W.J. Wong S.C. McKnight S.L. Immunity. 1995; 2: 321-329Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 20Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (717) Google Scholar, 21Liu X.L. Robinson G.W. Hennighausen L. Mol. Endocrinol. 1995; 10: 1496-1506Google Scholar, 22Mui A-L. Wakao H. O'Farrell A-M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (541) Google Scholar). Stat5 was originally identified as a prolactin-responsive mammary gland factor (20Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (717) Google Scholar) but has since been found to be regulated by a variety of cytokines including IL2–5, IL7, IL9, IL13, IL15, thrombopoietin, erythropoietin, growth hormone, and granulocyte-macrophage colony-stimulating factor (12Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1657) Google Scholar, 23Rolling C. Trenton D. Pelligrini S. Galanaud P. Richard Y. FEBS Lett. 1996; 393: 53-56Crossref PubMed Scopus (88) Google Scholar). Many of these factors also activate the Ras/MAPK pathway to regulate a number of cellular events including cell growth and differentiation as well as other physiological responses. However, the Ras/MAPK pathway does not appear to be crucial for IL2-induced cell proliferation or Stat5 activation (24Minami Y. Kono T. Yamada T. Kobayashi N. Kawahara A. Perlmutter R.M. Taniguchi T. EMBO J. 1993; 12: 759-768Crossref PubMed Scopus (123) Google Scholar, 25Beadling C. Ng J. Babbage J.W. Cantrell D.A. EMBO J. 1996; 15: 1902-1913Crossref PubMed Scopus (161) Google Scholar). It is therefore possible that Stat5a and Stat5b activities are regulated differently from other Stats and may not be inducibly phosphorylated on serine residues. Using IL2 (a potent activator of Stat5), the present study specifically set out to establish whether Stat5a and Stat5b are under the control of an IL2-activated Stat5 serine kinase. We now report that IL2 markedly induced phosphorylation of both Stat5a and Stat5b on serine and tyrosine but not on threonine residues in several target cell lines tested including normal human T lymphocytes. Moreover, we suggest that these phosphorylation events are mediated independently from several known IL2-activated signaling pathways. Polyclonal rabbit antisera were raised against peptides derived from the unique COOH termini of Stat5a and Stat5b as described previously (21Liu X.L. Robinson G.W. Hennighausen L. Mol. Endocrinol. 1995; 10: 1496-1506Google Scholar) or from R & D Systems (catalog no. PA-ST5A or PA-ST5B). These antibodies recognized mouse, rat, and human forms of Stat5a or Stat5b and were used for immunoprecipitation and immunoblotting. The rat T cell lymphoma cell line Nb2-11C or human T lymphocytes obtained from normal donors (26Wahl L.M. Katon I.M. Wilder R.L. Winter C. Haraoui B. Sher I. Wahl S.M. Cell. Immunol. 1984; 85: 373-383Crossref PubMed Scopus (162) Google Scholar) were grown in RPMI 1640 medium containing 10% fetal calf serum (Sigma, catalog no. F 2442), 2 mm l-glutamine, 5 mm HEPES buffer, pH 7.3, and penicillin/streptomycin (50 IU/ml and 50 μg/ml, respectively). The T lymphocytes were activated for 72 h with phytohemagglutinin (1 μg/ml) and were subsequently made quiescent by washing and incubating for 24 h in RPMI 1640 medium containing 1% fetal calf serum before exposure to cytokines. Nb2 cells were quieted for 24 h in the above medium except that 10% gelded horse serum was substituted for fetal calf serum. The IL3-dependent murine Ba/F3 cell clones expressing various IL2Rβ mutants were generated and cultured as described previously (27Howard O.M.Z. Kirken R.A. Garcia G.G. Hackett R.H. Farrar W.L. Biochem. J. 1995; 306: 217-224Crossref PubMed Scopus (22) Google Scholar) in RPMI 1640 medium with 10% fetal calf serum supplemented with 1,300 units/ml hygromycin B (Sigma, catalog no. H 3274) and 2% WEHI-3B supernatant as a source of IL3. Cells were stimulated with 100 nm recombinant human IL2 (Hoffmann-La Roche) at 37 °C as indicated in the corresponding figure legends. Cell pellets were frozen at −70 °C. Frozen cells were thawed on ice and solubilized in lysis buffer (108 cells/ml) containing 10 mm Tris-HCl, pH 7.6, 5 mm EDTA, 50 mm NaCl, 30 mm sodium pyrophosphate, 50 mm sodium fluoride, 200 mm sodium orthovanadate, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg/ml leupeptin. Cell lysates were rotated end over end at 4 °C for 60 min, and insoluble material was pelleted at 12,000 × gfor 20 min. Depending on the experiment, supernatants were incubated by rotating end over end for 2 h at 4 °C with either Stat5a or Stat5b antibodies (5 μl/ml). Antibodies were captured by incubation for 30 min with protein A-Sepharose beads (Pharmacia Biotech Inc.). Precipitated material was eluted by boiling in SDS-sample buffer for 4 min and was subjected to 7.5% SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membrane (Immobilon, Millipore, catalog no. 1PVH 00010) as described previously (28Kirken R.A. Rui H. Malabarba M.G. Farrar W.L. J. Biol. Chem. 1994; 269: 19136-19141Abstract Full Text PDF PubMed Google Scholar). Nb2-11C, phytohemagglutinin-activated human T lymphocytes, or Ba/F3 cells were metabolically labeled with 0.75 mCi/ml [32P]orthophosphate (DuPont NEN) for 2 h at 37 °C and stimulated with 100 nm IL2 for up to 60 min. Cells used for kinase inhibitor experiments were pre-incubated for 1 h with either Me2SO as a mock control, 100 μmPD98059 (New England Biolabs, Inc., catalog no. 9900L), 10 nm rapamycin (Calbiochem, catalog no. 553210-Q), or 100 nm wortmannin (Calbiochem, catalog no. 681675-Q). After treatment with IL2 (see figure legends for times), cells were lysed and immunoprecipitated as described above. Proteins were eluted from protein A-Sepharose beads, separated on SDS-PAGE (7.5% polyacrylamide), and transferred to polyvinylidene difluoride membranes. Labeled proteins were visualized by autoradiography and analyzed by phosphoamino acid analysis as described previously (29Rui H. Kirken R.A. Farrar W.L. J. Biol. Chem. 1994; 269: 5364-5368Abstract Full Text PDF PubMed Google Scholar). Labeled Stat5a and Stat5b proteins were excised from polyvinylidene difluoride membranes and exposed to limited hydrolysis in 6n HCl at 110 °C for 90 min. Samples were then dried, resuspended in water with phosphoamino acid standards, and spotted onto a thin layer cellulose acetate gel. One-dimensional thin layer electrophoresis was performed at 1500 V for 40 min in buffer containing pyridine:acetic acid:water at a 10:100:1890 ratio. Standards were visualized with ninhydrin, and samples were analyzed by autoradiography. Densitometric quantitation of individual phosphoamino acids were performed using a Molecular Dynamics PhosphorImager:SF. Counts/min volumes were normalized against the background and plotted as arbitrary units. Ba/F3 cell clones expressing various IL2Rβ mutants and treated as described were pelleted by centrifugation and immediately solubilized in EMSA lysis buffer (20 mm HEPES, pH 7.0, 10 mm KCl, 1 mm MgCl2, 20% glycerol, 0.2% Nonidet P-40, 1 mm orthovanadate, 25 mm sodium fluoride, 200 μm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg/ml leupeptin. Lysates were incubated on ice for 20 min and clarified by centrifugation at 20,000 ×g for 20 min at 4 °C. For the EMSA (30Wilson K.C. Finbloom D.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11964-11968Crossref PubMed Scopus (81) Google Scholar), 1 μg of32P-labeled oligonucleotide corresponding to the β-casein gene sequence (5′-AGATTTCTAGGAATTCAATCC-3′) were generated by end labeling and incubated with 10 μg of protein from cellular lysates in 30 μl of binding mixture (50 mm Tris-Cl, pH 7.4, 25 mm MgCl2, 5 mm dithiothreitol, 50% glycerol) at room temperature for 20 min, and samples were pre-incubated with 1 μl of either normal rabbit serum or antisera specific to Stat5a or Stat5b transcription factors as indicated. Polyacrylamide gels (5%) containing 5% glycerol and 0.25 × Tris borate/EDTA were pre-run in 0.25 × Tris borate/EDTA buffer at 4–10 °C for 1.5 h at 270 V. After loading of samples, the gels were run at room temperature for approximately 3 h at 250 V. Gels were dried by heating under vacuum conditions and exposed to x-ray film (X-Omat, Kodak). To establish whether Stat5a and Stat5b are under control of an IL2-activated serine kinase, the phosphorylation status of Stat5a and Stat5b was first analyzed in activated human T lymphocytes and the rat Nb2 lymphoma cell line. Cells were metabolically labeled with [32P]orthophosphate and incubated with or without IL2 for 10 min (Fig. 1). Stat5a and Stat5b were individually immunoprecipitated from cell lysates and separated by SDS-PAGE (Fig. 1, lower panel). This was followed by phosphoamino acid analysis after acid hydrolysis of the isolated Stat5a and Stat5b proteins (Fig. 1, upper panel). As seen in Fig. 1, IL2 stimulated incorporation of phosphate into Stat5a and Stat5b in either cell type, and the accompanying phosphoamino acid analysis specifically established that both Stat5a and Stat5b were inducibly phosphorylated on serine and tyrosine but not on threonine residues (Fig. 1, upper panel). This was also the case in the human natural killer cell line YT (data not shown) and the mouse pro-B cell line Ba/F3 that had been stably transfected with human IL2Rβ (see Fig. 3). We therefore conclude that Stat5a and Stat5b are under the control of an IL2-activated serine kinase. Furthermore, similar results were obtained when the same cells (Nb2 and T lymphocytes) were stimulated with either IL7 or IL9 (data not shown) thus suggesting that combined tyrosine and serine phosphorylation of Stat5a and Stat5b represents a general mechanism of cytokine regulation.Figure 1IL2-induced serine and tyrosine phosphorylation of Stat5a and Stat5b in human T lymphocytes and in the rat Nb2-11C cell line. Phytohemagglutinin-activated human T lymphocytes or Nb2-11C cells were metabolically labeled with [32P]orthophosphate and stimulated with (+) or without (−) 100 nm IL2 for 10 min. The lower panelshows an autoradiography of Stat5a or Stat5b isolated from clarified cell lysates by immunoprecipitation with specific polyclonal antibodies (lanes a, b, e, and f, αStat5a; lanes c, d, g, andh, αStat5b). The proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and exposed to film. The arrow denotes Stat5a or Stat5b proteins. The molecular mass marker (kDa) is indicated on the left. Theupper panel shows the phosphoamino acid analysis of Stat5a or Stat5b proteins excised from the blot shown in the lower panel that were subjected to acid hydrolysis and thin-layer electrophoresis. Migrational locations of phosphoserine (P-Ser), phosphothreonine (P-Thr), or phosphotyrosine (P-Tyr) are circled and indicated on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3IL2 activation of the Stat5a/b serine kinase does not require the Shc binding site (Tyr338), the acid-rich domain, or the COOH terminus of IL2Rβ. Panel Areviews the structure of three human IL2Rβ mutants stably introduced into the murine IL3-dependent Ba/F3 cell line. The full-length (FL) receptor was not modified, the AD mutant had an internal deletion corresponding to amino acids Gln315-Pro384, and the BD mutant was truncated at the amino acid position Leu385. The inactive mutant SD, which is truncated at Gln314 (27Howard O.M.Z. Kirken R.A. Garcia G.G. Hackett R.H. Farrar W.L. Biochem. J. 1995; 306: 217-224Crossref PubMed Scopus (22) Google Scholar), is not shown. The diagram also depicts the relative position of the homology boxes 1 (Box1) and 2 (Box2), the acid-rich region (Acid) as well as the conserved tyrosine residues. Ba/F3 cells expressing IL2Rβ mutants FL, AD, and BD were metabolically labeled with [32P]orthophosphate and stimulated with (+) or without (−) 100 nm IL2 for 10 min. The lower section of Panel B represents an autoradiography of Stat5a (lanes a, b, e, f,i, and j, αStat5a) or Stat5b (lanes c, d, g, h, k, andl, αStat5b) isolated from clarified cell lysates by immunoprecipitation with specific polyclonal antibodies and separated by SDS-PAGE. The arrow denotes Stat5a or Stat5b. The molecular mass marker (kDa) is indicated on theleft. The upper section of Panel B shows the phosphoamino acid analysis of Stat5a or Stat5b that had been excised from the blot shown underneath and subjected to acid hydrolysis and thin-layer electrophoresis. Migrational locations of phosphoserine (P-Ser), phosphothreonine (P-Thr), or phosphotyrosine (P-Tyr) are circled and indicated on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess whether IL2-regulated phosphorylation of serine and tyrosine residues of Stat5a and Stat5b differed in extent and kinetics, we compared time courses of Stat5a and Stat5b phosphorylation status during IL2 stimulation of activated human T lymphocytes. Cells were metabolically labeled with [32P]orthophosphate and were then stimulated with IL2 for up to 60 min. Cell pellets were lysed and immunoprecipitated with antibodies to either Stat5a or Stat5b. SDS-PAGE analysis (Fig. 2 A, lower panel) showed that IL2 stimulated general phosphorylation of Stat5a (lanes a–f) and Stat5b (lanes g–l) within 1 min. Whereas incorporation of phosphate into Stat5a reached maximal levels within 10 min, Stat5b continued to incorporate phosphate during the entire 60-min period (Fig. 2 A, lower panel). This difference in IL2-induced phosphorylation of Stat5a and Stat5b could be resolved further by phosphoamino acid analysis. The corresponding analysis of individual phosphoamino acids is shown in the upper panel of Fig. 2 A. First, the results demonstrated that IL2 induced rapid serine and tyrosine phosphorylation of both proteins. In contrast, no threonine phosphorylation of Stat5a or Stat5b was detected over the entire time course. The isotope incorporated into serine and tyrosine residues was quantitated by PhosphorImager:SF analysis, and the results are plotted as line diagrams in Fig. 2 B. The incorporation of phosphate into tyrosine residues of Stat5a and Stat5b was rapid and reached maximal levels within 1–5 min. In contrast, incorporation of phosphate into serine residues was more protracted and differed kinetically between Stat5a and Stat5b. Whereas serine phosphorylation of Stat5a reached a plateau after 10 min, Stat5b phosphoserine continued to accumulate over the entire 60-min period. The continued increase in total Stat5b phosphate content beyond 10 min (Fig. 2 A, lower panel) could be ascribed to increasing levels of phosphoserine (Fig. 2, A and B). The data shown in Fig. 2 are also representative of a series of experiments that suggest that the extent of IL2-induced incorporation of phosphate into Stat5b is higher than that of Stat5a in activated human T lymphocytes. Parallel immunoblot analysis showed that the levels of Stat5a and Stat5b proteins in activated human T lymphocytes were comparable (not shown). A similar preferential induction of phosphorylation of Stat5b by IL2 was seen in Nb2 pre-T lymphoma cells (Fig. 1, lanes e–h), the human natural killer cell line YT (not shown), and the mouse pro-B cell line Ba/F3 (Fig.3 B). Further studies are needed to determine if this differential phosphorylation of Stat5a and Stat5b by IL2 is due to preferential selectivity of IL2 receptor complexes for Stat5b or whether Stat5b contains more phosphorylated serine residues. JAK3 and JAK1 are presumed to be the IL2-activated Stat5 tyrosine kinases. The temporal relationship between IL2-induced phosphorylation of Stat5a and Stat5b on tyrosine versus serine residues with rapid tyrosine phosphorylation and more protracted serine phosphorylation kinetics is consistent with an activation of JAK3/JAK1 before activation of the Stat5 serine kinase. However, serine and tyrosine phosphorylation of Stat5 molecules may occur independently. Since we previously had established that the COOH-terminal region of IL2Rβ was not required for JAK3 activation, we next investigated whether this domain was required for activation of the Stat5a/b serine kinase. We stably introduced a series of IL2Rβ variants into the murine IL3-dependent lymphoblastoma line Ba/F3 (27Howard O.M.Z. Kirken R.A. Garcia G.G. Hackett R.H. Farrar W.L. Biochem. J. 1995; 306: 217-224Crossref PubMed Scopus (22) Google Scholar). The structures of wild-type (FL) IL2Rβ and mutants AD and BD are reviewed in Fig. 3 A. Previous analysis had shown that Ba/F3 cells expressing wild-type (FL) receptors or mutant receptor forms devoid of either the acid-rich region (AD) or the COOH terminus (BD) were capable of mediating IL2-induced proliferation and Jak3 activation (9Kirken R.A. Rui H. Malabarba M.G. Howard O.M.Z. Kawamura M. O'Shea J.J. Farrar W.L. Cytokine. 1995; 7: 689-700Crossref PubMed Scopus (79) Google Scholar, 27Howard O.M.Z. Kirken R.A. Garcia G.G. Hackett R.H. Farrar W.L. Biochem. J. 1995; 306: 217-224Crossref PubMed Scopus (22) Google Scholar). Stable cell clones expressing these forms of IL2Rβ were metabolically labeled with [32P]orthophosphate and incubated with or without IL2 for 10 min. The cells were then lysed, and Stat5a or Stat5b were immunoprecipitated with appropriate antibodies. Analysis of immunoprecipitated Stat5 proteins by SDS-PAGE (Fig. 3 B,lower panel) showed that IL2-induced Stat5a/b phosphorylation was mediated by FL (lanes a–d), AD (lanes e–h), and BD (lanes i–l) forms of IL2Rβ. On the other hand, a mutant of IL2Rβ (designated SD) that lacks both the acid-rich region and the COOH terminus failed to mediate inducible phosphorylation of Stat5a or Stat5b (not shown). We have previously demonstrated that the SD mutant does not mediate JAK1/JAK3 activation or proliferative signals (9Kirken R.A. Rui H. Malabarba M.G. Howard O.M.Z. Kawamura M. O'Shea J.J. Farrar W.L. Cytokine. 1995; 7: 689-700Crossref PubMed Scopus (79) Google Scholar, 27Howard O.M.Z. Kirken R.A. Garcia G.G. Hackett R.H. Farrar W.L. Biochem. J. 1995; 306: 217-224Crossref PubMed Scopus (22) Google Scholar). Specific phosphoamino acid analysis of inducibly phosphorylated Stat5a and Stat5b proteins from IL2-stimulated Ba/F3 cells expressing either FL, AD, or BD receptors revealed that each of these biologically active receptor variants were correspondingly competent to mediate IL2-induced serine and tyrosine phosphorylation (Fig. 3 B, upper panel). The present observation that BD is capable of mediating Stat5a/b phosphorylation (Fig. 3 B, lanes i–l) is consistent with the previously proposed roles of Tyr392 and Tyr510 of IL2Rβ as essential Stat5 docking sites (9Kirken R.A. Rui H. Malabarba M.G. Howard O.M.Z. Kawamura M. O'Shea J.J. Farrar W.L. Cytokine. 1995; 7: 689-700Crossref PubMed Scopus (79) Google Scholar, 31Fuji H. Nakagawa Y. Schindler U. Kawahara A. Mori H. Gouilleux F. Groner B. Minami Y. Miyazaki T. Taniguchi T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5482-5486Crossref PubMed Scopus (193) Google Scholar,32Friedmann M.C. Migone
The interleukin (IL)-2 receptor system has previously been shown to signal through the association and tyrosine phosphorylation of Shc. This study demonstrates that the IL-2 receptor β (IL-2Rβ) chain is the critical receptor component required to mediate this effect. The use of IL-2Rβ chain deletion mutants transfected into a Ba/F3 murine cell model describes a requirement for the IL-2Rβ “acid-rich” domain between amino acids 315 and 384 for Shc tyrosine phosphorylation and receptor association. COS cell co-transfection studies of IL-2Rβ chain constructs containing point mutations of tyrosine to phenylalanine along with the tyrosine kinase Jak-1 and a hemagglutinin-tagged Shc revealed that the motif surrounding phosphorylated tyrosine 338 within the acid-rich domain of the IL-2Rβ is a binding site for Shc. Deletion of this domain has previously been shown to abrogate the ability of IL-2 to activate Ras but does not affect IL-2-dependent mitogenesis in the presence of serum. Proliferation assays of Ba/F3 cells containing IL-2Rβ chain deletion mutants in serum-free medium with or without insulin shows that deletion of the acid-rich domain does not affect IL-2-driven mitogenesis regardless of the culture conditions. This study thus defines the critical domain within the IL-2Rβ chain required to mediate Shc binding and Shc tyrosine phosphorylation and further shows that Shc binding and phosphorylation are not required for IL-2-dependent mitogenesis. Neither serum nor insulin is required to supplement the loss of induction of the Shc adapter or Ras pathways, which therefore suggests a novel mechanism for mitogenic signal transduction mediated by this hematopoietin receptor. The interleukin (IL)-2 receptor system has previously been shown to signal through the association and tyrosine phosphorylation of Shc. This study demonstrates that the IL-2 receptor β (IL-2Rβ) chain is the critical receptor component required to mediate this effect. The use of IL-2Rβ chain deletion mutants transfected into a Ba/F3 murine cell model describes a requirement for the IL-2Rβ “acid-rich” domain between amino acids 315 and 384 for Shc tyrosine phosphorylation and receptor association. COS cell co-transfection studies of IL-2Rβ chain constructs containing point mutations of tyrosine to phenylalanine along with the tyrosine kinase Jak-1 and a hemagglutinin-tagged Shc revealed that the motif surrounding phosphorylated tyrosine 338 within the acid-rich domain of the IL-2Rβ is a binding site for Shc. Deletion of this domain has previously been shown to abrogate the ability of IL-2 to activate Ras but does not affect IL-2-dependent mitogenesis in the presence of serum. Proliferation assays of Ba/F3 cells containing IL-2Rβ chain deletion mutants in serum-free medium with or without insulin shows that deletion of the acid-rich domain does not affect IL-2-driven mitogenesis regardless of the culture conditions. This study thus defines the critical domain within the IL-2Rβ chain required to mediate Shc binding and Shc tyrosine phosphorylation and further shows that Shc binding and phosphorylation are not required for IL-2-dependent mitogenesis. Neither serum nor insulin is required to supplement the loss of induction of the Shc adapter or Ras pathways, which therefore suggests a novel mechanism for mitogenic signal transduction mediated by this hematopoietin receptor.
Abstract Human T cell leukemia virus type 1 (HTLV-1) transforms cytokine-dependent T lymphocytes and causes adult T cell leukemia. Janus tyrosine kinase (Jak)3 and transcription factors Stat5a and Stat5b are essential for the proliferation of normal T cells and are constitutively hyperactivated in both HTLV-1-transformed human T cell lines and lymphocytes isolated from HTLV-1-infected patients; therefore, a critical role for the Jak3-Stat5 pathway in the progression of this disease has been postulated. We recently reported that tyrphostin AG-490 selectively blocked IL-2 activation of Jak3/Stat5 and growth of murine T cell lines. Here we demonstrate that disruption of Jak3/Stat5a/b signaling with AG-490 (50 μM) blocked the proliferation of primary human T lymphocytes, but paradoxically failed to inhibit the proliferation of HTLV-1-transformed human T cell lines, HuT-102 and MT-2. Structural homologues of AG-490 also inhibited the proliferation of primary human T cells, but not HTLV-1-infected cells. Disruption of constitutive Jak3/Stat5 activation by AG-490 was demonstrated by inhibition of 1) tyrosine phosphorylation of Jak3, Stat5a (Tyr694), and Stat5b (Tyr699); 2) serine phosphorylation of Stat5a (Ser726) as determined by a novel phosphospecific Ab; and 3) Stat5a/b DNA binding to the Stat5-responsive β-casein promoter. In contrast, AG-490 had no effect on DNA binding by p50/p65 components of NF-κB, a transcription factor activated by the HTLV-1-encoded phosphoprotein, Tax. Collectively, these data suggest that the Jak3-Stat5 pathway in HTLV-1-transformed T cells has become functionally redundant for proliferation. Reversal of this functional uncoupling may be required before Jak3/Stat5 inhibitors will be useful in the treatment of this malignancy.
