Tyrosine Docking Sites of the Rat Prolactin Receptor Required for Association and Activation of Stat5
1997; Elsevier BV; Volume: 272; Issue: 40 Linguagem: Inglês
10.1074/jbc.272.40.25043
ISSN1083-351X
AutoresAlain Pezet, Fatima Ferrag, Paul A. Kelly, Marc Edery,
Tópico(s)Protein Tyrosine Phosphatases
ResumoProlactin (PRL) interacts with a single chain prolactin-specific receptor of the cytokine receptor superfamily. PRL triggers activation of Jak2 kinase which phosphorylates the PRL receptor itself and the mammary gland factor, Stat5, a member of the family of signal transducers and activators of transcription (Stat). Selection of the particular substrate (Stat 5), that is characterized by transcriptional responses to PRL, has been shown to be determined by specific tyrosine-based motifs common to many cytokine receptors. PRL-induced activation of Stat5 was abolished in 293 fibroblasts expressing PRL receptor mutants lacking all intracellular tyrosines. We have identified tyrosine phosphorylation sites of the PRL receptor (residues 580, 479, and 473) necessary for maximal Stat5 activation and subsequent Stat5-dependent gene transcription. Moreover, we have shown that none of the tyrosine residues of the PRL receptor are implicated in activation of Jak2. This study demonstrates that only specific tyrosines in the PRL receptor are phosphorylated and are in fact utilized differentially for Stat5-mediated transcriptional signaling. Prolactin (PRL) interacts with a single chain prolactin-specific receptor of the cytokine receptor superfamily. PRL triggers activation of Jak2 kinase which phosphorylates the PRL receptor itself and the mammary gland factor, Stat5, a member of the family of signal transducers and activators of transcription (Stat). Selection of the particular substrate (Stat 5), that is characterized by transcriptional responses to PRL, has been shown to be determined by specific tyrosine-based motifs common to many cytokine receptors. PRL-induced activation of Stat5 was abolished in 293 fibroblasts expressing PRL receptor mutants lacking all intracellular tyrosines. We have identified tyrosine phosphorylation sites of the PRL receptor (residues 580, 479, and 473) necessary for maximal Stat5 activation and subsequent Stat5-dependent gene transcription. Moreover, we have shown that none of the tyrosine residues of the PRL receptor are implicated in activation of Jak2. This study demonstrates that only specific tyrosines in the PRL receptor are phosphorylated and are in fact utilized differentially for Stat5-mediated transcriptional signaling. The prolactin receptor (PRLR) 1The abbreviations used are: PRLR, prolactin receptor; EPOR, erythropoietin receptor; GHR, growth hormone receptor; IL-2Rβ, interleukin-2 receptor β chain; Nb2R, PRLR form expressed in Nb2 cells; EPO, erythropoietin; hGH, human growth hormone; IL, interleukin; PRL, prolactin; oPRL, ovine prolactin; IRS-1, insulin receptor substrate 1; Jak2, Janus kinase 2; Stat, signal transducer and activator of transcription; SH2, Src homology domain 2; WT, wild type; TK, thymidine kinase. is a member of the cytokine receptor superfamily (1Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1881) Google Scholar). Members of this family are devoid of any catalytic domain in the cytoplasmic region and are known to associate with cytoplasmic tyrosine kinases of the Jak family that are necessary for signal transduction. The PRLR is constitutively associated with the tyrosine kinase Jak2 (2Rui H. Kirken R.A. Farrar W.L. J. Biol. Chem. 1994; 269: 5364-5368Abstract Full Text PDF PubMed Google Scholar). In BaF3 cells, prolactin (PRL) also induces activation of Jak1 (3Dusanter-Fourt I. Muller O. Ziemiecki A. Mayeux P. Drucker B. Djiane J. Wilks A. Harper A.G. Fischer S. Gisselbrecht S. EMBO J. 1994; 13: 2583-2591Crossref PubMed Scopus (134) Google Scholar) and in Chinese hamster ovary cells Jak3 may interact with Jak2. Downstream of Jak2 activation, PRL induces tyrosine phosphorylation and activation of other cytoplasmic signaling proteins including signal transducers and activators of transcription (Stats). Stat1, Stat3, and Stat5 can all be activated by PRL in Nb2, 32D, and T-47D cells (4DaSilva L. Rui H. Erwin R.A. Zack Howard O.M. Kirken R.A. Malabarba M.G. Hackett R.H. Larner A.C. Farrar W.L. Mol. Cell. Endocrinol. 1996; 117: 131-140Crossref PubMed Scopus (144) Google Scholar). Stat5, which was initially isolated as a factor that binds to DNA sequences essential for a lactogenic response (5Schmitt-Ney M. Doppler W. Ball R. Groner B. Mol. Cell. Biol. 1991; 11: 3745-3755Crossref PubMed Google Scholar), plays a critical role in regulating the expression of milk protein genes (6Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (717) Google Scholar). However, the expression of Stat5 is not restricted to the mammary gland. Recent studies indicate that there are at least two isoforms of Stat5 (a and b) (7Mui A.L-F. Wakao H. O'Farrell A-M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (541) Google Scholar, 8Silva C.M. Lu H. Day R.N. Mol. Endocrinol. 1996; 10: 508-518Crossref PubMed Scopus (62) Google Scholar, 9Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (462) Google Scholar) and that Stat5 can be activated upon stimulation with IL-2, IL-3, IL-5, granulocyte macrophage-colony stimulating factor, EPO, GH, and epidermal growth hormone. It has also been shown that Stat1 and Stat3 can be activated by Jak kinases (7Mui A.L-F. Wakao H. O'Farrell A-M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (541) Google Scholar, 10Wakao H. Harada N. Kitamura T. Mui A.L. Miyajima A. EMBO J. 1995; 14: 2527-2535Crossref PubMed Scopus (214) Google Scholar, 11Gouilleux F. Pallard C. Dusanter-Fourt I. Wakao H. Haldosen L-A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (333) Google Scholar). Jak2 is also able to phosphorylate Stat5 in vitro (12Gouilleux F. Wakao H. Mundt M. Groner B. EMBO J. 1994; 13: 4361-4369Crossref PubMed Scopus (529) Google Scholar). Stat proteins are presumed to be transiently recruited by activated cytokine receptor complexes via binding to specific phosphotyrosyl residues through their SH2 domain. Studies of the inhibitory activities of phosphotyrosine-containing peptides derived from the intracellular domain of the α chain of the interferon-γ receptor, the IL-4 receptor, the IL-10 receptor, or the EPO receptor provide evidence for a direct coupling of receptor and, respectively, Stat1, Stat6, Stat3, or Stat5 during the Stat activation cycle (13Greenlund A.C. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (376) Google Scholar, 14Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. Mcknight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (731) Google Scholar, 15Quelle F.W. Wang D. Nosaka T. Thierfelder W.E. Stravopodis D. Weinstein Y. Ihle J.N. Mol. Cell. Biol. 1996; 16: 1622-1631Crossref PubMed Scopus (242) Google Scholar, 16Weber-Nordt R.M. Riley J.K. Greenlund A.C. Moore K.W. Darnell J.E. Schreiber R.D. J. Biol. Chem. 1996; 271: 27954-27961Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In addition, Stat3 has been shown to associate with gp130, the signal transducing component common to IL-6, oncostatin M, ciliary neurotrophic factor, leukemia inhibitory factor, and IL-11 receptors. The regions of gp130 and leukemia inhibitory factor receptor β chain required for Stat3 activation have been defined as a small tyrosine containing motif YXXQ (17Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (869) Google Scholar). Studies of deleted or truncated mutant of IL-2, EPO, or GH receptor reported that cytoplasmic regions of this receptor other than the one required for Jak2 activation are necessary for a maximal Stat5 activation. This suggests that Stat5 activation may require binding of Stat5 to a receptor. Tyrosine phosphorylation of the PRLR is a necessary event for activation of gene transcription. A single phosphotyrosylated tyrosine in the Nb2 form of the PRLR (Nb2R), lacking 198 amino acids in the cytoplasmic domain, is required for PRL-mediated transcriptional induction of β-casein gene promoter. Indeed, mutation of the COOH-terminal tyrosine (Tyr382) of the Nb2R results in the absence of phosphorylation of the receptor and correlates with a complete loss of activation of gene transcription (18Lebrun J.J. Ali S. Goffin V. Ullrich A. Kelly P.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4031-4035Crossref PubMed Scopus (114) Google Scholar). However, mutation of the same residue in the long form of PRLR (Tyr580) leads to a variant receptor that retains ∼20% of transcriptional activity of wild type PRLR (18Lebrun J.J. Ali S. Goffin V. Ullrich A. Kelly P.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4031-4035Crossref PubMed Scopus (114) Google Scholar). These data are consistent with the hypothesis that the intermediary region of the cytoplasmic domain absent in the Nb2 form (amino acids 322–520) might contain other phosphorylated tyrosines involved in Stat5 recruitment, similar to the GH receptor where two regions of interaction with Stat5 have been identified; the region of residues 454–638 (19Sotiropoulos 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, 20Wang X. Darus C.J. Xu B.C. Kopchick J.J. Mol. Endocrinol. 1996; 10: 1249-1260PubMed Google Scholar, 21Xu B.C. Wang X. Darus C.J. Kopchick J.J. J. Biol. Chem. 1996; 271: 19768-19773Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 22Hansen L.H. Wang X. Kopchick J.J. Bouchelouche P. Nielsen J.H. Galsgaard E.D. Billestrup N. J. Biol. Chem. 1996; 271: 12669-12673Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and tyrosine 333 and 338 (23Smit L.S. Meyer D.J. Billestrup N. Norstedt G. Schwartz J. Carter-Su C. Mol. Endocrinol. 1996; 10: 519-533Crossref PubMed Scopus (184) Google Scholar). Furthermore, it has been reported that a region in the human GHR between amino acids 520 and 540 regulates the inactivation of the Jak/Stat pathway and appears to implicate SHP-1 (24Hackett R.H. Wang Y.D. Sweitzer S. Feldman G. Wood W.I. J. Biol. Chem. 1997; 272: 11128-11132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The cytoplasmic domain of the PRLR contains 9 tyrosines, among which 6 are found in the region that is deleted in the Nb2 form of the receptor and thus may be potentially specific to functions of the long form. In this paper, we have used site-directed mutagenesis to investigate the role of these individual tyrosine residues in the Jak-Stat pathway and their requirement for Stat5 activation. This study demonstrates that PRL stimulation of 293 cells transfected with the PRLR plasmid activates Stat5 and that three of the four COOH-terminal tyrosine residues of the intracellular domain of the PRLR are required for its complete activation. Each of these tyrosine residues is able to induce transcriptional activation of the luciferase reporter gene controlled by the lactogenic hormone response element that has been shown to bind Stat5 protein (11Gouilleux F. Pallard C. Dusanter-Fourt I. Wakao H. Haldosen L-A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (333) Google Scholar). Additionally, each of these mutant receptors co-immunoprecipitated with tyrosyl-phosphorylated Stat5, suggesting a direct interaction between Stat5 and the PRLR. However, in 293 cells overexpressing the kinase Jak2, receptor mutants expressing normally inactive forms are able to transmit a lactogenic signal, suggesting a direct activation and interaction between Stat5 and Jak2. Recombinant human GH was kindly provided by Dr. A. Ythier, Serono Aeres, Geneva, Switzerland; recombinant bovine GH was kindly provided by Dr. W. Brumbach, American Cynamile Co, Prinston, NJ. Ovine PRL was obtained from the National Hormone and Pituitary Program/National Institute of Diabetes and Digestive and Kidney Diseases, Baltimore, MD. The construction of mutants was carried out using the cDNA encoding the long form of the rat PRLR subcloned into pRc/CMV expression vector using its M13 origin of replication and the MK07 M13 helper phage. Single stranded DNA was generated for oligonucleotide-directed mutagenesis in CJ236 cells. The mutated cDNAs were confirmed by sequencing. Cells were grown in complete medium (Dulbecco's modified Eagle's medium nut F-12 medium containing 10% fetal calf serum). Six hours before transfection, cells were plated in a rich medium (two-thirds Dulbecco's modified Eagle's medium nut F-12, one-third Dulbecco's modified Eagle's medium 4.5 g/liter glucose containing 10% fetal calf serum). Cells were co-transfected using the calcium phosphate technique with the indicated amount of cDNA encoding either wild type or different mutant forms of PRLR and Jak2 and incubated overnight in 3% CO2 at 37 °C. After 24 h of expression, the cells were deprived of serum for an overnight period. 293 cells were split into 6-well plates (0.5 × 106 cells/well) before being transiently transfected with, for each well, 0.5 μg of PRLR cDNAs. Transfected cells were deprived of serum for an overnight period. Then cells were incubated with 100,000 cpm of125I-hGH and increasing concentrations (0 to 50 pm) of unlabeled ovine PRL (oPRL) in 1 ml of phosphate-buffered saline containing 0.5% BSA. Incubations were carried out at room temperature for 4 h. Cells were washed twice with ice-cold phosphate-buffered saline, solubilized in 1 ml of 0.5n NaOH and counted in a γ-counter. 125I-hGH was prepared using chloramine T; specific activity ranged from 75 to 120 μCi/μg. 293 cells were split into 6-well plates before being transiently transfected with 0.5 μg of pCH110 (β-galactosidase expression vector from Pharmacia), 0.1 μg of LHRE-TK-Luciferase (fusion gene carrying 6 copies of LHRE and the TK minimal promoter linked to the coding region of the luciferase gene) and 0.05 μg of plasmid pRc/CMV containing the different forms of PRLR cDNA. Co-transfected cells were incubated in a serum-free medium with or without 18 nmoPRL, 24 h, and then lysed. Luciferase activity was measured in relative light units and normalized for β-galactosidase activity. Each 100-mm culture dish of 293 cells was co-transfected with 2 μg of either wild type or mutant forms of PRLR cDNA, and 1 μg of the cDNA encoding the human tyrosine kinase Jak2. Cells were stimulated by oPRL (10−7m) for 10 min. Cells were subsequently lysed in 1 ml of lysis buffer (10 mm Tris-HCl, pH 7.5, 5 mm EDTA, 150 mm NaCl, 30 mm sodium pyrophosphate, 50 mm sodium fluoride, 1 mmNa3VO4, 10% glycerol, 0.5% Triton X-100) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 2 μg/ml leupeptin, 5 μg/ml aprotinin) for 15 min at 4 °C. Lysates were cleared of debris by centrifugation at 15,000 × g at 4 °C for 10 min. The lysates were incubated with anti-Jak2 antibody (1 μg/ml) (Upstate Biotechnology, Inc.) or with a monoclonal anti-PRLR antibody (U5, 5 μg/ml) and 15 μl of Protein A-Sepharose (Pharmacia Biotech, Inc.) overnight at 4 °C. Antibody complexes were washed in lysis buffer and boiled for 5 min in sample buffer (125 mm Tris, pH 6.8, 5% SDS, 10% β-mercaptoethanol, 20% glycerol). The amount of protein was equalized in all samples by measurement of protein concentration using the Bradford technique. Proteins, divided into 3 fractions, were separated on a SDS-polyacrylamide gel electrophoresis (7 or 6% gel), transferred onto polyvinylidene difluoride transfer membrane (Polyscreen™, NEN Life Science Products), and immunodetected with antibodies to anti-Jak2 (UBI, 1:5000), anti-PRLR (mAb U5, 5 μg/ml), anti-Stat5 (affinity 1:1000), or anti-phosphotyrosine (UBI, 1:4000) for 2 h at room temperature. Then, the membranes were incubated with an anti-rabbit or anti-mouse IgG-conjugated horseradish peroxidase (1:8000) for 1 h at room temperature and revealed by ECL detection system (Amersham). To investigate individually the role of each tyrosine residue in the cytoplasmic domain of the PRLR regarding Stat5 recruitment and the corresponding signaling pathway, several PRLR mutants were prepared (Fig.1): A, mutant 9F in which all tyrosines were replaced by phenylalanine; B, individual substitution of each phenylalanine for tyrosine (8F-Ynnn). The cDNA encoding the natural and mutant forms of the PRLR were transiently transfected in human 293 fibroblast cells to determine their binding capacity along with their ability to transmit a lactogenic signal by the activation of the LHRE-TK-Luciferase reporter construct. Additionally, mutants were assayed for PRL-stimulated tyrosyl phosphorylation of Jak2, which is associated with the PRLR and Stat5. To confirm that the PRLR constructs encoded proteins of the expected sizes, we analyzed the receptor proteins in solubilized membranes of transfected 293 cells. Western blots were performed after immnoprecipitation of the extracts with the anti-PRLR antibody U5. As shown in Fig. 2, the mutated PRLRs migrated as a protein of the expected size (∼92 kDa) and showed no evidence of unusual receptor cleavage that could contribute to any loss of activity. In order to determine the possible effect of tyosine replacement on cell surface PRLR expression, similar experiments using biotinylated PRL to purify specifically PRLR of cytoplasmic membrane were performed. The level of PRLR precipitated was similar for each PRLR variant (data not shown), suggesting that mutations of tyrosine residues have no effect on processing of PRLR. Specific binding of 125I-hGH to transiently transfected 293 cells demonstrated again that wild type and variant receptors were normally expressed. The number and affinity of PRL-binding sites were obtained with competition assays followed by Scatchard analysis (TableI). All clones exhibited a single class of binding sites with an apparent affinity of 2 to 5 nm−1 and expressed similar levels of cell surface receptor ranging from 33,000 to 66,000 sites per cell.Table IAssociation constants of PRL receptor mutants expressed in 293 cellsPRLRK a(nm−1)Sites/cellWT4.8 ± 0.555,800 ± 4,1009F6.3 ± 0.956,100 ± 3,8008F-Y5803.6 ± 0.941,700 ± 2,8008F-Y5154.4 ± 1.032,800 ± 2,9008F-Y4793.3 ± 1.345,600 ± 1,6008F-Y4735.8 ± 1.737,200 ± 3,7008F-Y4024.7 ± 0.545,500 ± 1,9008F-Y3463.7 ± 1.061,300 ± 3,1008F-Y3304.9 ± 1.151,400 ± 2,5008F-Y3094.4 ± 0.466,000 ± 2,4008F-Y2375.5 ± 0.837,200 ± 1,9004PA4.9 ± 0.633,800 ± 2,500The affinity constant K a and the number of of binding sites were calculated from the Scatchard analysis (Ligand program) of competition experiments using 293 cells expressing WT and mutant PRLR as described under "Materials and Methods." Values represent the mean ± S.E. of three independent experiments performed in duplicate. Open table in a new tab The affinity constant K a and the number of of binding sites were calculated from the Scatchard analysis (Ligand program) of competition experiments using 293 cells expressing WT and mutant PRLR as described under "Materials and Methods." Values represent the mean ± S.E. of three independent experiments performed in duplicate. To assess the transcriptional activity mediated by Stat5, we used the LHRE-TK-Luciferase reporter gene (19Sotiropoulos 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). In this construct, six copies of the lactogenic hormone response element (LHRE) were fused to the minimal promoter of the herpes simplex virus thymidine kinase gene (TK promoter) which governs the expression of the luciferase gene. LHRE is an element of the β-casein promoter that has been used for the affinity purification of Stat5 (6Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (717) Google Scholar), in addition, it is a PRL responsive enhancer (19Sotiropoulos 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). The cDNAs encoding mutated or wild type PRLRs were transiently cotransfected with the cDNA encoding the reporter gene in 293 cells. As shown in Fig.3, the activity associated with the wild type PRLR corresponds to a 27-fold luciferase induction in the presence of PRL. The mutated PRLR 8F-Y580 displayed ∼80% of the wild type receptor activity and the PRLR mutants 8F-Y479 and 8F-Y473 each retained ∼12% of the wild type receptor activity. In contrast, the other forms, particularly the 9F variant, failed to respond to PRL. The 4P-A mutant which is unable to interact with Jak2 (25Pezet A. Buteau H. Kelly P.A. Edery M. Mol. Cell. Endocrinol. 1997; 129: 199-208Crossref PubMed Scopus (56) Google Scholar) is not able to induce transcription of the reporter gene in response to PRL stimulation. To determine which PRLR tyrosines are involved in Stat5 activation, we analyzed the phosphorylation level of mutant PRLRs. In these experiments, the PRLR was immunoprecipitated with anti-PRLR antibody from cells expressing either the wild type or mutant receptors following PRL stimulation. The immunoprecipitates resolved by SDS-polyacrylamide gel electrophoresis were transferred to polyvinylidene difluoride filters and probed with the 4G10 anti-phosphotyrosine monoclonal antibody. As indicated in the Western blot in Fig. 4, two major bands of 95 and 92 kDa are co-immunoprecipitated with anti-PRLR antibody from PRL-treated 293 transfected cell lysates. To confirm that the 92- and 95-kDa tyrosine-phosphorylated bands are indeed phosphorylated PRLR and Stat5, respectively, and not other co-immunoprecipitated proteins, parallel samples of the same immunoprecipitate were probed with anti-PRLR or anti-Stat5. The PRLR monoclonal antibody revealed the 92-kDa protein (Fig. 2), while Stat5 antiserum detected a 95-kDa protein (Fig. 4, lower panel). The 8F-Y580 mutant, which retains 80% of transcriptional activity of wild type PRLR, also displayed a level of tyrosine phosphorylation of Stat5 that is comparable to that observed with the wild type PRLR. Stat5 phosphorylation was seen for mutants 8F-Y479 and 8F-Y473, which have reduced transcriptional activation, but to a lesser extent than that seen with wild type and 8F-Y580. No induction of Stat5 phosphorylation was detected with the other mutated receptors, which failed to induce transcriptional activity upon PRL stimulation. These results support the notion of an interaction between Stat5 and the PRLR, suggesting that 8F-Y479 and 8F-Y473 variants have a lower affinity for Stat5 than wild type and 8F-Y580 form. To determine whether Jak2 can restore PRL signaling of the mutated PRLRs, cDNAs for Jak2 were cotransfected with the receptor mutants and the LHRE-TK-Luciferase construct in 293 cells. As shown in Fig. 5 (upper panel), the basal level of luciferase activity was increased in cells overexpressing Jak2. However, the 4P-A mutant was still unable to transmit a lactogenic signal (50 versus 60 RLU/UβGal/h in response to PRL stimulation, representing a 1.2-fold induction in presence of Jak2). The other defective mutants, and most notably the 9F mutant, were able to partially activate the signaling pathway (fold induction ranged from 2.8 to 4.5). On the other hand, the three active mutants and the wild type receptor showed a lower fold induction when Jak2 was overexpressed, implying that the system was close to saturation. These results suggest that Jak2 is able to complement the absence of receptor tyrosines and interacts with and activates Stat5. To assess the importance of Jak2 for PRL-induced Stat5 activation, we compared the ability of wild type PRLR and 9F or 8F-Y479 mutant forms to mediate PRL-induced tyrosine phosphorylation of Stat5 (Fig. 5 lower panel). Overexpression of Jak2 in cells expressing mutated 8F-Y479 PRLR resulted in complexes that retained the ability to stimulate Stat5 at levels comparable to those observed with wild type receptor, while the 9F PRLR variant showed Stat5 activation, but at a decreased level. Based upon these observations, it appears that phosphotyrosyl residues of the receptor complex outside of the receptor, likely in Jak2, also contribute to the recruitment of Stat5 in response to PRL. However, the decrease in phosphorylation intensity of Stat5 in cells expressing 9F mutant suggests that Jak2 is less efficient than the receptor to activate Stat5. To further investigate the mechanisms involved in the Jak-Stat pathway, we evaluated whether different forms of the PRLR were able to phosphorylate Jak2. This was measured in 293 cells which were cotransfected with the cDNA encoding various PRLRs and Jak2. Lysates from unstimulated (−) or PRL-stimulated (+) 293 transfected cells were immunoprecipitated with Jak2 antibody, and further analyzed by Western blotting using anti-phosphotyrosine antibody. Fig.6 shows that Jak2 is tyrosyl-phosphorylated in response to PRL, except for the mutant 4P-A. As described previously (25Pezet A. Buteau H. Kelly P.A. Edery M. Mol. Cell. Endocrinol. 1997; 129: 199-208Crossref PubMed Scopus (56) Google Scholar), the presence of Jak2 in the immunoprecipitated complexes from unstimulated cells suggests preassociation of the kinase with the PRLR. Parallel samples of the same immunoprecipitates were immunoblotted with an antibody to Jak2, confirming the 130-kDa band as Jak2. In addition, it can be seen that comparable levels of Jak2 were present in all samples. Therefore, the defect associated with the 4P-A mutant is not due to an absence of Jak2. The prevailing hypothesis of signal transduction by members of the cytokine receptor family is that upon binding of ligand to its receptor, association and activation of one or more members of the Jak family of tyrosine kinases occurs (26Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (824) Google Scholar). The Jak kinases trans-phosphorylate each other as well as their associated membrane receptors. The latent cytoplasmic Stat proteins are recruited by the phosphotyrosines and serve as substrates for the Jak kinases. Subsequent to phosphorylation, the Stat proteins homo- or heterodimerize, translocate to the nucleus, and induce receptor-associated gene expression (27Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar). For various tyrosine kinase receptors such for epidermal growth hormone or platelet-derived growth factor, the tyrosine residues in the cytoplasmic region recruit specific signal transducers containing SH2 domains, (28Heldin C.H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1445) Google Scholar, 29Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar). Based on the ability of phosphotyrosine-containing peptides derived from the intracellular domain of different cytokine receptors to inhibit DNA complex formation with Stats, it has been proposed that activation of Stats occurs through direct receptor-Stat interactions. These include the activation of Stat1 by the interferon-γR (13Greenlund A.C. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (376) Google Scholar, 30Shuai K. Horvath C.M. Tsai Huang L.H. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (687) Google Scholar), Stat2 by the interferon-αR (31Leung S. Qureshi S.A. Kerr I.M. Darnell Jr., J.E. Stark G.R. Mol. Cell. Biol. 1995; 15: 1312-1317Crossref PubMed Google Scholar), Stat6 by the IL-4R (14Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. Mcknight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (731) Google Scholar), and Stat5 by the β chain of the IL-2R (32Lin J.-X. Migone T.-S. Tsang M. Friedman M. Weatherbee J.A. Zhou L. Yamauchi A. Bloom E.T. Mietz J. John S. Leonard W.J. Immunology. 1995; 2: 331-339Scopus (678) Google Scholar), or the EPOR (15Quelle F.W. Wang D. Nosaka T. Thierfelder W.E. Stravopodis D. Weinstein Y. Ihle J.N. Mol. Cell. Biol. 1996; 16: 1622-1631Crossref PubMed Scopus (242) Google Scholar). Functional analysis of systematic truncated and point mutated forms of receptors for GH and EPO have indicated that Stat proteins are required for hormone-induced gene transcription (19Sotiropoulos 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, 20Wang X. Darus C.J. Xu B.C. Kopchick J.J. Mol. Endocrinol. 1996; 10: 1249-1260PubMed Google Scholar, 21Xu B.C. Wang X. Darus C.J. Kopchick J.J. J. Biol. Chem. 1996; 271: 19768-19773Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 22Hansen L.H. Wang X. Kopchick J.J. Bouchelouche P. Nielsen J.H. Galsgaard E.D. Billestrup N. J. Biol. Chem. 1996; 271: 12669-12673Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 23Smit L.S. Meyer D.J. Billestrup N. Norstedt G. Schwartz J. Carter-Su C. Mol. Endocrinol. 1996; 10: 519-533Crossref PubMed Scopus (184) Google Scholar, 33Damen J.E. Wakao H. Miyajima A. Krosl J. Humphries R.K. Cutler R.L. Krystal G. EMBO J. 1995; 14: 5557-5568Crossref PubMed Scopus (264) Google Scholar, 34Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar). In addition, in vitroexperiments demonstrate an interaction between Stat5 and the COOH-terminal phosphorylated tyrosine residues (Tyr469, Tyr516) of the rbGH receptor (19Sotiropoulos A. Moutoussamy S. Renaudie F. Clauss M. Kayser C. Gouilleux F. Kelly P.A. Finidori J. Mol. Endocrinol. 1996; 10: 998-100
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