Prolactin receptor regulates Stat5 tyrosine phosphorylation and nuclear translocation by two separate pathways.

The SH2 domain containing signal transducers and activators of transcription (Stat proteins) are effector molecules downstream of cytokine receptors. Ligand/receptor engagement triggers Stat proteins tyrosine phosphorylation, dimerization, and translocation to the nucleus where they regulate gene transcription. Stat5, originally identified as a mammary gland growth factor, is an essential mediator of prolactin (PRL)-induced milk protein gene activation. Prolactin receptor (PRLR) is a member of the cytokine/growth hormone/PRL receptor superfamily. The mechanism through which PRLR modulates Stat5 tyrosine phosphorylation, nuclear translocation, and DNA binding was analyzed in HC11 cells, a mammary epithelial cell line, and 293-LA cells, a human kidney cell line stably overexpressing Jak2 kinase. We have found that in HC11 cells, Stat5 is specifically activated by PRL treatment, demonstrating that Stat5 is a physiological substrate downstream of PRLR. Furthermore, using different forms natural forms of the PRLR as well as receptor tyrosine to phenylalanine mutant forms, we determined that tyrosine phosphorylation of Stat5 is independent of PRLR phosphotyrosines. We established, however, that the C-terminal tyrosine of the PRLR Nb2 form, Tyr382, plays an essential positive role in PRLR-dependent Stat5 nuclear translocation and subsequently DNA binding. All together, our data propose a new model for activation of Stat5 through the PRLR, suggesting that Stat5 tyrosine phosphorylation and nuclear translocation are two separately regulated events.

Prolactin (PRL) 1 is a pituitary polypeptide hormone as well as a local growth factor that regulates several physiological functions such as reproduction, promotion of the growth and differentiation of the mammary gland, and immune function (1). PRL interacts with specific cell surface receptors expressed on different target tissues (reviewed in Ref. 2).The receptor for PRL (PRLR) belongs to a large group of receptors known as the cytokine/GH/PRL receptor superfamily, which includes the receptors for GH, EPO, granulocyte-macrophage colony-stimulating factor, and several interleukins (2,3). These receptors share common extracellular structural motifs such as two disulfide loops and intracellular such as the proline-rich Box1 homology domain. These receptors do not possess intrinsic kinase activity but signal through cytoplasmic protein tyrosine kinases of the Janus kinase family (Jak/Tyk kinases) and the Src-kinase family. Ligand binding to the cytokine/GH/PRL receptor family induces receptor dimerization and activation of the associated kinases. This leads in turn to tyrosine phosphorylation of multiple cellular proteins including the receptors themselves (4).
The downstream signaling molecules activated by this receptor family have not been completely elucidated. However, it has been shown that several of the SH2 domain containing molecules interact with cytokine receptors, e.g. phospholipid metabolizing enzymes, phospholipase C-␥, and the regulatory unit of phosphatidylinositol 3-kinase, protein tyrosine phosphatases SHP1 and SHP2, and adapter proteins Grb2, Shc, and IRS1 (5)(6)(7)(8)(9)(10)(11). Moreover, a family of SH2 domain containing transcription factors of 85-95 kDa, called signal transducers and activators of trasncription (Stat), have been identified as primary effector molecules for this receptor family (12). Rapidly upon receptor activation, tyrosine phosphorylation of Stat factors occurs leading to their homo-or heterodimerization and translocation to the nucleus where they induce transcription of cytokine responsive genes (13). The complete molecular events leading to Stat proteins activation are not fully understood. It has been suggested that phosphotyrosines on the receptor components may act as docking sites for the SH2 domains of Stat proteins (14,15), allowing them to be phosphorylated by Jak tyrosine kinase family, a process necessary for Stat proteins activation by cytokine receptors. Recent information, however, indicate that other signaling pathways might be involved for maximal induction of Stat proteins activity. For example, to fully activate Stat1␣, serine phosphorylation by mitogen-activated protein kinase is required (16).
The rat PRLR, a member of the cytokine/GH/PRL receptor family, exists in three natural forms; the long form, identified in the mammary epithelial and ovarian cells (17), the short form, characterized in liver cells (18), and a third intermediate form, found in rat T-lymphoma Nb2 cells called PRLR Nb2 form (19). The short form of the receptor results from alternative splicing of the long form, whereas the Nb2 form results from a deletion mutation of the long form. The membraneproximal events following PRLR activation have recently been clarified. PRL binding to its receptor triggers homodimerization of the PRLR and activation of the constitutively associated kinase, Jak2 (20 -22). Although this process lead to tyrosine phosphorylation of the PRLR long form and the PRLR Nb2 form, no tyrosine phosphorylation was observed for the PRLR short form (23). Furthermore, studies examining the mechanism through which PRLR regulate gene transcription have previously demonstrated that although the PRLR long and the Nb2 forms are able to transmit the signal of PRL to activate ␤-casein gene transcription, the short form was inactive in this biological assay system (24). We have further shown that PRLR signals through the coordinated action of Jak2 and a single tyrosine residue present on the PRLR long and Nb2 forms to activate ␤-casein gene transcription. Indeed, when the C-terminal tyrosine of the PRLR long form (Tyr 580 ) or of the PRLR Nb2 form (Tyr 382 ) was mutated to phenylalanine, signaling of the PRLR to ␤-casein gene promoter activation was impaired (23,25).
PRL has recently been shown to activate several Stat proteins such as Stat1, Stat3, and Stat5 (23,26,27). In particular, Stat5, for which two isoforms Stat5a and Stat5b differing in their C-terminal tail were characterized in mouse mammary gland (28), has been shown to be vital for hormonal induction of ␤-casein gene transcription in mammary gland of lactating animals (29) and in heterologous cell systems (30). Stat5 activation has also recently been shown to correlate with mitogenic signaling in response to PRL (31). Therefore, Stat5 appears to be a key player in PRL-induced gene activation and cell proliferation.
Stat5 has also been shown to be part of the signaling pathway for a number of cytokine receptors such as GHR, EPOR, IL3-R, granulocyte-macrophage colony-stimulating factor receptor, IL2-R, IL6-gp130, and epidermal growth factor receptor (reviewed in Ref. 32). The mechanism of activation of Stat5 through the different cytokine receptors remains elusive and controversial. Indeed, Stat5 tyrosine phosphorylation, DNA binding, and induction of transcription via the GHR requires certain phosphotyrosine residues on the receptor (33,34). Similarly, Stat5 activation downstream of the EPOR was found to be dependent on receptor phosphotyrosines (35)(36)(37). However, other studies have reported a mechanism of activation of Stat5 totally independent of receptor phosphotyrosines. For example, GHR phosphotyrosines-independent activation of Stat5 has been documented (38). Separate studies indicated that Stat5 tyrosine phosphorylation and Stat5-DNA binding activity through the gp130 subunit of the IL-6 receptor (39) and the granulocyte colony-stimulating factor receptor (40) is independent of receptor phosphotyrosines. Furthermore, direct Jak-Stat interaction has recently been implicated as an alternative mechanism for activation of Stat5 by cytokine receptors (39).Therefore, the mechanism of activation of Stat5 by the cytokine/GH/PRL receptor family and the possible role of receptor phosphotyrosines in this process remains to be elucidated.
In this paper, we examined the significance of tyrosine phosphorylation of the PRLR in activating Stat5 molecules. Our results indicate that although Stat5 tyrosine phosphorylation is regulated by PRLR-Jak2 activation, it is independent of PRLR tyrosine phosphorylation. We further found that the C-terminal tyrosine of the PRLR Nb2 form regulates Stat5 nuclear translocation. Together, our results indicate for the first time that Stat5 tyrosine phosphorylation and nuclear translocation are two separately regulated process.
Transient Transfection-Transfection was carried out as described earlier (23,25). Briefly, the human 293 clone stably expressing the tyrosine kinase Jak2 (clone LA) was grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium medium (Life Technologies, Inc.) containing 10% (v/v) fetal calf serum. Several hours before transfection, cells were plated in a rich medium (two parts Dulbecco's modified Eagle's medium/Ham's F-12 medium and one part Dulbecco's modified Eagle's medium containing glucose at 4.5 g/liter) containing 10% fetal calf serum. Approximately 5 ϫ 10 6 cells were then co-transfected with the cDNA encoding the different forms of RPLR (1 g each) and the cDNA for Stat5 (500 ng) by using the calcium phosphate technique. After 24 h of expression, the cells were starved by serum deprivation overnight.
Immunoprecipitations-Immunoprecipitations were carried out as described earlier (22). Briefly, protein extracts were immunoprecipitated for 2 h using polyclonal antibody to Stat5a and protein A-Sepharose beads. This antibody was used because immunoprecipitations were unsuccessful using the monoclonal antibody to Stat5. Precipitates were then separated on SDS-PAGE and probed with monoclonal antibody to phosphotyrosines (4G10) then stripped and reprobed with polyclonal antibody to Stat5a.
Total Cell Lysis and Western Blotting-Transiently co-trasfected 293-LA cells were stimulated with oPRL (1.5 g/ml) of for 5 min and then lysed in 300 l 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 mM sodium orthovanadate, 10% (v/v) glycerol, 0.5% Triton X-100) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, 5 g/ml aprotinin) for 5 min at 4°C. The lysates were then centrifuged at 12,000 ϫ g for 10 min at 4°C to remove insoluble material. Protein concentration was measured using the Bradford technique. Equal amounts of protein obtained by total lysis were loaded and run on an 8% SDS-PAGE. Western analysis was performed using monoclonal antibodies to phosphotyrosine, PRLR, or Stat5. Proteins were revealed using chemiluminescence (ECL kit from Amersham Corp.) following the manufacturer's instructions.
Nuclear Translocation-Procedure for obtaining nuclear extracts was described previously (30) with some modifications. Briefly, transiently co-transfected 293-LA cells or HC11 cells were collected by centrifugation, washed with phosphate-buffered saline, and then lysed in hypotonic buffer (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, 1 mM Na 3 VO 4 , 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, 2 g/ml leupeptin). Cells were incubated for 15 min and then vortexed vigorously and centrifuged at 12,000 ϫ g at 4°C. The pellet was washed once with cold phosphatebuffered saline, and then the nuclear extracts were obtained by adding a high salt buffer (25% glycerol, 420 mM NaCl,1.5 mM MgCl 2 , 0.2 mM EDTA, 1 mM Na 3 VO 4 , 20 mM NaF, 5 g/ml aprotinin, 2 g/ml leupeptin), shaken for 30 min at 4°C, and then centrifuged at 12,000 ϫ g for 5 min. Total nuclear proteins were separated on an 8% SDS-PAGE and transferred to nitrocellulose membrane, and Western blots were carried out using anti-Stat5 monoclonal antibody and revealed using chemiluminescence (ECL kit, Amersham Corp.).
Electrophoretic Mobility Shift Assay-EMSA was performed as described elsewhere (30). Briefly, nuclear extracts were prepared as described above. Binding reactions performed in binding buffer (10 mM HEPES-KOH, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol) contained nuclear extract (8 -10 g), end labeled double stranded DNA containing the Stat5 response element of the ␤-casein gene promoter (5 pmol) and nonspecific competitor polydeoxyinosinic-deoxycyctidylic acid (2 g). For supershifts, protein extracts were incubated on ice for 30 min with polyclonal antibody to Stat5a (1 g). We used this antibody because supershifts were unsuccessful when the monoclonal antibody to Stat5 was used. Samples were run on a 0.25ϫ TBE (45 mM Tris-borate, 1 mM EDTA), 5% polyacrylamide gel. The gel was dried and exposed to x-ray film at Ϫ80°C (Hyperfilm, Amersham Corp.).

RESULTS
PRL Activates Stat5a in HC11 Cells-Gene expression of milk proteins in mammary cells is under a complex hormonal control. A combination of insulin, glucocorticoids, and PRL is required for maximal hormonal induction of milk proteins, such as ␤-casein, in mammary organ and epithelial cell cultures (41,42). Stat5 activation has recently been shown to be the main intracellular mediator for activation of ␤-casein gene promoter (27). Therefore, we first evaluated the ability of PRL to induce Stat5 activation in HC11 cells, PRL-sensitive mouse mammary epithelial cells (27). Cells were starved in the presence of insulin and hydrocortisone, were either left untreated or treated with PRL (1.5 g/ml) for 10 min, and were lysed in lysis buffer. Cell lysates were immunoprecipitated using anti-Stat5a polyclonal antibody. Immune complexes were run on SDS-PAGE, transferred to nitrocellulose membranes, and immunodetected with monoclonal antibody to phosphotyrosines (4G10). As shown in Fig. 1A, no detectable tyrosine phosphorylation of Stat5 was observed under basal conditions. However, PRL stimulation of cells (10 min) rapidly induced tyrosine phosphorylation of Stat5. To confirm that equal amounts of Stat5 were immunoprecipitated, membrane was stripped and reprobed with polyclonal antibodies to Stat5a (Fig. 1B). These results indicate that in mammary cells Stat5 tyrosine phosphorylation is induced by PRL treatment. Furthermore, these results indicate that the combinations of insulin and hydrocortisone are not competent to induce Stat5 tyrosine phosphorylation in the absence of PRL.
We then examined the kinetics of Stat5 nuclear translocation in HC11 cells in response to PRL stimulation. Cells were grown and stimulated for the indicated times. Cytoplasmic (Fig. 1C) and nuclear (Fig. 1D) extracts were then prepared from the same culture. As shown in Fig. 1D, the amount of Stat5 in the nucleus increases rapidly following PRL stimulation. Meanwhile, the amount of Stat5 present in the cytoplasm (Fig. 1C) decreases until eventually no detectable Stat5 was found in the cytoplasm following 10 min of PRL hormone treatment.
We next examined the ability of Stat5 to bind to the ␤-casein gene promoter. It has been shown previously that Stat5 binds to the Ϫ75/Ϫ104 region of the ␤-casein gene promoter (27). EMSA was performed using a primer of the sequence 5Ј-TGT GGA CTT CTT GGA ATT AAG GGA C-3Ј, and nuclear extracts were prepared from unstimulated or PRL-stimulated HC11 cells. As shown in Fig. 1E, PRL stimulation of HC11 cells results in Stat5 binding to the GAS-like element present on the ␤-casein gene promoter. This complex was supershifted in the presence of anti-Stat5a polyclonal antibody (third lane), indicating the specificity of the DNA binding activity. All together, using the mammary epithelial cell system, we show here that Stat5 activation in mammary cells follows PRL inductive effects.
Tyrosine Phosphorylation of Stat5 Is Independent of Receptor Phosphotyrosines of the PRLR Nb2 Form and ⌬296 -322 Mutant Form-Having established that PRL regulates Stat5 tyrosine phosphorylation, nuclear translocation, and DNA binding activity in mammary cells, we were interested in defining the mechanism through which PRLR mediate this effect. We have previously shown that C-terminal tyrosine of the PRLR is necessary for PRL activation of ␤-casein gene promoter (25). When this tyrosine was mutated to phenylalanine in the PRLR Nb2 form, ␤-casein gene promoter induction was strongly inhibited. Similar mutation of the C-terminal tyrosine on the PRLR long form, however, had less notable effects on ␤-casein gene activation.
To investigate the role of PRLR tyrosine phosphorylation in Stat5 activation, we used a heterologous overexpression system consisting of 293-LA cells, human kidney cell line stably overexpressing Jak2 kinase (23,25). We have previously shown that transient overexpression of PRLR in these cells leads to ligand-independent constitutive activation of the receptor-kinase complex. Here we have used this system to investigate the role of PRLR tyrosine phosphorylation in activation of Stat5.
The receptor natural and mutant forms used in our studies were described previously (23,25). The PRLR Nb2 form, found in Nb2 T-lymphoma cells, has an inframe deletion mutation compared with the PRLR long form (19 1. PRL stimulation induces Stat5 tyrosine phosphorylation and binding to the GAS sequence of the ␤-casein gene promoter in HC11 cells. HC11 cells were either nonstimulated (Ϫ) or stimulated (ϩ) with oPRL (1.5 g/ml) for 10 min. Cells were lysed, and immunoprecipitations were performed using polyclonal antibody to Stat5a. Immunoprecipitated (IP) proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes. Membrane was probed with monoclonal antibody to phosphotyrosines (A) and then stripped and reprobed with polyclonal antibody to Stat5a (B). Serumstarved HC11 cells were stimulated with oPRL (1.5 g/ml) for 2, 5, 10, 20, and 50 min. Total cytoplasmic extracts (C) and nuclear extracts (D) were prepared. Proteins (15 g) from each extract were separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with monoclonal antibody to Stat5. E, HC11 cells were prepared the same way as above and stimulated with oPRL (1.5 g/ml) for 10 min. Total nuclear extracts were prepared, and EMSA was performed using Stat5 response element of the ␤-casein gene promoter. The Stat5-DNA complex was supershifted (S.S.) with polyclonal antibody to Stat5a.
Nb2 mutant form ⌬243-268 is also used in our studies (23). This mutant form is missing a 25-amino acid region encompassing the Box1 motif, important for PRLR-Jak2 complex formation (23). Therefore, this receptor form is unable to activate Jak2 and is used here as a negative control for receptor/ Jak2 activation.
Point mutation of the C-terminal tyrosine (Tyr 382 ) in the PRLR Nb2 form and ⌬296 -322 mutant form was shown to inhibit receptor tyrosine phosphorylation and ␤-casein gene promoter activation (25). Consequently, we intended to determine the role of this tyrosine residue in Stat5 tyrosine phosphorylation. 293-LA cells were co-transfected with cDNA encoding Stat5 (27) and the PRLR Nb2 wild type (Nb2-WT); the PRLR Nb2 form in which tyrosine 237 was substituted by phenylalanine, NbY237F; the PRLR Nb2 form in which tyrosine 382 was mutated to phenylalanine, NbY382F; or ⌬243-268 as a negative control (Fig. 2). In parallel, 293-LA cells were co-transfected with cDNA encoding Stat5 and with the ⌬296 -322 receptor form; ⌬296 -322 in which tyrosine 237 was exchanged with phenylalanine, ⌬Y237F; receptor form ⌬296 -322 in which tyrosine 382 was mutated to phenylalanine, ⌬Y382F; or ⌬243-268 as a negative control (Fig. 2). Cells were then stimulated with PRL for 10 min before lysis. Total protein extracts were separated on SDS-PAGE, transferred to membranes, and immunodetected with monoclonal antibody to phosphotyrosines ( Fig. 2A). We observed that all natural and mutant forms of the PRLR except mutant ⌬243-268, which act as a negative control, are fully capable of inducing Jak2 and Stat5 tyrosine phosphorylation ( Fig. 2A). However, not all receptor types are themselves tyrosine phosphorylated. The NbY382F and the ⌬Y382F mutant receptor forms were not tyrosine phosphorylated in this system, similar to what was shown previously (25). To confirm that we have equal expression of receptors and Stat5 in the different samples, the membranes were stripped and reprobed with monoclonal antibodies to PRLR that can recognize all receptor forms (Fig. 2B). This was followed by reprobing the membranes with monoclonal antibodies to Stat5 (Fig. 2C). Together, these findings indicate that Stat5 is tyrosine phosphorylated following PRLR/Jak2 activation independently of PRLR phosphotyrosines.

Stat5 Nuclear Translocation Is Regulated by Tyrosine 382 of the PRLR Nb2 Form and the ⌬296 -322 Mutant Form-Follow-
ing Stat protein tyrosine phosphorylation on the conserved tyrosine residue, it is thought that Stat proteins dimerize and translocate to the nucleus where they induce gene transcription (12). Because mutation of tyrosine 382 of the PRLR Nb2 form and of the receptor ⌬296 -322 form did not influence Stat5 tyrosine phosphorylation but it was shown to play a significant role in regulating ␤-casein gene promoter induction (25), we studied its influence on Stat5 nuclear transloction event. For this purpose, 293-LA cells were co-transfected with the cDNAs encoding the PRLR Nb2 form or one of the Tyr 3 Phe mutants, NbY237F and NbY382F, along with the cDNA encoding Stat5 (Fig. 3A). In parallel, 293-LA cells were co-transfected with the cDNAs encoding the receptor ⌬296 -322 form or one of its Tyr 3 Phe mutants, ⌬Y237F or ⌬Y382F, along with the cDNA encoding Stat5 (Fig. 3B). Cells were then stimulated with PRL for 10 min before lysis and total nuclear extracts were analyzed with monoclonal antibody to Stat5. As shown in Figs. 3 (A and  B, upper panels), Tyr 3 Phe mutations of Tyr 237 in the two receptor forms did not affect nuclear translocation of Stat5 compared with wild type receptor forms. However, the amount of nuclear Stat5 immunodetected in samples overexpressing NbY382F and ⌬Y382F was dramatically reduced compared with that observed for wild type receptors. Indeed, for the mutant ⌬Y382F, the level of nuclear Stat5 was similar to that observed in samples in which the inactive mutant form ⌬243-268 was overexpressed (Fig. 3, A and B, upper panels). This inhibition in Stat5 nuclear translocation was not due to differences in the overexpression of Stat5 as indicated by Western blots with monoclonal antibody to Stat5 of total cell extracts from the same transfections (Fig. 3, A and B, lower panels). Similarly, receptor expression was equal in the different samples (data not shown). These results indicate that the C-terminal tyrosine residue of the PRLR Nb2 form is required for Stat5 nuclear translocation.   1 g). Similarly, 293-LA cells were transiently co-transfected with cDNAs encoding Stat5 (500 ng) and with ⌬296 -322, ⌬Y237F, ⌬Y382F, or the mutant ⌬ 243-268 (1 g) (B). Cells were stimulated with oPRL (1.5 g/ml) for 5 min. Total protein extracts were separated on SDS-PAGE, transferred to nitrocellulose membrane, and probed with monoclonal antibody to phosphotyrosines. B. membranes were stripped and reprobed with monoclonal antibody to PRLR. C, membranes were stripped and immunodetected with monoclonal antibody to Stat5.

Element of the ␤-Casein
Gene Promoter-To confirm the role of tyrosine 382 of the PRLR Nb2 form in regulating Stat5 nuclear entry, we next studied the importance of this tyrosine in regulating Stat5 DNA binding activity. We co-transfected 293-LA cells with the cDNA encoding Stat5 and with the cDNAs encoding either PRLR Nb2 wild type or its Tyr 3 Phe mutants (Fig. 4A). Similarly, we co-overexpressed in 293-LA cells Stat5 with either ⌬296 -322 receptor form or its Tyr 3 Phe mutants (Fig. 4B). Nuclear extracts were then prepared, and an EMSA binding reaction containing the ␤-casein gene promoter GAS sequence was performed. We found (Fig. 4, A and B) that overexpression of PRLR Nb2 wild type, ⌬296 -322 receptor form, and their Y237F mutant forms lead to the appearance of Stat5 DNA binding activity to the ␤-casein gene promoter in gel shift assays. However, Stat5-DNA interactions were greatly reduced in samples overexpressing the mutant receptor forms, NbY382F and ⌬Y382F. This is judged from the absence of DNA bound Stat5 compared with the wild type PRLR Nb2 form and ⌬296 -322 mutant form. Therefore, tyrosine 382 of the PRLR Nb2 form is important and necessary for Stat5 activation.
Tyrosine 580 of the PRLR Long Form Is Not Required for Stat5 Tyrosine Phosphorylation-The PRLR long form contains nine tyrosine residues within its cytoplasmic domain. It was shown previously that individual Tyr 3 Phe mutations, Tyr 237 and Tyr 580 , in the PRLR long form had no effect on the level of tyrosine phosphorylation of the PRLR (25). This suggests that other tyrosine residues of the PRLR may undergo tyrosine phosphorylation following PRLR/Jak2 activation. Functional analysis of these mutant receptors indicate that Tyr 580 is required for ␤-casein gene promoter activation, albeit less apparent than that observed for NbY382F mutant (25). To assess the role of Tyr 580 of PRLR long form in mediating Stat5 tyrosine phosphorylation, 293-LA cells were co-transfected with cDNA encoding Stat5 and cDNAs encoding PRLR long wild type; PRLR long in which tyrosine 237 was exchanged with phenylalanine, LY237F; PRLR long in which tyrosine 580 was mutated to phenylalanine, LY580F; or ⌬243-268 as a negative control (Fig. 5). Cells were starved overnight before being stimulated with PRL for 10 min. Total cellular proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes for Western blot analysis. Immunodetections with antibodies to phosphotyrosine indicate that the PRLR long form as well as the two Tyr 3 Phe mutants were able to mediate both Jak2 and Stat5 tyrosine phosphorylation compared with the negative control samples in which ⌬243-268 were overexpressed (Fig. 5A). This is consistent with what we were transiently co-transfected with cDNAs encoding Stat5 (500 ng) and with long wild type (Long WT), LY237F, LY382F, or the mutant ⌬243-268. Cells were stimulated with oPRL (1.5 g/ml) for 5 min. Total protein lysates were separated on SDS-PAGE, transferred to nitrocellulose membrane, and probed with monoclonal antibody to phosphotyrosines (A). Membrane was stripped and reprobed with monoclonal antibody to PRLR (B). Membrane was then stripped and immunodetected with monoclonal antibody to Stat5 (C). observed for the PRLR Nb2 form and ⌬296 -322 mutant form ( Fig. 2A). Furthermore, Fig. 5A also indicates that PRLR long form as well as the two Tyr 3 Phe mutant forms, LY273F and LY580F, are themselves tyrosine phosphorylated as shown previously (25). To confirm that receptor expression was equal in all samples, the membrane was stripped and reprobed with monoclonal antibodies to the extracellular domain of the PRLR (Fig. 5B). Similarly, to verify that Stat5 was equally expressed in the different samples, the membrane was stripped and immunodetected by monoclonal antibodies to Stat5 (Fig. 5C). These results demonstrate that Stat5 tyrosine phosphorylation is not modulated by either tyrosine 237 or tyrosine 580 of the PRLR long form.
Tyrosine 580 of the PRLR Long Form Does Not Significantly Influence Stat5 Nuclear Translocation-We next investigated the influence of the same Tyr 3 Phe mutants of PRLR long form on Stat5 nuclear translocation. 293-LA cell co-transfections were carried out with cDNAs encoding Stat5 and PRLR long wild type, LY237F, LY580F, or ⌬243-268 (Fig. 6). Cells were starved overnight before being stimulated by PRL for 10 min. Total nuclear extracts were prepared, then loaded on an SDS-PAGE, and transferred to membranes, and Western blot analysis was performed using anti-Stat5 monoclonal antibody. As shown in Fig. 6, mutation of either Tyr 237 or Tyr 580 in the PRLR long form, had no or very little effect on Stat5 nuclear translocation (Fig. 6, upper panel). These results are in contrast to that observed for the PRLR Nb2 form and ⌬296 -322 mutant form (Fig. 3) and suggest that additional tyrosine residues specific to the PRLR long form might substitute the C-terminal tyrosine in mediating Stat5 nuclear translocation. Indeed, our data are consistent with the fact that mutation of the last tyrosine in the long form only partially inhibited ␤-casein gene promoter activation (25).
Tyrosine 580 of the PRLR Long Form Does Not Influence Stat5 Binding to the Response Element of the ␤-Casein Gene Promoter-We then used the PRLR long form and its Tyr 3 Phe mutants, LY237F and LY580F, to study their effects on the binding activity of Stat5 to the ␤-casein gene promotor. For this reason, 293-LA cells were co-transfected with cDNA encoding Stat5 and cDNAs encoding the PRLR long wild type, LY237F, or LY580F. Nuclear extracts were prepared, and electrophoretic mobility shift assay was performed using the GAS sequence of the ␤-casein gene promoter. As shown in Fig. 7, we did not detect any Stat5 DNA binding activity in samples overexpressing the negative control mutant ⌬243-268. However, Stat5 DNA binding activity was detected at similar levels in samples overexpressing the PRLR long form and the two tyrosine mutant forms LY237F and LY580F. These results indicate that mutation of tyrosine 580 of the PRLR long form does not affect PRLR-regulated Stat5-DNA interactions. DISCUSSION The initial steps in the signaling mechanisms of the PRLR, a member of the cytokine receptor superfamily, have recently been elucidated. Following PRL/receptor engagement there is activation of cytoplasmic tyrosine kinases of the Jak and Src families. This process leads to activation and tyrosine phosphorylation of the kinase, the receptor and cellular effector molecules resulting in the transmission of PRL biological responses.
Here we investigated the mechanism through which PRLR regulate the activation of the signal transducer and activator of transcription, Stat5. In the HC11 mammary epithelial model system that we used in this study, we find that PRL is able to induce Stat5 tyrosine phosphorylation, nuclear translocation, and DNA binding to the GAS-like element of the ␤-casein gene promoter. This clearly demonstrate that Stat5 is a physiological downstream mediator in PRLR intracellular signaling pathway in mammary epithelial cells. This is consistent with the fact that PRL induces activation of Stat5 in overexpression system (30) and in the Nb2 rat T-lymphoma cell line (43).
Stat5 is a member of the Stat family of transcription factors downstream of cytokine receptors. Our understanding of the mechanism of activation of these factors in the signaling relay of cytokine receptors is limited. It has been shown in different systems, however, that these factors undergo tyrosine phosphorylation on a conserved C-terminal tyrosine residue necessary for their homo-or heterodimerization. This process leads to their translocation to the nucleus where they bind to specific response elements on target genes to regulate gene expression (4,12).
Stat5 is activated by a number of cytokines and growth factors (32). Studies on the role of the cytokine receptors to mediate Stat5 protein activation focused on events like Stat5 association to the receptor, tyrosine phosphorylation, and DNA binding. These studies indicated that Stat5 tyrosine phosphorylation and DNA binding via the GHR, EPOR, and IL-2R␤ chain requires receptor phosphotyrosines (33-37, 44 -46), whereas similar events mediated through the IL-6-gp130 receptor system and granulocyte colony-stimulating factor receptor were found to be independent of receptor phosphotyrosines Cultures were then stimulated with oPRL (1.5 g/ml) for 10 min. Nuclear extracts or total cell extracts were prepared from two setsϩ of cells that were simultaneously transfected. Proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes. Both membranes were probed with monoclonal antibody to Stat5.

FIG. 7.
Tyrosine 580 of the PRLR long form does not affect significantly Stat5 binding to the GAS sequence of the ␤-casein gene promoter. 293-LA cells (5 ϫ 10 5 cells) were transiently cotransfected with cDNAs encoding Stat5 and long wild type (Long-WT), LY237F, LY382F, or the mutant ⌬243-268 as described in legend to Fig. 6. Cells were stimulated with oPRL (1.5 g/ml) for 10 min. Nuclear extracts were prepared, and EMSA was performed using Stat5 response element of the ␤-casein gene promoter. The Stat5-DNA complex was supershifted (S.S.) with polyclonal antibody to Stat5a. (38 -39). Limited attempts, however, have been performed to study the effect of receptor phosphotyrosine residues on Stat protein nuclear translocation event and its regulation mechanism.
We report here for the first time that the C-terminal tyrosine residue of the PRLR does not influence Stat5 tyrosine phosphorylation, but it is required for regulating Stat5 nuclear translocation and DNA binding. Our data suggest a possible Jak2-Stat5 interaction that is direct or that is through an adapter protein may be taking place, allowing Stat5 tyrosine phosphorylation by the kinase Jak2 itself independently of PRLR tyrosine phosphorylation. This would be consistent with what was demonstrated for the Il-6-gp130 receptor system (38). We observed that Stat5 tyrosine phosphorylation did not grant its nuclear translocation. This implies that Stat5 activation via the PRLR involves at least two separately regulated events; tyrosine phosphorylation and nuclear translocation. Whether other members of the cytokine receptor superfamily share the same feature remains to be determined.
We also report that tyrosine 580 of the PRLR long, which is homologous to tyrosine 382 of the PRLR Nb2 form, has no effect on Stat5 nuclear translocation or DNA binding. A redundancy may be implicated in this tyrosine activity because the PRLR long form has six other tyrosine residues that can potentially substitute for the mutated tyrosine 580. Indeed, in contrast to the PRLR Nb2 form, mutation of tyrosine 580 of the PRLR long form does not lead to inhibition of receptor tyrosine phosphorylation, indicating that other tyrosines on the PRLR long form may undergo phosphorylation following receptor activation. In addition, mutation of the C-terminal tyrosine in the PRLR long form only partially inhibited ␤-casein gene promoter activation, suggesting again that other alternative tyrosine residues specific to the receptor long form might mediate PRL effects. Moreover, it has been recently suggested that tyrosines 509 and 496 of the PRLR long form in the context of gp130 truncation mutant to be potential sites for mediating Stat5 activation and DNA binding (47). Using myeloid cell system, however, a previous report suggested a mechanism for activation of Stat5 independent of PRLR phosphotyrosines (26). These seemingly controversial observations might be due to differences in the GAS-like element used in the study. In addition, it is possible that the PRL-induced DNA binding activity observed is that of Stat5-related proteins in myeloid cells (48).
The mechanism by which tyrosine 382 of the PRLR Nb2 form exerts its effect on Stat5 nuclear translocation is not known. We speculate that this phosphotyrosine residue is involved in the activation of a certain cellular component that modulates Stat5 complexes in the cytoplasm controlling their nuclear entry.
Accumulating evidence suggests that Stat proteins do not act in seclusion; rather they are a part of complex interactions with a number of other cellular components. For example, Stats1 and 3 are shown to be phosphorylated by mitogen-activated protein kinase on a serine residue adjacent to the conserved tyrosine phosphorylation site (49,50). Furthermore, Stat5 has been shown to be serine phosphorylated following PRL stimulation (43); however, the physiological significance of this phosphorylation is not known. Potentially it can play a role in Stat5 activation including nuclear translocation. Stat proteins have also been shown to interact with other cellular components besides mitogen-activated protein kinase; examples are phosphatidylinositol 3-kinase (51), glucocorticoid receptor (52), and a number of nuclear transcription factors (53). Any one of these molecules, or even possibly a new cellular protein, potentially influence Stat5 activation by the PRLR. Further investigation is required to determine how receptor phosphotyrosines are affecting Stat proteins nuclear translocation.
In conclusion, we demonstrate in this study for the first time that Stat5 activation involves two separately regulated events, Stat5 tyrosine phosphorylation and nuclear translocation. We further report that Stat5 tyrosine phosphorylation induced by the PRLR is independent of receptor phosphotyrosines, and we establish that tyrosine 382 of the PRLR Nb2 form positively regulates Stat5 nuclear translocation.