Interaction of Growth Hormone-activated STATs with SH2-containing Phosphotyrosine Phosphatase SHP-1 and Nuclear JAK2 Tyrosine Kinase

Abstract

Growth hormone (GH) rapidly stimulates tyrosine phosphorylation followed by serine/threonine phosphorylation of multiple cytoplasmic STAT transcription factors, including one, STAT5b, that is uniquely responsive to the temporal pattern of plasma GH stimulation in rat liver and is proposed to play a central role in the activation of male-expressed liver genes by GH pulses in vivo (Waxman, D. J., Ram, P. A., Park, S. H., and Choi, H. K. (1995) J. Biol. Chem. 270, 13262–13270). We now show that JAK2, the GH receptor-associated tyrosine kinase, is present both in the cytosol and in the nucleus in cultured liver cells and in rat liver in vivo and that GH-activated STAT3 but not STAT5b becomes associated with nuclear JAK2. GH is also shown to activate by 3–4-fold SHP-1, a phosphotyrosine phosphatase that contains twosrc homology 2 (SH2) domains. GH also induces nuclear translocation and binding of SHP-1 to tyrosine-phosphorylated STAT5b, suggesting that this GH-activated phosphatase may play a role in dephosphorylation leading to deactivation of nuclear STAT5b following the termination of a plasma GH pulse in male rat liver in vivo. No such association of SHP-1 with GH-activated STAT3 was detected, a finding that could help explain the marked desensitization of STAT3, but not STAT5b, to subsequent GH pulses following an initial GH activation event. Growth hormone (GH) rapidly stimulates tyrosine phosphorylation followed by serine/threonine phosphorylation of multiple cytoplasmic STAT transcription factors, including one, STAT5b, that is uniquely responsive to the temporal pattern of plasma GH stimulation in rat liver and is proposed to play a central role in the activation of male-expressed liver genes by GH pulses in vivo (Waxman, D. J., Ram, P. A., Park, S. H., and Choi, H. K. (1995) J. Biol. Chem. 270, 13262–13270). We now show that JAK2, the GH receptor-associated tyrosine kinase, is present both in the cytosol and in the nucleus in cultured liver cells and in rat liver in vivo and that GH-activated STAT3 but not STAT5b becomes associated with nuclear JAK2. GH is also shown to activate by 3–4-fold SHP-1, a phosphotyrosine phosphatase that contains twosrc homology 2 (SH2) domains. GH also induces nuclear translocation and binding of SHP-1 to tyrosine-phosphorylated STAT5b, suggesting that this GH-activated phosphatase may play a role in dephosphorylation leading to deactivation of nuclear STAT5b following the termination of a plasma GH pulse in male rat liver in vivo. No such association of SHP-1 with GH-activated STAT3 was detected, a finding that could help explain the marked desensitization of STAT3, but not STAT5b, to subsequent GH pulses following an initial GH activation event. Growth hormone (GH) 1The abbreviations used are: GH, growth hormone; STAT, signal transducer and activator of transcription; SHP,src homology 2 domain-containing phosphotyrosine phosphatase; PTP, phosphotyrosine phosphatase; GST, glutathioneS-transferase; GST-GHR, GST-GH receptor fusion protein; PBS, phosphate-buffered saline; JAK2, Janus protein tyrosine kinase 2. 1The abbreviations used are: GH, growth hormone; STAT, signal transducer and activator of transcription; SHP,src homology 2 domain-containing phosphotyrosine phosphatase; PTP, phosphotyrosine phosphatase; GST, glutathioneS-transferase; GST-GHR, GST-GH receptor fusion protein; PBS, phosphate-buffered saline; JAK2, Janus protein tyrosine kinase 2. regulates the transcription of a variety of genes that mediate its diverse effects on body growth and metabolic function. GH actions at the cellular level include direct mitogenic effects (1Isaksson O.G. Eden S. Jansson J.O. Annu. Rev. Physiol. 1985; 47: 483-499Crossref PubMed Google Scholar), insulin-like metabolic effects, and gene regulatory actions (2Carter-Su C. Schwartz J. Smit L.S. Annu. Rev. Physiol. 1996; 58: 187-207Crossref PubMed Scopus (275) Google Scholar). In liver, a major target of GH action, GH exerts both stimulatory and inhibitory effects on the expression of a wide range of gene products, including hormone receptors, secretory products, and enzymes such as cytochrome P450 (3Waxman D.J. J. Steroid Biochem. Mol. Biol. 1992; 43: 1055-1072Crossref PubMed Scopus (89) Google Scholar). An important early cellular response to GH is the rapid tyrosine phosphorylation of GH receptor at the cell surface catalyzed by JAK2, a Janus family protein tyrosine kinase (4Argetsinger L.S. Carter-Su C. Physiol. Rev. 1996; 76: 1089-1107Crossref PubMed Scopus (246) Google Scholar). This leads to the activation by tyrosine phosphorylation of intracellular signaling molecules termed STATs, SH2 domain-containing latent cytoplasmic transcription factors (5Darnell Jr., J. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4948) Google Scholar). The initial GH-activated STAT tyrosine phosphorylation event is followed by a STAT serine or threonine phosphorylation reaction that modulates the DNA-binding activity of the GH-activated STATs (6Ram P.A. Park S.-H. Choi H.K. Waxman D.J. J. Biol. Chem. 1996; 271: 5929-5940Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). STAT tyrosine phosphorylation is associated with STAT homo- or heterodimerization and translocation to the nucleus (5Darnell Jr., J. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4948) Google Scholar, 7Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1262) Google Scholar), enabling the hormone-activated STATs to interact with DNA enhancer elements that have been identified in several GH-responsive promoters (8Sliva D. Wood T.J.J. Schindler C. Lobie P.E. Norstedt G. J. Biol. Chem. 1994; 269: 26208-26214Abstract Full Text PDF PubMed Google Scholar, 9Subramanian A. Teixeira J. Wang J. Gil G. Mol. Cell. Biol. 1995; 15: 4672-4682Crossref PubMed Google Scholar, 10Galsgaard E.D. Gouilleux F. Groner B. Serup P. Nielsen J.H. Billestrup N. Mol. Endocrinol. 1996; 10: 652-660Crossref PubMed Scopus (91) Google Scholar).In liver, three distinct STAT proteins respond to GH, STATs 1, 3, and 5b (6Ram P.A. Park S.-H. Choi H.K. Waxman D.J. J. Biol. Chem. 1996; 271: 5929-5940Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 11Gronowski A.M. Rotwein P. J. Biol. Chem. 1994; 269: 7874-7878Abstract Full Text PDF PubMed Google Scholar, 12Waxman D.J. Ram P.A. Park S.-H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 13Gronowski A.M. Zhong Z. Wen Z. Thomas M.J. Darnell Jr., J.E. Rotwein P. Mol. Endocrinol. 1995; 9: 171-177Crossref PubMed Google Scholar), albeit with distinct dependence on GH concentration and with a differential sensitivity to the temporal pattern of plasma GH stimulation (6Ram P.A. Park S.-H. Choi H.K. Waxman D.J. J. Biol. Chem. 1996; 271: 5929-5940Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Moreover, whereas STAT5b can be repeatedly phosphorylated by the regular, repeated pulses of GH that characterize adult male rodents, STAT3 and STAT1 become desensitized with respect to GH-induced tyrosine phosphorylation following a single GH pulsein vivo in the hypophysectomized rat liver model (6Ram P.A. Park S.-H. Choi H.K. Waxman D.J. J. Biol. Chem. 1996; 271: 5929-5940Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). These differences between the STATs may, in part, relate to their distinct mechanisms of activation; STAT5b appears to be activated by JAK2 kinase after docking to one or more phosphotyrosine residues along GH receptor's cytoplasmic tail, whereas STAT3 activation is less reliant on interactions between the STAT and GH receptor's intracellular domain and may involve direct binding of STAT3 to tyrosine-phosphorylated residues on JAK2 kinase (14Yi W. Kim S.O. Jiang J. Park S.H. Kraft A.S. Waxman D.J. Frank S.J. Mol. Endocrinol. 1996; 10: 1425-1443PubMed Google Scholar, 15Smit L.S. Meyer D.J. Billestrup N. Norstedt G. Schwartz J. Carter-Su C. Mol. Endocrinol. 1996; 10: 519-533Crossref PubMed Scopus (181) Google Scholar, 16Sotiropoulos A. Moutoussamy S. Renaudie F. Clauss M. Kayser C. Gouilleux F. Kelly P.A. Finidori J. Mol. Endocrinol. 1996; 10: 998-1009Crossref PubMed Scopus (125) Google Scholar).Although several components of the GH signaling pathway have thus been identified, the precise mechanisms whereby GH-induced intracellular signals are terminated remain to be determined. These signal termination events are of particular biological importance in the case of GH, given the need for GH target cells to respond to intermittent plasma GH pulses, which stimulate a male pattern of long bone and whole body growth in male rodents (17Jansson J.O. Albertsson-Wikland K. Eden S. Thorngren K.G. Isaksson O. Acta Physiol. Scand. 1982; 114: 261-265Crossref PubMed Scopus (102) Google Scholar, 18Waxman D.J. Pampori N.A. Ram P.A. Agrawal A.K. Shapiro B.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6868-6872Crossref PubMed Scopus (232) Google Scholar). GH pulses also induce a pattern of liver gene transcription in male rat liver that is distinct from the one induced in female liver in response to continuous plasma GH activation (3Waxman D.J. J. Steroid Biochem. Mol. Biol. 1992; 43: 1055-1072Crossref PubMed Scopus (89) Google Scholar, 19Sundseth S.S. Alberta J.A. Waxman D.J. J. Biol. Chem. 1992; 267: 3907-3914Abstract Full Text PDF PubMed Google Scholar, 20Legraverend C. Mode A. Westin S. Strom A. Eguchi H. Zaphiropoulos P.G. Gustafsson J.-A. Mol. Endocrinol. 1992; 6: 259-266Crossref PubMed Scopus (117) Google Scholar). Of the GH-responsive STATs, STAT5b is uniquely responsive to the pulsatile pattern of plasma GH found specifically in adult male rats and is proposed to be a key intracellular mediator of the physiological effects of GH pulses on male liver gene expression (12Waxman D.J. Ram P.A. Park S.-H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). This hypothesis is given strong support by our recent finding that targeted disruption of the STAT5b gene in mice leads to complete loss of male-specific liver gene expression, in addition to the feminization of whole body growth rates, a second important GH pulse-regulated biological response. 2Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,7239–7244 2Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,7239–7244Recent studies in CWSV-1 cells, a GH-responsive liver cell model (21Kempe K.C. Isom H.C. Greene F.E. Biochem. Pharmacol. 1995; 49: 1091-1098Crossref PubMed Scopus (21) Google Scholar), have revealed that phosphotyrosine phosphatases (PTPs) play an important role in GH signal termination (22Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar). Rapid deactivation of GH-activated STAT5b following cessation of a GH pulse was shown to involve STAT5b phosphotyrosine dephosphorylation as an essential first step. This phosphatase reaction is required to reset the GH receptor/JAK2/STAT5b signaling apparatus so that it may respond to subsequent rounds of GH pulse activation (22Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar). However, the PTPs involved in this key regulatory step have not been identified. PTPs that contain SH2 (phosphotyrosine-binding) domains have been proposed to play a role in signaling from activated receptors belonging to the cytokine receptor superfamily (23Streuli M. Curr. Opin. Cell Biol. 1996; 8: 182-188Crossref PubMed Scopus (164) Google Scholar), of which GH receptor is a member (24Goffin V. Kelly P.A. Clin. Endocrinol. 1996; 45: 247-255Crossref PubMed Scopus (52) Google Scholar). The PTP designated SHP-1 3Other designations used in the literature are: SHP-1: PTP-1C, SHPTP1, HCP, SHP; SHP-2: PTP-1D, SHPTP2, Syp, PTP-2C. 3Other designations used in the literature are: SHP-1: PTP-1C, SHPTP1, HCP, SHP; SHP-2: PTP-1D, SHPTP2, Syp, PTP-2C. negatively regulates signaling by several cytokine and growth factor receptors, apparently by promoting dephosphorylation of the receptor's intracellular domain and/or the receptor-associated JAK tyrosine kinase (25Klingmuller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (837) Google Scholar, 26D'Ambrosio D. Hippen K.L. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-297Crossref PubMed Scopus (507) Google Scholar, 27Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). A second SH2 domain PTP, SHP-2,3 acts as a positive signal transducer in several cytokine and growth factor receptor pathways, including prolactin receptor (28David M. Zhou G. Pine R. Dixon J.E. Larner A.C. J. Biol. Chem. 1996; 271: 15862-15865Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 29Ali S. Chen Z. Lebrun J.J. Vogel W. Kharitonenkov A. Kelly P.A. Ullrich A. EMBO J. 1996; 15: 135-142Crossref PubMed Scopus (128) Google Scholar, 30Sun H. Tonks N.K. Trends Biochem. Sci. 1994; 19: 480-485Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 31Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (490) Google Scholar). In the present study, we examine the role of PTPs in regulating GH pulse-dependent activation of STAT5b. We find that GH activates SHP-1, induces its translocation to the nucleus, and stimulates its association with GH-activated, tyrosine-phosphorylated STAT5b. We also show that a significant portion of JAK2 tyrosine kinase is nuclear, rather than cytoplasmic in liver cells, and that this nuclear JAK2 binds specifically to GH-activated STAT3 but not STAT5b in rat liver. The importance of these findings is discussed in the context of the signaling pathways induced by pulsatile GH in hepatocytes.DISCUSSIONGH is presently shown to activate the phosphotyrosine-specific phosphatase SHP-1, to stimulate its nuclear translocation, and to induce the selective binding of SHP-1 to GH-activated, tyrosine-phosphorylated STAT5b. These studies, carried out in liver cells in vivo and in the liver-derived cell culture model CWSV-1, suggest that SHP-1 may catalyze the phosphotyrosine dephosphorylation reaction which serves as the initial step of STAT5b deactivation (22Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar). SHP-1, an SH2 domain-containing protein-tyrosine phosphatase preferentially expressed in hematopoietic and epithelial cells, acts as a negative regulator of cell signaling by several cytokines and growth factors, including erythropoietin (25Klingmuller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (837) Google Scholar), interferon α (36David M. Chen H.E. Goelz S. Larner A.C. Neel B.G. Mol. Cell. Biol. 1995; 15: 7050-7058Crossref PubMed Scopus (317) Google Scholar), and antigen receptor (26D'Ambrosio D. Hippen K.L. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-297Crossref PubMed Scopus (507) Google Scholar), although instances of positive regulatory effects have also been reported (37Su L. Zhao Z. Bouchard P. Banville D. Fischer E.H. Krebs E.G. Shen S.-H. J. Biol. Chem. 1996; 271: 10385-10390Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In the case of erythropoietin, SHP-1 inhibits the signaling cascade by docking to the receptor's tyrosine-phosphorylated cytoplasmic tail, enabling SHP-1 to interact with and dephosphorylate, and thereby deactivate, JAK2 kinase (25Klingmuller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (837) Google Scholar). This inhibition by SHP-1 of erythropoietin signaling at the level of the cell surface receptor-JAK kinase complex would appear to be distinct from the effects of SHP-1 on GH signaling, where we were unable to detect a direct GH receptor-SHP-1 binding interaction. Rather, SHP-1 is proposed to down-regulate GH signals by dephosphorylation of STAT5b, a process that is critical for the rapid deactivation of STAT5b between plasma GH pulses in vivo and that enables STAT5b to recycle back to the cytoplasm in time to respond to an incoming GH pulse (12Waxman D.J. Ram P.A. Park S.-H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 22Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar). SHP-1 may thus participate in two fundamentally distinct signal desensitization processes. SHP-1-catalyzed dephosphorylation of cell surface receptors and/or JAK2 kinase blocks a catalytic cascade, whereby one growth factor receptor or JAK kinase dephosphorylation event prevents multiple downstream STAT5b activation events. By contrast, the dephosphorylation of GH-activated STAT5b by SHP-1 would correspond to a downstream inactivation event that is stoichiometric with respect to the STAT transcription factor. This deactivation of GH signaling at the level of STAT5b dephosphorylation does not, however, preclude the need for upstream step(s) to dephosphorylate GH receptor and to down-regulate GH-activated JAK2, which dephosphorylates within 45 min after GH addition in the case of liver cells (Fig. 6). These upstream deactivation events may be mediated by SHP-2, as suggested by the weak association that we have detected between SHP-2 and the tyrosine- phosphorylated GH receptor COOH terminus. 6P. A. Ram and D. J. Waxman, unpublished experiments. The mechanism by which SHP-1 binds to STAT5b is not yet known, but given the requirement for STAT5b tyrosine phosphorylation for binding to occur, this interaction is likely to involve a direct binding of phosphotyrosine residue 694 of STAT5b (38Gouilleux F. Wakao H. Mundt M. Groner B. EMBO J. 1994; 13: 4361-4369Crossref PubMed Scopus (524) Google Scholar) by one of the SH2 domains of SHP-1. SH2 domain occupancy leads to activation of catalytic activity for both SHP-1 and SHP-2 (26D'Ambrosio D. Hippen K.L. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-297Crossref PubMed Scopus (507) Google Scholar, 32Pei D. Lorenz U. Klingmuller U. Neel B.G. Walsh C.T. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (185) Google Scholar, 39Pluskey S. Wandless T.J. Walsh C.T. Shoelson S.E. J. Biol. Chem. 1995; 270: 2897-2900Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), and thus the binding of activated STAT5b per se could directly stimulate the GH-dependent increase in SHP-1 enzyme activity that we observed. Alternatively, other cellular molecules that undergo GH-induced tyrosine phosphorylation could be responsible for SHP-1 activation. SHP-1-STAT5b binding was seen with tyrosine-phosphorylated STAT5b and also with tyrosine + serine/threonine-diphosphorylated STAT5b, indicating that the secondary serine/threonine phosphorylation reaction is not required for binding to SHP-1. Further studies are needed to ascertain whether STAT5b directly activates SHP-1, and if so, whether the two phosphorylated STAT5b forms both activate SHP-1 to the same extent. A possible differential effect of the two phosphorylated STAT5b forms is suggested by the partial inhibitory effect that the serine/threonine kinase inhibitor H7 had on GH-induced SHP-1 activation (Fig. 2 A2). This possibility is consistent with our earlier conclusion that a cellular serine/threonine kinase activity is required for rapid STAT5b deactivation (22Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar).Only a portion of the cellular pool of activated STAT5b appears to be bound by SHP-1, as judged by the incomplete association of STAT5b with SHP-1 (Fig. 3 B and data not shown). This is not unexpected, insofar as the interaction of these two signaling molecules may be transient. Moreover, given the proposed catalytic role of SHP-1, whereby one molecule of SHP-1 could, in principle, dephosphorylate many STAT5b molecules, SHP-1 levels in the nucleus are likely to be sub-stoichiometric with respect to STAT5b. In this case, only a portion of the STAT5b pool would be stably associated with SHP-1 at any one time, allowing for the remaining STAT5b molecules to comprise a substrate pool.GH stimulated a 3–4-fold increase in SHP-1 activity, as judged by phosphotyrosine phosphatase assay of SHP-1 immunoprecipitates from GH-activated CWSV-1 cells. This activity increase was reversed within 1–2 h and may reflect activation resulting from the binding of STAT5b's phosphotyrosine residue to the amino-terminal SH2 domain of SHP-1. The observed activity increase could be a minimum estimate of the fold activation of SHP-1 in intact cells, as there may be some loss of STAT5b, or perhaps other bound activators during the immunoprecipitation step. In vitro activation assays using synthetic phosphotyrosine-containing peptides derived from cell surface receptors and other signaling molecules that bind to SHP-1 have revealed intrinsic fold activation values that range up to >25-fold (32Pei D. Lorenz U. Klingmuller U. Neel B.G. Walsh C.T. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (185) Google Scholar, 33Pei D. Wang J. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1141-1145Crossref PubMed Scopus (127) Google Scholar). However, this potential for a high degree of SHP-1 activation, evident in vitro, does not always translate into a corresponding major enhancement of SHP-1 activity in intact cells, as seen in the case of SHP-1 bound to the receptor for the Fc region of IgG (26D'Ambrosio D. Hippen K.L. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-297Crossref PubMed Scopus (507) Google Scholar). The 3–4-fold activation observed for SHP-1 in GH-treated cells could similarly underestimate the intrinsic fold activation of this PTP by the STAT5b-derived phosphotyrosine peptide. Recruitment of STAT5b to both the NH2-terminal and the COOH-terminal SH2 domain of SHP-1 would be expected to moderate the extent of SHP-1 activation compared with NH2-terminal SH2 binding interactions alone (33Pei D. Wang J. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1141-1145Crossref PubMed Scopus (127) Google is only stimulated by a GH pulse in liver in vivo (12Waxman D.J. Ram P.A. Park S.-H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). This for of plasma GH to induce of STAT5b activation. This response of liver STAT5b to GH pulses does not on protein (22Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar), of STAT5b dephosphorylation followed by The translocation of SHP-1 to the nucleus following GH stimulation and the association of SHP-1 with tyrosine-phosphorylated STAT5b in the present are thus likely to be not only for of the phosphatase to nuclear STAT5b, but also for of phosphatase activity in a that for the repeated activation of STAT5b stimulation of by plasma GH pulses in vivo. we have observed that GH pulses also to of SHP-1 activation and deactivation (Fig. 2 the hypophysectomized rat liver SHP-1 in the nucleus following GH after the deactivation and loss of nuclear STAT5b were complete (Fig. This is consistent with our that SHP-1 is present in the nucleus of male rat both at the time of a plasma GH pulse and during the GH STAT5b been and is present in the is also present in female liver albeit at a level than in the A and the that the STAT5b signaling pathway is and is in these in the case of GH rat liver or CWSV-1 cells cultured in the of we levels of nuclear SHP-1 and high levels of cytoplasmic SHP-1. We that the GH-induced nuclear translocation of SHP-1 is not on a nuclear translocation of STAT5b and thus may by a mechanism that is distinct from the direct tyrosine phosphorylation mechanism that induces STAT5b nuclear although tyrosine phosphorylation of SHP-1 and SHP-2 been observed in other 29Ali S. Chen Z. Lebrun J.J. Vogel W. Kharitonenkov A. Kelly P.A. Ullrich A. EMBO J. 1996; 15: 135-142Crossref PubMed Scopus (128) Google Scholar, 31Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (490) Google M. Pani G. Siminovitch K.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, and Science. 1993; 259: PubMed Scopus Google Scholar), we were unable to detect tyrosine phosphorylation SHP-1 in our experiments. it is that GH may induce SHP-1 tyrosine phosphorylation that could not be detected using our it more likely that SHP-1 nuclear translocation and its activation not involve SHP-1 tyrosine phosphorylation directly but rather a of SHP-1 following GH could involve its direct binding to tyrosine-phosphorylated signaling molecules, as by the SHP-1-STAT5b binding interaction observed in the present This possibility is an one, it a mechanism for deactivation of SHP-1 its tyrosine-phosphorylated substrate been GH pulse of cultured liver cells leads to a of SHP-1 activation, followed by deactivation within 1–2 h a time that both the of STAT5b activation and deactivation (Fig. 3 and the binding of STAT5b to SHP-1 (Fig. 3 studies have shown that the three GH-activated liver STATs, STATs 1, 3 and in their to GH in several important These include the dependence of STAT5b, but not STATs and 3, on the temporal pattern of GH activation, the activation of STAT5b, as compared with the other STATs, at physiological GH and the marked desensitization of STATs and 3, but not STAT5b, following a single GH pulse (6Ram P.A. Park S.-H. Choi H.K. Waxman D.J. J. Biol. Chem. 1996; 271: 5929-5940Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). These differences may, in part, from differences in the mechanisms which these STATs become and following GH STAT5b becomes tyrosine-phosphorylated as a of its direct binding to the COOH-terminal cytoplasmic of GH receptor (Fig. (14Yi W. Kim S.O. Jiang J. Park S.H. Kraft A.S. Waxman D.J. Frank S.J. Mol. Endocrinol. 1996; 10: 1425-1443PubMed Google Scholar, 15Smit L.S. Meyer D.J. Billestrup N. Norstedt G. Schwartz J. Carter-Su C. Mol. Endocrinol. 1996; 10: 519-533Crossref PubMed Scopus (181) Google Scholar, 16Sotiropoulos A. Moutoussamy S. Renaudie F. Clauss M. Kayser C. Gouilleux F. Kelly P.A. Finidori J. Mol. Endocrinol. 1996; 10: 998-1009Crossref PubMed Scopus (125) Google Scholar), STAT3 is more reliant on interactions with JAK2 for activation. This model is now by the association of JAK2 with STAT3 but not STAT5b (Fig. and by studies using JAK2 receptor (14Yi W. Kim S.O. Jiang J. Park S.H. Kraft A.S. Waxman D.J. Frank S.J. Mol. Endocrinol. 1996; 10: 1425-1443PubMed Google Scholar). A between the GH-activated STATs is from the present SHP-1 binding studies, where SHP-1 binding was observed in the case of activated STAT5b, but not activated SHP-1 likely to play an important role in the deactivation of STAT5b and its subsequent to the GH-activated STAT3 may be by a distinct leading to for activated STAT1 Science. 1996; PubMed Scopus Google Scholar), is a possible deactivation mechanism for of STATs and 3 could help explain the marked desensitization that both STAT proteins undergo following an initial GH activation event in vivo (6Ram P.A. Park S.-H. Choi H.K. Waxman D.J. J. Biol. Chem. 1996; 271: 5929-5940Abstract Full Text Full Text PDF PubMed Scopus (203) Google studies that a significant of JAK2 is expressed in the nucleus of liver cells in vivo (Fig. studies that following GH a portion of the cellular JAK2 from the cytoplasm to the nucleus these studies were in Lobie P.E. B.