Differential Effects of Prolactin andsrc/abl Kinases on the Nuclear Translocation of STAT5B and STAT5A*

In this study, DNA binding and tyrosine phosphorylation of STAT5A and STAT5B were compared with their subcellular localization determined using indirect immunofluorescence microscopy. Following prolactin activation, both STAT5A and STAT5B were rapidly translocated into the nucleus and displayed a detergent-resistant, punctate nuclear staining pattern. Similar to prolactin induction, src activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B. However, nuclear translocation of only STAT5B but not STAT5A was observed. This selective nuclear translocation appears to be mediated via the carboxyl-terminal sequences in STAT5B. Furthermore, overexpression of a dominant negative kinase-inactive mutant of JAK2 prevented prolactin-induced tyrosine phosphorylation and nuclear translocation of STAT5A and STAT5B but did not block src kinase activation and nuclear translocation of STAT5B. In co-transfection assays, prolactin-mediated activation but not src kinase-mediated activation of STAT5B resulted in the induction of a β-casein promoter-driven reporter construct. These results suggest that STAT5 activation by src may occur by a mechanism distinct from that employed in cytokine activation of the JAK/STAT pathway, resulting in the selective nuclear translocation of STAT5B.


From the Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030-3498
In this study, DNA binding and tyrosine phosphorylation of STAT5A and STAT5B were compared with their subcellular localization determined using indirect immunofluorescence microscopy. Following prolactin activation, both STAT5A and STAT5B were rapidly translocated into the nucleus and displayed a detergentresistant, punctate nuclear staining pattern. Similar to prolactin induction, src activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B. However, nuclear translocation of only STAT5B but not STAT5A was observed. This selective nuclear translocation appears to be mediated via the carboxyl-terminal sequences in STAT5B. Furthermore, overexpression of a dominant negative kinase-inactive mutant of JAK2 prevented prolactin-induced tyrosine phosphorylation and nuclear translocation of STAT5A and STAT5B but did not block src kinase activation and nuclear translocation of STAT5B. In co-transfection assays, prolactin-mediated activation but not src kinasemediated activation of STAT5B resulted in the induction of a ␤-casein promoter-driven reporter construct. These results suggest that STAT5 activation by src may occur by a mechanism distinct from that employed in cytokine activation of the JAK/STAT pathway, resulting in the selective nuclear translocation of STAT5B.
Cytokines influence a variety of cellular functions including proliferation, growth arrest, and differentiation. The neuroendocrine hormone prolactin (Prl) 1 plays a central role in the development and differentiation of the mammary gland. Binding of Prl to its cell surface receptor, a member of the cytokine receptor superfamily, regulates the transcription of several milk protein genes, including the whey acidic protein (1), ␤-lactoglobulin (2), and ␤-casein (3,4) genes. The Prl receptor (PrlR) transmits signals in part via activation of the JAK/signal transducers and activators of transcription (STAT) pathway. Interaction of Prl with PrlR induces receptor dimerization, activation of the JAK2 protein-tyrosine kinase (3,(5)(6)(7), and tyrosine phosphorylation of transcription factors that belong to the STAT family.
Among the seven mammalian STAT proteins that have been discovered (8), STAT1, STAT3, and STAT5 are capable of activation by the PrlR (9). STAT5, however, plays a key role in Prl-induced milk protein gene expression and mammary gland differentiation (10,11). Tyrosine 700 of rat STAT5 is the site of phosphorylation by JAK2 and the primary regulator of STAT5 DNA binding (3). After tyrosine phosphorylation, STAT5 dimerizes and translocates into the nucleus, where it binds to specific DNA elements and activates transcription of target genes, such as the milk protein genes.
Two different STAT5 genes encoding STAT5A and STAT5B have been identified that share 93% identity at the amino acid level with the primary differences occurring at their carboxyl termini (12)(13)(14). STAT5A was discovered as a mediator of Prl response in mammary epithelial cells and was originally designated as mammary gland-specific factor, or MGF (4,15). STAT5B was cloned from hematopoietic cells, mammary gland, and liver tissue (12,14,16,17). STAT5A and STAT5B are ubiquitously expressed in most cell lines and tissues at comparable levels with a few exceptions (14).
In addition to Prl, both STAT5A and STAT5B are activated by many other cytokines, including growth hormone, erythropoietin, granulocyte-macrophage colony-stimulating factor (17,18), IL-2 (19,20), IL-3 (16,17), , IL-7, IL-15 (20), and thrombopoietin (21). STAT5 can also be activated by certain growth factors, like epidermal growth factor, through their respective receptor tyrosine kinases (22) as well as by certain nonreceptor tyrosine kinases like src and bcr-abl (23,24). STAT5A and STAT5B can, therefore, participate in many different signaling pathways leading to cell growth and differentiation. The targeted knockout of the individual genes in mice has suggested that they play essential but often redundant roles in the physiological responses associated with Prl (25). Despite their homology, there is some evidence suggesting that STAT5A and STAT5B may be differentially activated (26) and even exhibit distinct DNA binding specificities (27). However, functional differences between STAT5A and STAT5B and their precise roles in normal mammary gland development and cancer are just beginning to be elucidated.
In this study, we have compared the kinetics of DNA binding and tyrosine phosphorylation of STAT5A and STAT5B following either Prl treatment or src/abl kinase activation with their subcellular localization determined by indirect immunofluorescence microscopy. src kinase activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B, but unlike prolactin induction, nuclear translocation of only STAT5B but not STAT5A was observed. This selective nuclear translocation appears to be mediated via the carboxylterminal sequences in STAT5B and was not prevented but instead stimulated by a dominant-negative kinase-inactive mutant of JAK2. These observations establish another mechanism for STAT5 activation in response to the src/abl kinase family that results in the selective nuclear translocation of STAT5B and possibly activation of unique gene targets.

EXPERIMENTAL PROCEDURES
Expression and Reporter Constructs-Rat STAT5A cDNA was cloned and characterized in our laboratory (13). Rat STAT5B was cloned by Guoyang Luo and Dr. Li-yuan Yu-Lee at Baylor College of Medicine (28). Both STAT5A and STAT5B were subcloned into the pRCCMV expression vector (Invitrogen, Carlsbad, CA). src kinase-active (srcKϩ) and dominant negative src (srcKϪ) constructs (29) were kindly provided by Dr. Sara Courtneidge at Sugen Corp. The JAK2 mutant expression vector (JAK829) has been described previously and was kindly provided by Dr. Nelson Horseman (30). A chimeric STAT5A:B expression construct was made by ligation of the 5Ј segment of STAT5A (HindIII/XhoI 1.7-kilobase fragment) to the 3Ј portion of STAT5B (XhoI/SpeI 0.9kilobase fragment) in the HindIII/XbaI sites of the pCDNA3 vector. The Ϫ2300/ϩ490 ␤-casein gene promoter-CAT construct and the expression vector for the long form of PrlR have been described previously (31). All DNA plasmids were purified using a QIAGEN maxiprep kit (Qiagen, Valencia, CA).
Cell Culture and Transfections-The majority of the experiments were performed using COS-1 cells maintained in Dulbecco's modified Eagle's medium (JRH, Lenexa, KS), which was supplemented with 10% fetal bovine serum (JRH), glutamine (2 mM), and gentamicin (50 g/ml) in a 37°C and 5% CO 2 incubator. DNA constructs (10 g) were transiently transfected into COS cells using LipofectAMINE reagent (Life Technologies, Inc.) at a working concentration of 5 g/ml, according to the manufacturer's protocol. HeLa cells were grown in Opti-MEM media (Life Technologies), supplemented with 3% fetal bovine serum and gentamicin (50 g/ml); transfections were performed by using a calcium phosphate precipitation technique (5 Prime 3 3 Prime, Boulder, CO). For the co-transfection experiments, 2 g of STAT5A or STAT5B expression vector, 3 g of PrlR or src kinase constructs, and 5 g of JAK829 expression vector were utilized. For the reporter gene assays, 4 g of ␤-casein promoter construct was used. Before Prl or src induction, the cells were switched overnight to medium containing 10% charcoal-stripped horse serum, insulin (5 g/ml), gentamicin (50 g/ ml), and hydrocortisone (1 g/ml) and then (for the Prl experiments only) stimulated with ovine Prl (1 g/ml, lot; AFP-10677C kindly provided by the National Hormone and Pituitary Program, NIDDK, National Institutes of Health) as indicated. CAT assays were performed using a CAT enzyme-linked immunoassay kit (Roche Molecular Biochemicals) as specified by the manufacturer.
Antibodies-We have generated specific polyclonal antiserum in rabbits to the carboxyl-terminal region of STAT5A (13) and STAT5B (28) and to the activated forms that are phosphorylated on tyrosine 700 using the peptides indicated in Fig. 1. Antibodies were purified against corresponding antigenic peptides by affinity chromatography. For phosphotyrosine-specific antibodies, antisera were first purified by using an affinity column containing the nonphosphorylated peptide to remove antibodies that could react with nonphosphorylated forms. Antisera were then purified using an affinity column containing the phosphorylated peptide to enrich for fractions of the desired specificity. Enzymelinked immunoassay and Western blot analyses were used to confirm that the resulting antibodies did not recognize nonphosphorylated forms of STAT5. NH 2 -terminal STAT5 (N-20) and c-abl antibodies were purchased from Santa Cruz Biotechnology. Anti-mouse monoclonal antibody to STAT5 and phosphotyrosine antibody (PY-20) were purchased from Transduction Laboratories (Lexington, KY). v-src antibody was purchased from Calbiochem.
Electrophoretic Mobility Shift Assays (EMSA), Immunoprecipitation, and Western Blot Analysis-EMSA were performed as described previously (32). For immunoprecipitations, protein A-trysacryl (Pierce) was washed three times with RIPA buffer and diluted in RIPA buffer containing protease inhibitors in the volume twice the original volume. For each immunoprecipitation 400 g of whole cell lysate was used. The cell extracts were first precleared by incubation with 40 l of protein Atrysacryl for 15 min at 4°C. After that, they were incubated with respective antibodies at a 1:100 dilution for 2 h at 4°C, followed by incubation with protein A-trysacryl overnight at 4°C. The immunoprecipitates were then collected by centrifugation, washed three times with RIPA, and dissociated by boiling in 2ϫ denaturing buffer.
For Western blot analyses, we used 100 g of total cell lysate per lane. Protein samples were separated by 7.5% SDS-polyacrylamide gel electrophoresis and transferred overnight to an Immunobilon-P membrane (Millipore Corp.). The membranes were blocked in 3% milk for 1 h at room temperature and incubated with primary antibody for 1 h at room temperature. They were then washed three times with TBS-T (20 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween 20) and incubated with secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. After washing three times with TBS-T, the membranes were incubated for 30 min with streptavidin-horseradish peroxidase (1:2500 dilution) (Calbiochem), followed by five washes with TBS-T. Immunoreactive bands were visualized with Super Signal Chemiluminescent Substrate (Pierce). The primary antibodies, used for Western blot analysis were as follows: anti-Stat5a (COOH-terminal), diluted 1:1000; anti-Stat5b (COOH-terminal), diluted 1:2000; anti-Stat5YP (5A,5B:Y-700), diluted 1:500; and anti-phosphotyrosine (PY-20), diluted 1:1000.
For immunostaining, coverslips were blocked in 5% milk in TBS-T overnight at 4°C and incubated with primary antibodies for 1 h at room temperature. After washing five times with TBS-T, cells were incubated with secondary antibodies conjugated with Texas Red (Molecular Probes, Inc., Eugene, OR) or fluorescein isothiocyanate (Santa Cruz Biotechnology) for 30 min in the dark at room temperature and then washed with TBS-T five times and stained by DAPI by using VECTASHIELD mounting media (VECTOR, Burlingame, CA). Images were obtained using a Zeiss Axiophot fluorescent microscope.
Analysis of STAT5 Nuclear Localization-Two sets of coverslips were set up in the dish. One of them was pretreated with 0.5% Triton X-100 in CSK buffer in order to remove the soluble proteins from the cytoplasm and nuclei prior fixing the cells, while another set was fixed without pretreatment with Triton-CSK buffer. Both sets of coverslips were subjected to immunostaining. Nuclear staining was detected only in cells pretreated with Triton-CSK buffer, while the untreated cells exhibited both cytoplasmic and nuclear staining. The percentage of nuclear localization was measured by dividing the total number of cells stained for STAT5 following Triton-CSK pretreatment by the total number of cells exhibiting STAT5 staining without pretreatment relative to the average number of transfected cells. For each time point, 4 -6 fields of view were analyzed.

RESULTS
Although there have been numerous studies following the kinetics of STAT tyrosine phosphorylation and DNA binding as a function of cytokine activation, there are relatively few reports in which the subcellular distribution of STATs following activation by either cytokines or nonreceptor tyrosine kinases such as src has been investigated. In fact, little is known about the mechanisms governing nuclear import and export of STATs. To perform these studies, it was necessary to generate highly specific, affinity-purified antisera raised against either the carboxyl termini of STAT5A and STAT5B proteins or to the phosphotyrosine 700 epitope (Fig. 1).
Using these specific reagents, the tyrosine phosphorylation and DNA binding activity was initially compared with the nuclear localization of STAT5A and STAT5B following both Prl and src activation. Transfection experiments with cDNA expression plasmids encoding STAT5A or STAT5B and the PrlR were performed in COS-1 cells. For the src experiments, both constitutively active and dominant negative src kinase constructs were employed in similar transient transfection experiments but in the absence of the PrlR.
As illustrated in Fig. 2, Prl treatment as expected led to a rapid increase in tyrosine phosphorylation and DNA binding within 30 min for both STAT5A (Fig. 2, lane 2) and STAT5B (Fig. 2, lane 9). STAT 5A and STAT5B tyrosine phosphorylation was detected using both the specific STAT5A phosphotyrosine 700(YP) antibody by direct Western blotting and by the more conventional method of immunoprecipitating with anti-STAT5A or anti-STAT5B followed by Western blotting with a general anti-phosphotyrosine antibody. Prl induction of DNA binding activity of STAT5 has been shown previously to be accompanied by phosphorylation on Tyr 700 (Tyr 694 in sheep STAT5A (3)). The results illustrated in Fig. 2 suggest that Tyr 700 is the primary epitope on STAT5 for PY-20 and indicate that the kinetics of total tyrosine phosphorylation of STAT5A and STAT5B parallel the phosphorylation of Tyr 700 . For both STAT5A and STAT5B, the maximal level of tyrosine phosphorylation was observed after 30 min of Prl exposure and then appeared to decrease with similar kinetics. Direct Western blot analysis with carboxyl-terminal antibodies revealed that the total expression level of STAT proteins was relatively uniform in the transiently transfected COS-1 cells (Fig. 2, second panel). However, in the case of STAT5B, the appearance of slower mobility hyperphosphorylated forms that can be resolved from the nonphosphorylated forms by 7.5% SDS-polyacrylamide gel electrophoresis was observed to parallel the changes in tyrosine phosphorylation.
To establish the time course of DNA binding activity for STAT5A and STAT5B, EMSAs were performed using whole cell extracts and a double-stranded oligonucleotide correspond-ing to a 34-base pair region of the ␤-casein proximal promoter containing the STAT5 binding site (Fig. 2, bottom panel, lanes  1-5 and lanes 8 -12, respectively). Similar to the tyrosine phosphorylation results, STAT5A and STAT5B DNA binding activity reached a maximum after 30 min of Prl induction and subsequently decreased at longer times of Prl treatment. As expected, the phosphorylation status of STAT5A and STAT5B in response to Prl induction generally correlated with their DNA binding activity Tyrosine phosphorylation and DNA binding activity of STAT5A and STAT5B were also increased by a constitutively active src (srcKϩ, Fig. 2, lanes 7 and 14) but were not observed following transfection of a src kinase, dominant negative (srcKϪ, Fig. 2, lanes 6 and 7) construct. Direct Western blot analysis revealed phosphorylation of both STAT5A and STAT5B (Fig. 2, top two panels, lanes 7 and 14) on Tyr 700 as a result of co-transfection of srcKϩ. Similar results were obtained from immunoprecipitation experiments for STAT5A and STAT5B (Fig. 2, third panel, lanes 7 and 14). The DNA binding activity of STAT5A and STAT5B in response to src kinase activation was also demonstrated by EMSA (Fig. 2, bottom  panel, lanes 7 and 14). No significant differences in the phosphorylation status or DNA binding activity in response to src were detected between STAT5A and STAT5B using these techniques.
Cytoplasmic-Nuclear Transport of STAT5A and STAT5B in Response to Prl Induction-Using indirect immunofluorescence, it was possible to examine if the decrease in STAT5A and STAT5B tyrosine phosphorylation and DNA binding activity ( Fig. 2) with time after Prl treatment was correlated with the changes in subcellular localization of these transcription factors. In cells transfected with PrlR and STAT5A or STAT5B without Prl treatment, both STAT5A and STAT5B appear to be predominantly localized in the cytoplasm (Fig. 3, A and B, respectively). Using deconvolution confocal microscropy, some staining for both STAT5A and STAT5B could be detected in the nucleus in the absence of prolactin activation (data not shown). Similar observations using GFP-STAT5B constructs and confocal microscropy have been reported recently in human fibrosarcoma cells (35). However, the nuclear staining observed in the absence of Prl in our studies was completely removed by extraction with O.5% Triton in CSK buffer, suggesting a loose association of nonactivated STAT5A and STAT5B in the nucleus. After 30 min of Prl stimulation, both STAT5A and STAT5B translocate into the nucleus and generate detergentresistant complexes (Fig. 3, C and D, respectively). For both STAT5A and STAT5B, a specific punctate pattern of nuclear staining was observed that was resistant to Triton-CSK extraction.
Differential Localization of STAT5A and STAT5B in Re- sponse to src/abl Kinase Activation-Tyrosine phosphorylation and DNA binding of STAT5A and STAT5B appear similar in response to Prl and srcKϩ activation (Fig. 2). Thus, it was expected that STAT5A and STAT5B would also exhibit similar nuclear translocation in response to SrcKϩ activation. However, surprisingly when transiently transfected COS cells co- expressing srcKϩ and STAT5A or STAT5B were examined by indirect immunofluorescent microscopy, it was discovered that only STAT5B was localized in the nucleus following src activation. STAT5A remained in the cytoplasm (Fig. 3, compare E (for STAT5A) and F (for STAT5B)). This was confirmed in experiments using double immunofluorescence staining in which STAT5A or STAT5B was localized using specific carboxyl-terminal antibodies with corresponding secondary antibodies conjugated with Texas Red and srcKϩ was localized with an antiv-src antibody recognized with a corresponding secondary antibody conjugated with fluorescein isothiocyanate. In cells co-transfected with cDNA encoding STAT5A and srcKϩ, predominantly yellow cytoplasmic staining formed by the combination of the green and red fluorescence was observed (Fig. 4A). In contrast, in cells co-expressing STAT5B and srcKϩ, green cytoplasmic staining for src and punctated red nuclear staining for STAT5B was observed (Fig. 4B). The punctate nuclear pattern observed following src activation was similar to that seen for STAT5B after stimulation with Prl. Furthermore, a similar pattern of selective nuclear translocation for STAT5B activated by c-abl, another member of the same nonreceptor tyrosine kinase family was also detected (Fig. 4C).
Identification of the Sequences Required for the Selective Nuclear Translocation of STAT5B in Response to src Activation-Because the sequence differences between the two isoforms of STAT5 are most pronounced in the carboxyl terminus, we constructed a chimeric STAT5A:B mutant, that consisted of 545 amino acids from the NH 2 -terminal end of STAT5A and 241 amino acids from the COOH-terminal end of STAT5B. Based upon the structural analysis of the STAT proteins, the STAT5A/B chimeric construct was made in the linker domain between the DNA binding and Src homology 2 domains of STAT5A and STAT5B (36) and presumably does not affect the ability of STAT5 to dimerize or form tetramers on the appropriate DNA response elements (37). This chimera was co-transfected in HeLa cells with the PrlR or srcKϩ. In parallel, STAT5A and STAT5B were expressed with the PrlR or srcKϩ as positive and negative controls. The control experiments showed similar patterns for STAT5A (data not shown) and STAT5B localization in HeLa cells (Fig. 5, A-C) as observed previously in COS cells, confirming that the localization patterns did not result from marked overexpression of these proteins in COS cells. Remarkably, the chimeric STAT5A:B mutant translocated to the nucleus in response to both Prl induction and srcKϩ activation (Fig. 5, E and F, respectively). This result suggests that STAT5B contains a unique sequence in its carboxyl terminus that could be responsible for nuclear translocation in response to src activation.
Association of STAT5 with src Kinase and the Role of JAK2 in Nuclear Translocation-Although src has been shown to associate with and phosphorylate STAT3 both in vivo and in vitro (38) it was unclear whether src could associate with and phosphorylate either STAT5A or STAT5B. Accordingly, immunoprecipitation with an anti-src antibody followed by Western blotting with an antibody that will recognize either STAT5A or STAT5B was performed using extracts prepared from transiently transfected COS cells. These experiments demonstrated that src kinase is capable of in vivo association with both STAT5A and STAT5B (Fig. 6). However, this association appeared not to be dependent upon the enzymatic activity of src kinase, since it was observed in both srcKϩ and srcKϪ cells. Furthermore, in vitro kinase assays failed to reveal an increase in STAT5A or STAT5B tyrosine phosphorylation (data not shown) in the co-immunoprecipitated complex, suggesting that this was not a direct kinase-substrate interaction.
Having determined that the activation of both STAT5A and STAT5B by src kinase is not a direct enzyme-substrate reaction, we wished to investigate whether srcKϩ might activate either STAT5 indirectly through the activation of JAK2. For this purpose, a dominant negative mutant of JAK2 consisting of a carboxyl-terminal truncation deleting the entire kinase domain (JAK829) (30) was utilized. STAT5A or STAT5B and PrlR or srcKϩ were co-transfected in COS cells with or without JAK829. Whole cell extracts were prepared for immunoprecipitation, Western blotting, and immunofluorescent analyses (Fig.  7). As expected (39), overexpression of the kinase-inactive, dominant negative mutant of JAK2 blocked Prl-inducible tyrosine phosphorylation of STAT5A and STAT5B (Fig. 7, A and B,  lanes 1-3). However, the JAK2 dominant negative mutant did inhibit block src-inducible tyrosine phosphorylation of STAT5A (Fig. 7A, lanes 4 and 5) or STAT5B (Fig. 7B, lanes 4 and 5). Immunofluorescence analyses were consistent with these results. In cells co-transfected with STAT5A or STAT5B, PrlR, and JAK829 and treated with Prl for 30 min, exclusive cytoplasmic staining for both STAT5s was observed, similar to cells that did not receive Prl treatment (data not shown). In cells co-expressing STAT5B, srcKϩ, and JAK829, punctated nuclear staining similar to that seen in cells without JAK829 was observed (data not shown). The analysis of numerous fields was performed in order to calculate the percentage of cells displaying nuclear localization (Fig. 8). These results indicate that JAK829 significantly reduced the nuclear translocation of STAT5B induced by Prl, but not by src kinase. In contrast, in cells co-expressing STAT5B and srcKϩ, an increased level of tyrosine phosphorylation and nuclear localization of STAT5B was detected in the presence of JAK829 compared with cells not expressing the dominant-negative JAK2 mutant. These results suggest that src activation of STAT5B may occur by a mechanism independent of the conventional ligand-dependent activation of the JAK/STAT pathway ( Fig. 10 and "Discussion").
Gene Promoter after src Kinase-mediated Nuclear Translocation-Since both Prl and srcKϩ activation result in STAT5B tyrosine phosphorylation, nuclear translocation, and DNA binding, it was reasonable to examine if these pathways are transcriptionally equivalent. ␤-Casein is a well characterized STAT5 target gene. Prl induces ␤-casein gene expression through STAT5A and STAT5B both in mammary epithelial cells (15,40)

and in COS cells (3) and CHO cells (4). STAT5A
and STAT5B bind to the region between Ϫ105 and Ϫ75 in the ␤-casein gene promoter (40,41), which contains the recognition site for STAT5, a highly conserved sequence 5Ј-CTTCTTG-GAATT-3Ј. A ␤-casein gene promoter fragment (Ϫ2300/ϩ490) linked to the CAT gene as a reporter was transfected into COS cells with the PrlR or srcKϩ expression vectors. Cellular extracts were prepared, and CAT activity was determined. In cells transfected with the promoter-reporter gene construct, STAT5B, and PrlR and treated with Prl overnight, an expected 4.5-fold increase (3) in CAT protein was detected as compared with cells treated with only insulin and glucocorticoids (Fig. 9). However, STAT5B co-expressed with srcKϩ was unable to cause transactivation of the ␤-casein gene promoter (Fig. 9). Western blot and indirect immunofluorescent analyses of STAT5B were performed in parallel to these transactivation experiments to demonstrate src activation of STAT5B tyrosine phosphorylation and nuclear translocation (data not shown). These results suggest that src activation is not transcriptionally equivalent to Prl activation.

DISCUSSION
Differential Effects of src/abl Kinases on the Nuclear Localization of STAT5A and STAT5B-The general paradigm for JAK/STAT signaling is that ligand binding to cytokine receptors leads to JAK kinase activation and STAT tyrosine phosphorylation, followed by dimerization and obligatory nuclear translocation. However, as reported in this study, signals from the src/abl family of protein kinases also led to STAT5 activation but did not result in the equivalent nuclear translocation of STAT5A and STAT5B. This appears to represent a novel property of cytokine-independent pathways for STAT activation (Fig. 10). Furthermore, recent observations suggest that there are distinct biochemical differences between the closely related STAT5A and STAT5B proteins (37) that could potentially result in the differential activation of STAT5 gene targets (27). Thus, the preferential nuclear translocation of STAT5B as well as other STAT proteins provides another level of gene regulation that may have profound biological consequences. These studies also illustrate the importance of analyzing the subcellular distribution of STAT proteins following activation in addition to merely assessing their tyrosine phosphorylation and in vitro DNA binding activity by EMSA. For example, discrepancies between cell fractionation and immunofluorescence results have been reported when analyzing the nuclear translocation of STAT chimeras (42).
Little is known about the mechanisms regulating the nuclear import and export of STAT proteins. STAT1 nuclear import following activation by interferon-␥ is mediated by a Ran GTPasedependent process involving a nuclear pore-targeting complex with NPI-1 (43,44). However, no conventional nuclear localization signals have been identified in the STAT proteins. In fact, it has been hypothesized that a ligand-receptor complex might function as a chaperone to facilitate STAT nuclear import (45,46). Such a mechanism appears unlikely to account for the selective nuclear import of STAT5B by the src/abl kinases.
While little is known about the mechanisms of nuclear import, even less is known about the mechanisms regulating nuclear export of STATs. Limited studies have suggested that nuclear export appears to be dependent upon a nuclear tyrosine phosphatase (47). Pulse-chase studies have indicated that STAT1 cycles into the nucleus as tyrosine-phosphorylated molecules and quantitatively returns to the cytoplasm as nonphosphorylated molecules (47).
Although STAT5A and STAT5B display 93% identity at the amino acid level, the chimeric construct in which the aminoterminal region of STAT5A was fused to the carboxyl terminus of STAT5B still exhibited src kinase-dependent nuclear translocation, and, as expected, responded to Prl activation (Fig. 5). Recent analysis of STAT chimeras has suggested that STAT amino termini provide a signal that is important for nuclear translocation and subsequently deactivation (42). However, the analysis of STAT5A/5B chimeras in our study suggests that differences at the amino termini cannot account for the selective effects of src on the nuclear import of STAT5B as compared with STAT5A. There are two regions in the carboxyl-terminal regions of STAT5A and STAT5B that display significant differences (Fig. 1). The principal difference between these two STATs is at the carboxyl terminus in a region thought to be a transcriptional transactivation domain (48). In addition, there is a six-amino acid difference in STAT5B immediately aminoterminal to tyrosine 700. We thought that the sequence, PCE-PAT, might be important for the selective effects on STAT5B nuclear localization and generated the respective in frame deletion in STAT5B. Preliminary results indicate that this deletion did not inhibit src-dependent nuclear translocation (data not shown). More detailed analysis of carboxy-deleted naturally occurring splice variants of STAT5B (14) and additional chimeric constructs may be informative for the identification of the precise determinants that discriminate between the selec-  2 and 3). STAT5 was immunoprecipitated with an NH 2 -terminal anti-Stat5 antibody. The immunoprecipitated proteins were subjected to Western blot analysis using the PY-20 antiphosphotyrosine antibody. The blots were then stripped and probed with the anti-Stat5 monoclonal antibody. Biological Consequences of STAT5B Activation-The src family of protein-tyrosine kinases are involved in signal transduction pathways that result in growth and differentiation (49,50) and when dysregulated can result in a variety of pathological conditions including cancer (51)(52)(53). A number of primary tumors and tumor-derived cell lines including breast and colon cancer, melanoma, and sarcoma have been shown to possess elevated src tyrosine kinase activity (54 -57). src and its family members are required for mitogenesis initiated by several growth factor receptors, including epidermal growth factor receptor (58 -60), platelet-derived growth factor receptor (50,(61)(62)(63), basic fibroblast growth factor receptor (64,65), and colony-stimulating factor-1 receptor (49,60). A number of different substrates have been identified for src kinase including contractin (66 -68), p125FAK (69), and p130 cas (70), but how src contributes to the mitogenic response is still not well understood.
It has been demonstrated that in addition to their signaling functions in normal cells, STATs can also participate in oncogenesis (71). A number of reports correlate STAT activation with the activity of nonreceptor proto-oncogenic tyrosine kinases such as v-src (38,72,73), v-abl (74), lyn (75), lsk (76), bcr/abl (77), and fes (78). Thus, constitutive, non-cytokine activation of STATs may play an important role in the etiology of primary lymphoid and myeloid leukemias as well as in breast cancer (24,72,79). In support of this hypothesis, recent functional studies have demonstrated that STAT3 activation is required for transformation of mammalian fibroblasts (80). A reciprocal pattern of STAT5 and STAT3 activation has been observed during mammary gland development (10), suggesting that these STAT proteins may play different or even opposite roles in the regulation of cell proliferation, differentiation, and apoptosis (10). Conceivably, src activation may disrupt these processes.
Although it has been reported that src-transformed cells exhibit constitutive activation of JAK1 and possibly JAK2 (81), there is some evidence that v-src and bcr/abl may, in addition, be directly associated with STATs (23,24). This is consistent with the observation that dominant negative mutant of JAK2 did not prevent STAT5B activation by src (Fig. 7). In fact, in the presence of the dominant negative JAK2 mutant, increased STAT5B nuclear translocation was observed (Fig. 8). Thus, it is conceivable that JAK2 and src might compete for activation of STAT5B, and expression of the dominant negative JAK2, therefore, resulted in a small increase in src activation of STAT5B. Interestingly, a dominant negative JAK2 has also been reported not to prevent IL-3-mediated activation of STAT3, which is thought to be mediated in part by c-src (82).
Our results provide evidence that the interaction of the src kinase with STAT5 is most likely not a kinase-substrate interaction and that activation of STAT5B occurs through an undetermined indirect mechanism. The interaction of src and STAT5 observed in the co-immunoprecipitation/Western blotting experiments is most likely due to direct or indirect interaction with its Src homology 2 domains. It is possible that STAT5B is a target of tyrosine kinases activated by src and/or that src may serve as an adaptor protein facilitating STAT5B association as part of a multiprotein complex. For example, src kinase has been shown to bind to and phosphorylate the adapter protein p130 cas , an important modulator of signal transduction.
In addition to the JAK/STAT pathway, PrlR signaling may be mediated via the MAP kinase pathway and by activation of members of the src kinase family (83)(84)(85). However, despite these observations, the activation of STAT5B by src failed to activate a ␤-casein-CAT reporter construct that contains a consensus mammary gland-specific factor-binding site for STAT5 (Fig. 9). Prl activation of STAT5B has been reported to be sufficient for the activation of ␤-casein promoter-driven reporter constructs in COS cells (12). The ␤-casein gene pro- FIG. 9. Src-activated STAT5B does not activate a ␤-casein promoter-driven reporter construct. COS cells were co-transfected with a ␤-casein CAT construct, STAT5B and with constitutively active src kinase or with PrlR without or with induction by Prl for 24 h. CAT concentrations were determined by an enzyme-linked immunoassay assay.
FIG. 10. STAT5 activation mechanisms. A, cytokine-dependent pathway. Binding of Prl to its membrane receptor activates the tyrosine kinase JAK2. This kinase catalyzes the tyrosine phosphorylation of STAT5A and STAT5B. After tyrosine phosphorylation, STAT5 isoforms form homo-or heterodimers, which translocate into the nucleus, where they bind to specific DNA response elements and activate transcription of target genes presumably facilitating the proliferation and terminal differentiation of mammary epithelial cells. B, cytokine-independent pathway. STAT5B is activated by src kinase via an indirect mechanism. After tyrosine phosphorylation, STAT5B homodimers translocate into the nucleus, where they may facilitate the selective regulation of genes involved in proliferation and/or apoptosis. moter, however, contains binding sites for a number of other transcription factors that comprise a composite response element responsible for both lactogenic hormone and developmental regulation (86). Thus, it is conceivable that src kinase influences either directly or indirectly the activity of these other factors, leading to the inhibition of casein gene expression independently of activated STAT5B. A similar inhibition of lactogenic hormone signaling by other proto-oncogenes has been reported (87).
In conclusion, these studies have suggested that ligand-dependent and -independent signaling pathways may differentially regulate STAT5A and STAT5B nuclear translocation. One potential consequence may be the differential activation of gene targets involved in differentiation, proliferation, or apoptosis.