Although it has been well documented that the biological activities of gamma interferon (IFN-gamma) are initiated through interaction with its cell surface receptor, the signal transduction mechanisms which mediate the effects of this cytokine have remained unclear. In order to facilitate a better understanding of IFN-gamma signaling, we have designed an assay using human fibroblast cell homogenates in which IFN-gamma activates the formation of the IFN-gamma activation factor (GAF) transcription complex. GAF mediates the rapid transcriptional activation of the guanylate-binding protein gene by IFN-gamma. Activation of GAF in homogenates required ATP, but not Ca2+ or GTP. Fractionation of homogenates indicated that both the pellet (18,000 x g) and the remaining cytoplasmic fraction were required for GAF activation by IFN-gamma. In intact cells and cell homogenates, the activation of GAF was prevented by the specific tyrosine kinase inhibitor genistein. Treatment of GAF-containing nuclear extracts with either monoclonal antiphosphotyrosine antibody or protein tyrosine phosphatase prevented the assembly of the transcription complex, indicating that its formation required phosphorylation of tyrosine residues. Furthermore, the tyrosine phosphatase inhibitors phenylarsine oxide and zinc chloride also inhibited GAF formation in vitro, but only if these agents were added to cell homogenates before IFN-gamma was added. The addition of either agent 5 min after IFN-gamma had no effect. These results provide the first evidence for an IFN-gamma-regulated tyrosine phosphatase/kinase signaling cascade that permits this cytokine to activate the transcription of an early-response gene.
We have previously shown that in rat H4 hepatoma cells insulin enhances the nuclear transcription of p33 mRNA in a dose- and time-dependent manner, with no alteration in mRNA half-time (t½). Presumably, this effect is mediated by the cell surface receptor. In this report, we have investigated the effect of putative insulin mediator fractions which act to control metabolic events on p33 mRNA accumulation in these cells. Initial experiments originally demonstrated an insulin-like effect of an added putative metabolic fraction to enhance p33 mRNA concentrations. However, when the fetal calf serum supply was changed, the effect of insulin remained, but that of added mediator was no longer observed. After a series of experimental approaches designed to alter the permeability of the cell membrane, it was found that in the presence of increased Ca2+, the effect of mediator could again be observed. The present data demonstrate that the partially purified c AMP -dependent protein kinase/adenylate cyclase inhibitory putative mediator fractions from liver and muscle enhance p33 mRNA accumulation in intact H4 hepatoma cells by a mechanism that is differentiated from that of insulin. The action of the putative mediator is inhibited by cycloheximide, while the action of insulin itself is not. These results suggest that insulin may control nuclear transcription by multiple signaling mechanisms. Alternatively, the added putative metabolic mediator may not enter the cell in the presence of cycloheximide or is inactive as such within the cell and must first be converted to an active species by a step requiring protein synthesis. (Endocrinology123: 1559–1564, 1988)
The signal pathways that control effector function in human natural killer (NK) cells are little known. In this study, we have identified the critical role of the mitogen-activated protein kinase (MAPK) pathway in NK lysis of tumor cells, and this pathway may involve the mobilization of granule components in NK cells upon interaction with sensitive tumor target cells. Evidence was provided by biological, biochemical, and gene transfection methods. NK cell binding to tumor cells for 5 min was sufficient to maximally activate MAPK/extracellular signal–regulatory kinase 2 (ERK2), demonstrated by its tyrosine phosphorylation and by its ability to function as an efficient kinase for myelin basic protein. MAPK activation was achieved in NK cells only after contact with NK-sensitive but not NK-resistant target cells. In immunocytochemical studies, cytoplasmic perforin and granzyme B were both maximally redirected towards the tumor contact zone within 5 min of NK cell contact with tumor cells. A specific MAPK pathway inhibitor, PD098059, could block not only MAPK activation but also redistribution of perforin/granzyme B in NK cells, which occur upon target ligation. PD098059 also interfered with NK lysis of tumor cells in a 5-h 51Cr-release assay, but had no ability to block NK cell proliferation. Transient transfection studies with wild-type and dominant-negative MAPK/ERK2 genes confirmed the importance of MAPK in NK cell lysis. These results document a pivotal role of MAPK in NK effector function, possibly by its control of movement of lytic granules, and clearly define MAPK involvement in a functional pathway unlinked to cell growth or differentiation.
Transcription factors of the Stat gene family are selectively activated by many hormones and cytokines. Stat5 originally was cloned as a prolactin-stimulated DNA-binding protein, but is also activated by non-lactogenic cytokines in many cell types. The recent identification of two distinct Stat5 genes, which encode a 94-kDa Stat5a and a 92-kDa Stat5b as well as several lower molecular weight isoforms, suggests additional complexity and combinatorial possibilities for transcriptional regulation. We now report a biochemical analysis of prolactin activation of Stat proteins in Nb2 lymphocytes, which was associated with: 1) rapid tyrosine phosphorylation of Stat5a, Stat5b, a COOH-terminally truncated 80-kDa Stat5 form, Stat1α, and Stat3; 2) rapid and selective formation of Stat5a/b heterodimers, without involvement of Stat1α or Stat3; 3) marked serine, but not threonine phosphorylation of Stat5a and Stat5b; and 4) the appearance of two qualitatively distinct Stat5 protein complexes, which discriminated between oligonucleotides corresponding to the prolactin response elements of the β-casein and interferon regulatory factor-1 gene promoters. Collectively, our analyses showed that Stat5a and Stat5b respond similarly to prolactin receptor activation, but also suggested that the two genes have evolved unique properties that may contribute to the specificity of receptors that utilize Stat5 signaling proteins. Transcription factors of the Stat gene family are selectively activated by many hormones and cytokines. Stat5 originally was cloned as a prolactin-stimulated DNA-binding protein, but is also activated by non-lactogenic cytokines in many cell types. The recent identification of two distinct Stat5 genes, which encode a 94-kDa Stat5a and a 92-kDa Stat5b as well as several lower molecular weight isoforms, suggests additional complexity and combinatorial possibilities for transcriptional regulation. We now report a biochemical analysis of prolactin activation of Stat proteins in Nb2 lymphocytes, which was associated with: 1) rapid tyrosine phosphorylation of Stat5a, Stat5b, a COOH-terminally truncated 80-kDa Stat5 form, Stat1α, and Stat3; 2) rapid and selective formation of Stat5a/b heterodimers, without involvement of Stat1α or Stat3; 3) marked serine, but not threonine phosphorylation of Stat5a and Stat5b; and 4) the appearance of two qualitatively distinct Stat5 protein complexes, which discriminated between oligonucleotides corresponding to the prolactin response elements of the β-casein and interferon regulatory factor-1 gene promoters. Collectively, our analyses showed that Stat5a and Stat5b respond similarly to prolactin receptor activation, but also suggested that the two genes have evolved unique properties that may contribute to the specificity of receptors that utilize Stat5 signaling proteins. A variety of polypeptide hormones and cytokines use cytoplasmicsignal transducers and activators of transcription (Stats) 1The abbreviations used are:Statsignal transducers and activators of transcriptionPRLprolactinoPRLovine prolactinMAPKmitogen-activated protein kinasePAGEpolyacrylamide gel electrophoresismAbmonoclonal antibodyEMSAelectrophoretic mobility shift assay to regulate expression of specific genes (1Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1139) Google Scholar, 2Larner A.C. Finbloom D.S. Biochim. Biophys. Acta. 1995; 1266: 278-287Crossref PubMed Scopus (55) Google Scholar). The ability of individual cytokine receptors to activate overlapping, but distinct sets of homo- and heterodimerizing Stat proteins contributes to their signal specificity. For example, interferon-α activates Stat1, Stat2, and Stat3 and exerts antiviral and growth-inhibitory effects in target cells (3Davis E. Krishnan K. Yan H. Newcomb E.W. Krolewski J.J. Leukemia. 1996; 10: 543-551PubMed Google Scholar). Prolactin (PRL), on the other hand, activates Stat1, Stat3, and Stat5 (4Sidis Y. Horseman N.D. Endocrinology. 1994; 134: 1979-1985Crossref PubMed Scopus (28) Google Scholar, 5David M. Petricoin III, E.F. Igarashi K. Feldman G.M. Finbloom D.S. Larner A.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7174-7178Crossref PubMed Scopus (70) Google Scholar, 6DaSilva L. Rui H. Erwin R.A. Howard O.M.Z. Kirken R.A. Malabarba M.G. Hackett R.H. Larner A.C. Farrar W.L. Mol. Cell. Endocrinol. 1996; 117: 131-140Crossref PubMed Scopus (143) Google Scholar), and stimulates β-casein synthesis in mammary epithelial cells and proliferation of Nb2 lymphocytes (7Groner B. Altiok S. Meier V. Mol. Cell. Endocrinol. 1994; 100: 109-114Crossref PubMed Scopus (43) Google Scholar, 8Gout P.W. Beer C.T. Noble R.L. Cancer Res. 1980; 40: 2433-2436PubMed Google Scholar, 9O'Neal K.D. Yu-Lee L.-Y. Lymph. Cytokine Res. 1993; 12: 309-312PubMed Google Scholar).Two distinct Stat5 genes have recently been identified which encode the highly homologous 94-kDa Stat5a and 92-kDa Stat5b proteins, as well as shorter 78–80-kDa isoforms of each gene product (10Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (455) Google Scholar, 11Hou J. Schindler U. Henzel W.J. Wong S.C. McKnight S.L. Immunity. 1995; 2: 321-329Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 12Mui A.-L. Wakao H. O'Farrell A.-M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (537) Google Scholar, 13Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (712) Google Scholar, 14Azam 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 (299) Google Scholar). Interestingly, the COOH-terminally truncated forms of Stat5a and Stat5b have transdominant negative effects on transcription (15Moriggl R. Gouilleux-Gruart V. Jahne R. Berchtold S. Gartman C. Liu X.W. Hennighausen L. Sotiropoulos A. Groner B. Gouilleux F. Mol. Cell. Biol. 1996; 16: 5691-5700Crossref PubMed Scopus (246) Google Scholar, 16Wang D. Stravopodis D. Teglund S. Kitazawa J. Ihle J.N. Mol. Cell. Biol. 1996; 16: 6141-6148Crossref PubMed Scopus (225) Google Scholar). Similarly, shorter forms of Stat1 and Stat3 have been identified, and result from alternative splicing of mRNA (11Hou J. Schindler U. Henzel W.J. Wong S.C. McKnight S.L. Immunity. 1995; 2: 321-329Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 12Mui A.-L. Wakao H. O'Farrell A.-M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (537) Google Scholar, 17Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar, 18Schaefer T.S. Sanders L.K. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9097-9102Crossref PubMed Scopus (324) Google Scholar). This multiplicity of Stat isoforms adds further combinatorial possibilities to receptor-mediated transcriptional regulation. Thus, preferential activation of Stat5a over Stat5b by granulocyte colony-stimulating factor has suggested receptor selectivity in Stat5 recruitment (19Rosen R.L. Weinstock K.D. Chen G. Liu X.W. Hennighausen L. Finbloom D.S. Blood. 1996; 88: 1206-1214Crossref PubMed Google Scholar). Knowledge of how different combinations of Stat5 isoforms may be used by distinct receptors therefore becomes critical to our understanding of gene regulation by a large number of hormones and cytokines.Thus far all Stat proteins have been shown to require phosphorylation of a positionally conserved tyrosine residue corresponding to Tyr-701 of human Stat1, which in turn facilitates dimerization and binding to DNA response elements (20Shuai K. Stark G.R. Kerr I.M. Darnell Jr., J.E. Science. 1993; 259: 1694-1695Crossref PubMed Scopus (7) Google Scholar, 21Shuai K. Horvath C.M. Huang L.H. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (677) Google Scholar). Furthermore, inducible serine phosphorylation of Stat1α or Stat3 is needed for full transcriptional activation (22Wen Z.L. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1728) Google Scholar, 23Zhang X.K. Blenis J. Li H.C. Schindler C. Chenkiang S. Science. 1995; 267: 1990-1994Crossref PubMed Scopus (521) Google Scholar), and the mitogen-activated protein kinase (MAPK) p42ERK2 has been implicated in the phosphorylation of Stat1α (22Wen Z.L. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1728) Google Scholar, 24David M. Petricoin III, E. Benjamin C. Pine R. Weber M.J. Larner A.C. Science. 1995; 269: 1721-1723Crossref PubMed Scopus (528) Google Scholar). However, Stat5 homologues lack the putative MAPK phosphorylation site (X-Pro-X-Ser-Pro) corresponding to Ser-727 of human Stat1α (22Wen Z.L. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1728) Google Scholar). This finding raises the possibility that Stat5 activities are regulated different than other Stats.The present study specifically set out to define and compare the molecular activation of different Stat5 gene products by PRL in Nb2 lymphocytes, a well characterized model of PRL-induced cell proliferation and signal transduction. We now show that multiple forms of Stat5, including p94Stat5a, p92Stat5b, and a COOH-terminally truncated 80-kDa Stat5 isoform, undergo marked phosphorylation on tyrosine and serine residues in response to PRL. Furthermore, PRL induced rapid and selective heterocomplex formation without involving Stat1α and Stat3, which also became tyrosine-phosphorylated. Finally, we demonstrate the formation of a Stat5a/b-containing complex that bound equally well to oligonucleotide probes corresponding to the PRL-response elements of the β-casein and interferon regulatory factor (IRF1) gene promoters. In contrast, a slower migrating complex that contained Stat5b and not Stat5a bound exclusively to the β-casein promoter and not to the IRF-1 probe. Collectively, these observations demonstrate that the highly homologous Stat5a and Stat5b proteins respond biochemically in a similar manner to PRL receptor activation. However, the data also suggest that the two gene products, which differ most in their COOH-terminal transactivation domains, have evolved unique properties that may contribute specificity to the large number of Stat5-activating receptors. A variety of polypeptide hormones and cytokines use cytoplasmicsignal transducers and activators of transcription (Stats) 1The abbreviations used are:Statsignal transducers and activators of transcriptionPRLprolactinoPRLovine prolactinMAPKmitogen-activated protein kinasePAGEpolyacrylamide gel electrophoresismAbmonoclonal antibodyEMSAelectrophoretic mobility shift assay to regulate expression of specific genes (1Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1139) Google Scholar, 2Larner A.C. Finbloom D.S. Biochim. Biophys. Acta. 1995; 1266: 278-287Crossref PubMed Scopus (55) Google Scholar). The ability of individual cytokine receptors to activate overlapping, but distinct sets of homo- and heterodimerizing Stat proteins contributes to their signal specificity. For example, interferon-α activates Stat1, Stat2, and Stat3 and exerts antiviral and growth-inhibitory effects in target cells (3Davis E. Krishnan K. Yan H. Newcomb E.W. Krolewski J.J. Leukemia. 1996; 10: 543-551PubMed Google Scholar). Prolactin (PRL), on the other hand, activates Stat1, Stat3, and Stat5 (4Sidis Y. Horseman N.D. Endocrinology. 1994; 134: 1979-1985Crossref PubMed Scopus (28) Google Scholar, 5David M. Petricoin III, E.F. Igarashi K. Feldman G.M. Finbloom D.S. Larner A.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7174-7178Crossref PubMed Scopus (70) Google Scholar, 6DaSilva L. Rui H. Erwin R.A. Howard O.M.Z. Kirken R.A. Malabarba M.G. Hackett R.H. Larner A.C. Farrar W.L. Mol. Cell. Endocrinol. 1996; 117: 131-140Crossref PubMed Scopus (143) Google Scholar), and stimulates β-casein synthesis in mammary epithelial cells and proliferation of Nb2 lymphocytes (7Groner B. Altiok S. Meier V. Mol. Cell. Endocrinol. 1994; 100: 109-114Crossref PubMed Scopus (43) Google Scholar, 8Gout P.W. Beer C.T. Noble R.L. Cancer Res. 1980; 40: 2433-2436PubMed Google Scholar, 9O'Neal K.D. Yu-Lee L.-Y. Lymph. Cytokine Res. 1993; 12: 309-312PubMed Google Scholar). signal transducers and activators of transcription prolactin ovine prolactin mitogen-activated protein kinase polyacrylamide gel electrophoresis monoclonal antibody electrophoretic mobility shift assay Two distinct Stat5 genes have recently been identified which encode the highly homologous 94-kDa Stat5a and 92-kDa Stat5b proteins, as well as shorter 78–80-kDa isoforms of each gene product (10Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (455) Google Scholar, 11Hou J. Schindler U. Henzel W.J. Wong S.C. McKnight S.L. Immunity. 1995; 2: 321-329Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 12Mui A.-L. Wakao H. O'Farrell A.-M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (537) Google Scholar, 13Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (712) Google Scholar, 14Azam 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 (299) Google Scholar). Interestingly, the COOH-terminally truncated forms of Stat5a and Stat5b have transdominant negative effects on transcription (15Moriggl R. Gouilleux-Gruart V. Jahne R. Berchtold S. Gartman C. Liu X.W. Hennighausen L. Sotiropoulos A. Groner B. Gouilleux F. Mol. Cell. Biol. 1996; 16: 5691-5700Crossref PubMed Scopus (246) Google Scholar, 16Wang D. Stravopodis D. Teglund S. Kitazawa J. Ihle J.N. Mol. Cell. Biol. 1996; 16: 6141-6148Crossref PubMed Scopus (225) Google Scholar). Similarly, shorter forms of Stat1 and Stat3 have been identified, and result from alternative splicing of mRNA (11Hou J. Schindler U. Henzel W.J. Wong S.C. McKnight S.L. Immunity. 1995; 2: 321-329Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 12Mui A.-L. Wakao H. O'Farrell A.-M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (537) Google Scholar, 17Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar, 18Schaefer T.S. Sanders L.K. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9097-9102Crossref PubMed Scopus (324) Google Scholar). This multiplicity of Stat isoforms adds further combinatorial possibilities to receptor-mediated transcriptional regulation. Thus, preferential activation of Stat5a over Stat5b by granulocyte colony-stimulating factor has suggested receptor selectivity in Stat5 recruitment (19Rosen R.L. Weinstock K.D. Chen G. Liu X.W. Hennighausen L. Finbloom D.S. Blood. 1996; 88: 1206-1214Crossref PubMed Google Scholar). Knowledge of how different combinations of Stat5 isoforms may be used by distinct receptors therefore becomes critical to our understanding of gene regulation by a large number of hormones and cytokines. Thus far all Stat proteins have been shown to require phosphorylation of a positionally conserved tyrosine residue corresponding to Tyr-701 of human Stat1, which in turn facilitates dimerization and binding to DNA response elements (20Shuai K. Stark G.R. Kerr I.M. Darnell Jr., J.E. Science. 1993; 259: 1694-1695Crossref PubMed Scopus (7) Google Scholar, 21Shuai K. Horvath C.M. Huang L.H. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (677) Google Scholar). Furthermore, inducible serine phosphorylation of Stat1α or Stat3 is needed for full transcriptional activation (22Wen Z.L. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1728) Google Scholar, 23Zhang X.K. Blenis J. Li H.C. Schindler C. Chenkiang S. Science. 1995; 267: 1990-1994Crossref PubMed Scopus (521) Google Scholar), and the mitogen-activated protein kinase (MAPK) p42ERK2 has been implicated in the phosphorylation of Stat1α (22Wen Z.L. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1728) Google Scholar, 24David M. Petricoin III, E. Benjamin C. Pine R. Weber M.J. Larner A.C. Science. 1995; 269: 1721-1723Crossref PubMed Scopus (528) Google Scholar). However, Stat5 homologues lack the putative MAPK phosphorylation site (X-Pro-X-Ser-Pro) corresponding to Ser-727 of human Stat1α (22Wen Z.L. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1728) Google Scholar). This finding raises the possibility that Stat5 activities are regulated different than other Stats. The present study specifically set out to define and compare the molecular activation of different Stat5 gene products by PRL in Nb2 lymphocytes, a well characterized model of PRL-induced cell proliferation and signal transduction. We now show that multiple forms of Stat5, including p94Stat5a, p92Stat5b, and a COOH-terminally truncated 80-kDa Stat5 isoform, undergo marked phosphorylation on tyrosine and serine residues in response to PRL. Furthermore, PRL induced rapid and selective heterocomplex formation without involving Stat1α and Stat3, which also became tyrosine-phosphorylated. Finally, we demonstrate the formation of a Stat5a/b-containing complex that bound equally well to oligonucleotide probes corresponding to the PRL-response elements of the β-casein and interferon regulatory factor (IRF1) gene promoters. In contrast, a slower migrating complex that contained Stat5b and not Stat5a bound exclusively to the β-casein promoter and not to the IRF-1 probe. Collectively, these observations demonstrate that the highly homologous Stat5a and Stat5b proteins respond biochemically in a similar manner to PRL receptor activation. However, the data also suggest that the two gene products, which differ most in their COOH-terminal transactivation domains, have evolved unique properties that may contribute specificity to the large number of Stat5-activating receptors.
It has been previously demonstrated that growth hormone (GH)-stimulated tyrosine phosphorylation of Jak2 and Stat5a and Stat5b occurs in FDP-C1 cells expressing either the entire GH receptor or truncations of the cytoplasmic domain expressing only the membrane-proximal 80 amino acids. However, other receptor domains that might modulate rates of GH activation and inactivation of this cascade have not been examined. Here we have defined a region in the human GH receptor between amino acids 520 and 540 in the cytoplasmic domain that is required for attenuation of GH-activated Jak/Stat signaling. Immunoprecipitations with antibodies to Jak2 indicate that the protein tyrosine phosphatase SHP-1 is associated with this kinase in cells exposed to GH. To address the possibility that SHP-1 could function as a negative regulator of GH signaling, liver extracts from motheaten mice deficient in SHP-1 or unaffected littermates were analyzed for activation of Stats and Jak2. Extracts from motheaten mice displayed prolonged activation of the Stat proteins as measured by their ability to interact with DNA and prolonged tyrosine phosphorylation of Jak2. These results delineate a novel domain in the GH receptor that regulates the inactivation of the Jak/Stat pathway and appears to be modulated by SHP-1. It has been previously demonstrated that growth hormone (GH)-stimulated tyrosine phosphorylation of Jak2 and Stat5a and Stat5b occurs in FDP-C1 cells expressing either the entire GH receptor or truncations of the cytoplasmic domain expressing only the membrane-proximal 80 amino acids. However, other receptor domains that might modulate rates of GH activation and inactivation of this cascade have not been examined. Here we have defined a region in the human GH receptor between amino acids 520 and 540 in the cytoplasmic domain that is required for attenuation of GH-activated Jak/Stat signaling. Immunoprecipitations with antibodies to Jak2 indicate that the protein tyrosine phosphatase SHP-1 is associated with this kinase in cells exposed to GH. To address the possibility that SHP-1 could function as a negative regulator of GH signaling, liver extracts from motheaten mice deficient in SHP-1 or unaffected littermates were analyzed for activation of Stats and Jak2. Extracts from motheaten mice displayed prolonged activation of the Stat proteins as measured by their ability to interact with DNA and prolonged tyrosine phosphorylation of Jak2. These results delineate a novel domain in the GH receptor that regulates the inactivation of the Jak/Stat pathway and appears to be modulated by SHP-1.
Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3 â/â cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the protein's function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.
The epidermal growth factor (EGF) receptor activates several signaling cascades in response to the ligands EGF and amphiregulin (AR). One of these signaling events involves the tyrosine phosphorylation of STATs (signal transducers and activators of transcription), a process believed to require the activation of a tyrosine kinase of the JAK family. In this report we demonstrate that EGF- and AR-induced STAT activation requires the intrinsic kinase activity of the receptor but not the presence of Jak1. We show that both wild type (WT) and truncated EGF receptors lacking all autophosphorylation sites activate STAT 1, 3, and 5 in response to either EGF or AR. Furthermore, relative to cells expressing WT receptor, ligand-induced tyrosine phosphorylation of the STATs was enhanced in cells expressing only the truncated receptor. These results provide the first evidence that (i) EGF receptor-mediated STAT activation occurs in a Jak1-independent manner, (ii) the intrinsic tyrosine kinase activity of the receptor is essential for STAT activation, and (iii) tyrosine phosphorylation sites within the EGF receptor are not required for STAT activation. The epidermal growth factor (EGF) receptor activates several signaling cascades in response to the ligands EGF and amphiregulin (AR). One of these signaling events involves the tyrosine phosphorylation of STATs (signal transducers and activators of transcription), a process believed to require the activation of a tyrosine kinase of the JAK family. In this report we demonstrate that EGF- and AR-induced STAT activation requires the intrinsic kinase activity of the receptor but not the presence of Jak1. We show that both wild type (WT) and truncated EGF receptors lacking all autophosphorylation sites activate STAT 1, 3, and 5 in response to either EGF or AR. Furthermore, relative to cells expressing WT receptor, ligand-induced tyrosine phosphorylation of the STATs was enhanced in cells expressing only the truncated receptor. These results provide the first evidence that (i) EGF receptor-mediated STAT activation occurs in a Jak1-independent manner, (ii) the intrinsic tyrosine kinase activity of the receptor is essential for STAT activation, and (iii) tyrosine phosphorylation sites within the EGF receptor are not required for STAT activation.
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