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Molecular Endocrinology 20 (10): 2392-2405
Copyright © 2006 by The Endocrine Society

Modulation of Signal Transducer and Activator of Transcription 5b Activity in Breast Cancer Cells by Mutation of Tyrosines within the Transactivation Domain

Amanda M. Weaver and Corinne M. Silva

Departments of Microbiology and Internal Medicine, and the Cancer Center, University of Virginia Health System, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Corinne Silva, P.O. Box 800578, University of Virginia Health System, Charlottesville, Virginia 22908. E-mail: cms3e{at}virginia.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The signal transducer and activator of transcription (STAT) proteins are latent transcription factors activated by a variety of cytokines and growth factors. Activation leads to phosphorylation on a conserved tyrosine residue. Although phosphorylation of STAT5b on Y699 is required for activation, it was previously shown that in epidermal growth factor receptor (EGFR)-overexpressing cell lines, three tyrosines (Y725, Y740, and Y743) in the STAT5b transactivation domain are also phosphorylated upon epidermal growth factor stimulation. The significance of these additional tyrosine phosphorylation sites was analyzed in the context of the human breast cancer cell line SKBr3, which overexpresses the EGFR and c-Src. When compared with wild-type STAT5b, mutation of Y725 decreased basal and epidermal growth factor-induced DNA synthesis. In contrast, mutation of Y740 and/or Y743 enhanced basal STAT5b Y699 phosphorylation, basal transcriptional activity, and basal DNA synthesis compared with wtSTAT5b. This indicates that Y699 and Y725 are positive regulators and Y740 and Y743 are negative regulators for STAT5b activity. Anti-phospho-Y740/743-specific antibodies demonstrated that the c-Src tyrosine kinase inhibits the phosphorylation of these two sites. Furthermore, Y740 and Y743 were not detectably phosphorylated in breast cancer cells overexpressing c-Src, but the Y740/743F mutant increased basal activity suggesting that the conformation of the transactivation domain is important in regulating STAT5b activity. Mechanistic insight into the inhibitory action of Y740 and Y743 may lead to the development of therapeutics that specifically modulate the activity of STAT5b in breast cancer and potentially other EGFR/c-Src-overexpressing cancers.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SIGNAL TRANSDUCERS AND activators of transcription (STATs), although originally discovered as mediators of cytokine-induced gene expression, are also involved in growth factor signaling pathways (1, 2). STATs remain latent in the cytoplasm until the binding of a cytokine or growth factor to its receptor, resulting in recruitment of the STAT to the ligand receptor complex. The STAT protein is then phosphorylated by receptor tyrosine kinases or nonreceptor tyrosine kinases, such as Janus kinases or c-Src. This phosphorylation results in SH2 domain-mediated dimerization of STATs and their translocation to the nucleus. In the nucleus, STAT dimers bind to consensus DNA sequences and recruit additional transcription machinery to initiate specific gene regulation. To date, seven members of the STAT family have been identified (STATs 1, 2, 3, 4, 5a, 5b, and 6), and they have been shown to be involved in cellular differentiation, proliferation, and survival (1, 2, 3, 4).

Initially identified for their involvement in lactogenesis and GH responses, STAT5a and STAT5b are expressed in most tissues and are activated by a wide variety of cytokines including GH, as well as the growth factor, epidermal growth factor (EGF) (5, 6, 7). Although encoded by two separate genes, STAT5a and STAT5b are 94% similar at the amino acid level (8). Except for scattered amino acid differences, the two proteins differ mainly in their C terminus, implying that the C terminus mediates STAT5a- and STAT5b-specific gene regulation. The importance of the C terminus is best illustrated by the observation that truncation of the C-terminal transactivation domain results in a dominant-negative STAT5 (dnSTAT5) (9, 10). Published data suggest that the C-terminally truncated dominant negative mediates its inhibitory effects by hindering the recruitment of necessary transcriptional machinery to the DNA bound dnSTAT5-wtSTAT5 heterodimer (9).

Phosphorylation of a single tyrosine in the C terminus of the STAT protein is required for activation, dimerization, and subsequent translocation to the nucleus and transcriptional activity. This tyrosine is 694 for STAT5a and 699 for STAT5b (11, 12). Recently, a number of studies have provided evidence for additional tyrosine phosphorylation sites in the STAT5 proteins. Olayioye et al. (13) demonstrated that the Y694F STAT5a mutant is further tyrosine phosphorylated upon EGF, but not prolactin treatment. More recently, Kabotyanski et al. (14) demonstrated a role for the c-Src kinase in the Y679 and Y725 phosphorylation in STAT5b. Our previously published studies demonstrate that the Y699F STAT5b mutant is tyrosine phosphorylated upon EGF, but not GH treatment (15). Using a combination of tryptic peptide mapping and site-directed mutagenesis, we identified three additional tyrosines (Y725, Y740, and Y743) in the transactivation domain of STAT5b that are phosphorylated upon EGF treatment (15). The location of the three tyrosines in comparison to Y699 is shown in the STAT5b structural schematic (Fig. 1Go). Furthermore, mutation of these three tyrosines to phenylalanines increases the activity of STAT5b compared with wild-type STAT5b, as measured by DNA binding and transcriptional activity assays (15). Although these data illustrate the ability of these three tyrosines to modulate STAT5b activity, the phosphorylation mechanism of these three tyrosines and the ability of each tyrosine to influence STAT5b function have not been elucidated.


Figure 1
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  		Fig. 1. STAT5b Schematic

Schematic of STAT5b structure illustrating the conserved domains of the STAT proteins: amino terminus (N-term), DNA binding domain (DBD), Src homolog domain 2 (SH2), transactivation domain (TAD). The Y indicates where tyrosines are located.

 
Not only do STAT5a and -5b play an important role in normal growth and development of the mammary gland, but both proteins are also overexpressed or constitutively activated in breast cancer (5, 6, 7, 16, 17, 18, 19, 20). There is evidence that increased activation of STAT5a and -5b in the context of a cancer cell results from the overexpression or constitutive activation of other signaling molecules, such as the EGF receptor (EGFR) and c-Src tyrosine kinases and the fusion protein Bcr/Abl (17, 19, 20). Due to their ability to regulate the expression of genes involved in cell cycle regulation (cyclin D1, c-myc, and p21) and cellular survival (Bcl-xL), STAT5a and -5b have emerged as possible targets for cancer therapeutics (18, 20, 21, 22, 23). However, recent studies have shown that STAT5b, but not STAT5a, mediates a pro-proliferative signal in cancer cells (24, 25, 26). Our previous studies demonstrate that STAT5b plays an integral signaling role in EGFR/c-Src co-overexpressing breast cancer cells (27).

In the studies presented here, the functional role of the C-terminal tyrosine phosphorylation sites of STAT5b in a breast cancer cell model was investigated. Exogenous expression of STAT5b mutants demonstrated that EGF induced phosphorylation of Y699, Y725, Y740, and Y743. However, use of anti-phospho-Y740/743-specific antibodies demonstrated that phosphorylation of Y740 and Y743 on endogenous STAT5b was only detected when c-Src was inhibited. Furthermore, the mutation of Y740 and Y743 resulted in a basally active STAT5b in breast cancer cells supporting their role as negative regulatory sites. These results provide mechanistic insight into the function and activation of the STAT5b protein, setting the foundation for targeting the transactivation domain of this protein to modulate its function in a breast cancer cellular context.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
STAT5a and STAT5b Expression and Activation in Breast Cancer Cells
Although STAT5a and STAT5b are widely expressed in tissues, STAT5a is expressed at a higher level than STAT5b in normal mammary tissue (28). To investigate the relative expression of STAT5a and STAT5b in human breast cancer cell lines, lysates from six cell lines were analyzed by immunoblotting with an antibody that detects both STAT5a and STAT5b. Although both STAT5a and STAT5b were equally expressed in the positive control human IM-9 lymphocyte cell line, STAT5b was predominantly expressed compared with STAT5a in the human breast cancer cell lines analyzed (Fig. 2AGo). The greater expression of STAT5b compared with STAT5a was seen in the EGFR-overexpressing cell lines (SKBr3, MDA-MB-468, MDA-MB-231), and the estrogen receptor-positive cell lines that do not overexpress the EGFR (T47D, MCF-7). In agreement with reports by others (24, 29), STAT5a was not detected in two other breast cancer cell lines, BT-20 and BT-549 (data not shown). To investigate this relative expression further and to determine whether the STAT5 proteins could be activated by tyrosine phosphorylation in this context, we used specific antibodies to immunoprecipitate STAT5a or STAT5b from SKBr3 cell lysates. Figure 2BGo demonstrates that EGF induced STAT5b tyrosine phosphorylation in SKBr3 cells. In agreement with the immunoblotting results, there were barely detectable levels of STAT5a in immunoprecipitates from SKBr3 cells, and no EGF-induced tyrosine phosphorylation. This same result was confirmed in MDA-MB231 cells (data not shown). Finally, Fig. 2CGo demonstrates that transfection of STAT5b, but not STAT5a, into the SKBr3 cell line mediated EGF-induced DNA synthesis. Although the STAT5b-expressing cells mediated higher EGF-induced DNA synthesis compared with the nontransfected cells, it was not statistically significant. However, the expression of STAT5a inhibited the EGF-induced DNA synthesis compared with the nontransfected cells. STAT5a has previously been shown to inhibit invasion and migration in breast cancer cell lines (24). Together, these data suggest that STAT5b is the predominant form in breast cancer cell lines, that it is activated by EGF, and that it can, in contrast to STAT5a, convey a pro-proliferative signal.


Figure 2
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  		Fig. 2. SKBr3 Cells Predominantly Express STAT5b

A, Whole-cell lysates were prepared from the breast cancer cell lines (SKBr3, MDA-MB-231 and -468, T47D, and MCF-7) as well as the positive control IM-9 human lymphocyte cell line. Lysates were immunoblotted with an antibody that detects both STAT5a and STAT5b. B, Cells were treated with medium alone (–) or with 100 ng/ml EGF (+) for 15 min at 37 C, and lysates were prepared. Immunoprecipitations were performed using antibodies directed specifically against STAT5b (left panel) or STAT5a (right panel) and analyzed by immunoblotting with antibodies directed toward STAT5b or STAT5a and phosphotyrosine (P-Tyr). C, Twenty-four hours after SKBr3 cells were either not transfected (NT) or transfected with HA-STAT5b or HA-STAT5a, BrdU was added to the medium with or without EGF (100 ng/ml) for 8 h. The cells were fixed and stained with fluorescent antibodies against the HA-tag and BrdU. The cells that were transfected with the HA-tagged construct were scored for BrdU incorporation and graphed. The nontransfected cells were scored for BrdU incorporation. Between 80 and 120 cells were counted for each treatment group. Results are expressed as the mean BrdU ± SE. The average fold of BrdU incorporation ± SE from three independent experiments are as follows: nontransfected, con (12.34 ± 2.30%) and EGF (20.73 ± 2.01%); STAT5b, con (18.02 ± 3.61%) and EGF (29.97 ± 4.05%); STAT5a, con (11.22 ± 1.40%) and EGF (12.03 ± 2.26%). Student’s t test was used to determine statistical significance between the following: NT EGF treated and wtSTAT5a EGF treated; EGF-treated STAT5b and EGF-treated STAT5a. {diamondsuit}, P = 0.0452; *, P = 0.0026.

 
Phosphorylation of Tyrosine Sites by EGF in Breast Cancer Cells
Although phosphorylation of STAT5b on Y699 is required for transcriptional activation, our previous studies led to the identification of three additional tyrosines (Y725, Y740, and Y743) located in the carboxyl-terminal transactivation domain of STAT5b that become phosphorylated upon EGF, but not GH stimulation. These additional tyrosine phosphorylation sites were originally identified in a 293 human embryonic kidney cell overexpression system and in STAT5a and STAT5b knockout mouse embryo fibroblast (MEF5–/–) cells into which STAT5b constructs were transiently transfected. In these cell systems, transient overexpression of the EGFR leads to tyrosine phosphorylation of the Y699F STAT5b mutant upon EGF, but not GH stimulation (15). To investigate whether these novel tyrosine phosphorylations occur in the context of a breast cancer model that overexpresses HER family members (EGFR and HER2) and c-Src, the SKBr3 cell line was used. Cells were transfected with wtSTAT5b or the Y699F STAT5b mutant and then treated with medium alone or EGF. As seen in Fig. 3AGo, both wtSTAT5b and the Y699F mutant were tyrosine phosphorylated in response to EGF treatment. To examine whether any tyrosines in addition to those previously identified were phosphorylated upon EGF treatment in SKBr3 cells, the cells were transfected with either wtSTAT5b or the Y699/725/740/743F mutant. Whereas wtSTAT5b was tyrosine phosphorylated in response to EGF stimulation, mutation of all four tyrosines, Y699/725/740/743F, completely abrogated detectable tyrosine phosphorylation in SKBr3 cells upon EGF stimulation (Fig. 3BGo). Thus, these four tyrosines, but no others, were detectably phosphorylated upon EGF stimulation in the EGFR/HER2-overexpressing SKBr3 breast cancer cell line. Because these phosphorylations occur in models of HER family overexpression, as seen in aggressive breast cancers, we pursued their characterization in the SKBr3 cell line as a means to better mechanistically understand the activation of STAT5b in breast cancer.


Figure 3
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  		Fig. 3. Phosphorylation of Tyrosines 699, 725, 740, and 743 upon EGF Stimulation

A, After transient transfection of SKBr3 cells with His-wtSTAT5b or His-Y699F, the cells were either not treated (–) or stimulated with 100 ng/ml EGF (+). After isolation using nickel-NTA magnetic agarose beads (Qiagen) as described in Materials and Methods, the STAT5b proteins were analyzed via immunoblotting with anti-phosphotyrosine (top) and anti-STAT5b (bottom) antibodies. B, SKBr3 cells were transfected with His-wtSTAT5b or Y699/725/740/743F with (+) or without (–) 100 ng/ml EGF. Proteins were isolated and analyzed as in A. C, SKBr3 cells were transfected with the various His-tagged STAT5b mutants. After stimulation, His-tagged proteins were isolated. Proteins were analyzed by immunoblotting with anti-phosphotyrosine antibody (top). The blot was then stripped and reprobed with anti-STAT5b antibody (bottom). Constructs are labeled at the top of the blot with the tyrosines mutated (e.g. Y699/725/740F) and by the tyrosine(s) left intact (P-Y743) at the bottom of the blot. D, To determine the relative amount of EGF-induced tyrosine phosphorylation, three experiments were quantitated using densitometric analysis. The values are fold induction with the EGF-treated Y699/725/740F STAT5b mutant set at 1. Values are as follows: wtSTAT5b con (2.05 ± 0.89), EGF (4.89 ± 1.99); Y725/740/743F con (2.14 ± 0.76), EGF (8.63 ± 0.76); Y699/740/743F con (0.98 ± 0.24), EGF (3.81 ± 0.83); Y699/725/743F con (0.74 ± 0.08), EGF (1.85 ± 0.35); Y699/725/740F con (0.75 ± 0.06), EGF (1.00 ± 0.00); Y699/725F con (0.91 ± 0.19), EGF (3.58 ± 0.13). Note that only the EGF-treated values are graphed.

 
Individual Phosphorylation of Tyrosines 699, 725, 740, and 743 in SKBr3 Cells in Response to EGF
To examine whether each of the three C-terminal tyrosines could be phosphorylated individually or whether they functioned in an interdependent manner, SKBr3 cells were transfected with STAT5b mutants in which all but one of the four tyrosines was mutated. Figure 3CGo shows that each tyrosine (Y699, Y725, Y740, or Y743) was individually phosphorylated in response to EGF. Although phosphorylation of Y725, Y740, and Y743 each occurred individually, their extent of phosphorylation varied: Y725 was greater than Y740, and Y740 was greater than Y743. Figure 3DGo demonstrates that phosphorylation of Y740 and Y743 together (P-Y740/743) was comparable to the combined phosphorylation of Y740 and Y743 individually. Figure 3CGo also demonstrates that mutation of any of these four tyrosines resulted in an apparent alteration in STAT5b electrophoretic mobility. Most notably, the Y725/740/743F mutant appeared as a faster migrating protein. It is well documented that phosphorylation of STAT5b results in a slower migrating band compared with unphosphorylated STAT5b (30, 31). Thus, the altered banding pattern of tyrosine-mutated STAT5b could potentially reflect a change in phosphorylation and/or the conformation of STAT5b. Importantly, an apparent increase was observed in the basal and EGF-induced phosphorylation of Y699 when the other three tyrosines were not present (compare Y725/740/743F to wt5b). This result suggests that Y725, Y740, and/or Y743 may function to inhibit Y699 phosphorylation.

The Effect of Y725, Y740, and Y743 on the Y699 Phosphorylation
To more directly determine the effect of mutating these C-terminally located tyrosines on Y699 phosphorylation, SKBr3 cells were transfected with the various STAT5b tyrosine mutants, and the STAT5b constructs were isolated from cell lysates. STAT5b phosphorylation was analyzed by immunoblotting with a commercially available antibody specific for phosphorylated Y699. Figure 4Go shows each immunoblot and the densitometric analysis of three experiments. Immunoblotting with ant anti-phospho-Y699-specific antibody (Fig. 4AGo) illustrates that although wtSTAT5b and each of the tyrosine mutants were phosphorylated on Y699 in response to EGF, the Y740F and Y743F STAT5b mutants had a greater basal Y699 phosphorylation than wtSTAT5b. In contrast, the Y725F STAT5b mutant had decreased basal Y699 phosphorylation compared with wtSTAT5b.


Figure 4
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  		Fig. 4. Mutation of Y740 or Y743 Increases Basal Y699 Phosphorylation

A, Thirty-six hours after SKBr3 cells were transfected with various His-tagged STAT5b tyrosine mutants, the cells were treated for 15 min with medium alone (–) or 100 ng/ml EGF (+). Nickel-NTA magnetic agarose beads were used to isolate the His-tagged proteins. Proteins were analyzed by immunoblotting with anti-phospho-Y694/699 (STAT5a/b) (top) and anti-STAT5b (bottom). Averages and SD values were calculated from densitometric analysis of three experiments. The values are fold induction with the control wtSTAT5b set at 1: wtSTAT5b con (1.00 ± 0.00), EGF (5.00 ± 1.36); Y725F con (0.59 ± 0.17), EGF (5.65 ± 2.96); Y740F con (2.75 ± 0.47), EGF (8.44 ± 2.46); Y743F con (2.34 ± 0.45), EGF (7.33 ± 2.49). Inset, Graph of untreated (con) values. B, His-tagged STAT5b tyrosine mutants were isolated as described above from untreated (–) or EGF-treated (+) cells. Proteins were analyzed by immunoblotting with anti-phospho-Y694/699 (STAT5a/b) (top) and anti-STAT5b antibody (bottom). Averages and SD values were calculated from densitometric analysis of three experiments. The values are fold induction with the control wtSTAT5b set at 1: wtSTAT5b con (1.00 ± 0.00), EGF (3.92 ± 0.34); Y740/743F con (3.63 ± 0.46), EGF (7.98 ± 0.63); Y725/740/743F con (1.95 ± 1.53), EGF (5.74 ± 3.08). Inset, Graph of values from untreated (con) cells.

 
The immunoblot results of the single site mutants, shown in Fig. 4AGo, suggest a positive role of Y725 and a negative role of Y740 or Y743 on STAT5b activity. Therefore, the effect of the combined mutation of Y740 and Y743 was more directly investigated. The STAT5b constructs were transfected into SKBr3 cells and analyzed by immunoblotting with an anti-phospho-Y699 antibody. As expected, the wtSTAT5b, Y740/743F, and Y725/740/743F mutants were all phosphorylated on Y699 in response to EGF, whereas the Y699/725/740/743F STAT5b mutant was not (Fig. 4BGo). The Y740/743F mutant had increased basal Y699 phosphorylation, demonstrating that mutation of these two tyrosines together influenced the basal Y699 phosphorylation. However, the basal Y699 phosphorylation of the Y725/740/743F mutant was not significantly different from wtSTAT5b or the Y740/743F mutant. Because Y699 phosphorylation is required for STAT5b activation, these data suggest that Y740 and Y743 potentially alter STAT5b biological activity.

Role of C-Terminal Tyrosines in DNA Synthesis
The importance of STAT5b in DNA synthesis in SKBr3 cells is illustrated by the observation that the dominant negative (dnSTAT5b) inhibits EGF-induced bromodeoxyuridine (BrdU) incorporation (27). Because mutation of Y740 and/or Y743 increased the basal phosphorylation of Y699, we investigated the potential role of these tyrosines on STAT5b-mediated DNA synthesis in breast cancer cells. The SKBr3 cells were transfected with the various STAT5b mutants, and BrdU incorporation was measured with or without EGF stimulation. Mutation of Y725 decreased basal and EGF-induced DNA synthesis compared with wtSTAT5b, again reflecting the positive role of Y725 in STAT5b activity (Fig. 5AGo). In contrast, mutation of either Y740 or Y743 increased the basal level of DNA synthesis, equal to that of the EGF-induced response, indicating that Y740 and Y743 both negatively regulate STAT5b activity (Fig. 5AGo).


Figure 5
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  		Fig. 5. Mutation of Y740 or Y743 Increases Basal Level of DNA Synthesis and Transcriptional Activity

Twenty-four hours after SKBr3 cells were transfected with His-tagged constructs, BrdU was added to the medium with or without EGF (100 ng/ml) for 8 h. The cells were fixed and stained with fluorescent antibodies against the His-tag and BrdU. The cells that were transfected with the His-tagged construct were scored for BrdU incorporation and graphed. Between 80 and 120 cells were counted for each treatment group. Results are expressed as the mean percentage ± SE of STAT5b-positive cells that were positive for BrdU incorporation. A, The average percent of BrdU incorporation ± SE from three experiments are as follows: for wtSTAT5b, con (10.83 ± 0.75) and EGF (25.95 ± 1.68); Y725F, con (8.15 ± 0.51) and EGF (16.68 ± 2.39); Y740F, con (20.15 ± 2.51) and EGF (20.81 ± 1.93); Y743F, con (19.50 ± 2.53) and EGF (21.22 ± 3.54). Student’s t test was used to determine statistical significance between transfected wt5b and transfected Y-F mutants. bullet, P = 0.041; {diamondsuit}, P = 0.034; *, P < 0.02. B, The average percent of BrdU incorporation ± SE for three experiments are as follows: for wtSTAT5b, con (10.91 ± 0.66) and EGF (17.83 ± 0.74); Y740/3F, con (24.89 ± 1.79) and EGF (22.54 ± 1.36); Y725/40/43F, con (16.79 ± 1.46) and EGF (17.84 ± 1.76); Y699/725/740/743F, con (9.97 ± 0.70) and EGF (13.46 ± 0.18). Student’s t test was used to determine the statistical significance between transfected wt5b and transfected Y-F mutants. *, P = 0.0002; {diamondsuit}, P = 0.0384; bullet, P = 0.0037. C, SKBr3 cells were transfected with the Spi 2.1 promoter luciferase construct and either wtSTAT5b or the Y740/3F STAT5b mutant. Cells were treated for 16 h with either medium alone (con) or EGF (100 ng/ml). Luciferase activity was measured as described in Materials and Methods. The graph represents three independent experiments with each group in each experiment done in triplicate. Values are reported as fold induction over wtSTAT5b (con) ± SE. They are as follows: wtSTAT5b, con (1.00 ± 0.00) and EGF (4.07 ± 1.74); Y740/743F, con (3.90 ± 0.66) and EGF (7.60 ± 2.49). Student’s t test was used to determine statistical significance between wtSTAT5b con and Y740/743F con. *, P = 0.001.

 
Given that the results of Fig. 5AGo suggest a positive role of Y725 and a negative role of Y740 and Y743 on STAT5b activity, the effect of the double mutant Y740/743F on basal and EGF-induced DNA synthesis in SKBr3 cells was investigated. As shown in Fig. 5BGo, the Y740/743F STAT5b mutant increased basal DNA synthesis compared with wtSTAT5b. Furthermore, EGF-induced DNA synthesis was significantly higher in Y740/743F-expressing cells than the EGF-induced DNA synthesis in the wtSTAT5b-expressing cells. These results support the previous conclusion that Y740 and Y743 negatively regulate STAT5b-mediated DNA synthesis.

To further establish a positive role for Y725 in STAT5b activity, the ability of the Y725/740/743F STAT5b mutant to affect DNA synthesis was compared with wtSTAT5b and the Y740/743F STAT5b mutant. Although the Y725/740/743F and Y740/743F STAT5b mutants both increased basal DNA synthesis compared with wtSTAT5b, the basal DNA synthesis of the Y740/743F STAT5b mutant was significantly higher than that seen with the Y725/740/743F STAT5b mutant (Fig. 5BGo). These results suggest that although Y725 was not required for the increase in basal DNA synthesis, its presence can further enhance STAT5b activity. Importantly, the Y699/725/740/743F mutant was not able to increase basal DNA synthesis compared with wtSTAT5b or the His-vector (Fig. 5BGo and data not shown), indicating the continued requirement for Y699 phosphorylation. In other words, the increased activity of STAT5b by mutation of Y740 and Y743 did not overcome the requirement for phosphorylation of Y699, and thus the basic paradigm for STAT5b activation has not been altered.

Y740/743F Mutant and STAT5b Transcriptional Activity
Increases in the basal Y699 phosphorylation of the Y740/743F STAT5b and in basal DNA synthesis, suggest that this mutant has increased transcriptional activity. To investigate this function more directly, SKBr3 cells were transfected with either wtSTAT5b or the Y740/743F STAT5b mutant in addition to a reporter plasmid containing STAT5 response elements (Spi 2.1-luciferase). Cells were treated with either EGF or with medium alone (to assess basal activity). Figure 5CGo demonstrates that the Y740/743F STAT5b mutant significantly increased the basal transcriptional activity compared with wtSTAT5b. Together, the results of Fig. 5Go support a mechanism by which mutation of Y740 and Y743 increases the Y699 phosphorylation, leading to increased basal transcriptional activity of STAT5 response elements, and a subsequent increase in basal DNA synthesis.

Role of c-Src on STAT5b Tyrosine Phosphorylation
Previously published work (14, 15) demonstrates a role for the c-Src tyrosine kinase in mediating the phosphorylation of Y699 and Y725. In studies not shown here, we confirmed that overexpression of c-Src, in the absence of functional EGFR, mediated Y699 and Y725 phosphorylation. In contrast, studies in MEFs from Src, Yes, and Fyn knockout mice (SYF) (32) demonstrated that overexpression of the EGFR resulted in EGF-induced phosphorylation of all four tyrosines. During the course of these studies, it was noted that Y740 and Y743 phosphorylation more readily occurred in the SYF cells compared with the SKBr3 cells, suggesting that c-Src kinase activity may negatively influence Y740 and Y743 phosphorylation (data not shown).

Because EGF-induced Y740 and Y743 phosphorylation was more readily seen in the SYF cells than SKBr3 cells, the potential role of c-Src to inhibit EGF-induced Y740 and Y743 phosphorylation was examined. The Src++ cell line, which expresses endogenous wild type c-Src, was used in comparison to the SYF cells. The SYF cells and Src++ cells were transfected with various STAT5b constructs and stimulated with EGF (Fig. 6Go). All of the STAT5b constructs demonstrated EGF-induced tyrosine phosphorylation. Whereas wtSTAT5b and the Y725/740/743F mutant were phosphorylated similarly in the two cell lines, the Y740 and Y743 sites demonstrated greater phosphorylation in the SYF cells than in the Src++ cells (Fig. 6Go, A and B). This difference in EGF-induced phosphorylation is best illustrated in the densitometric analysis graph (Fig. 6CGo). One possible explanation for the increased phosphorylation of Y740 and Y743 in the SYF cells is the lack of the c-Src kinase, suggesting that c-Src kinase activity is inhibiting Y740 and Y743 phosphorylation.


Figure 6
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  		Fig. 6. Phosphorylation of Y740 and Y743 Is Reduced in Src-Expressing Cells

A and B, SYF cells (A) or Src++ cells (B) were transfected with various STAT5b constructs, treated with (+) or without (–) EGF, and nickel-NTA magnetic agarose beads were used to isolate the His-tagged proteins. C, To determine the relative amount of EGF-induced tyrosine phosphorylation, three experiments were quantitated using densitometric analysis. The values are fold induction with the EGF-treated SYF cells expressing wtSTAT5b set at 1. Values are as follows and EGF-treated values are graphed. For SYF cells: wtSTAT5b (1.00 ± 0.00); Y725/740/743F EGF (1.031 ± 0.108); Y699/725/743F EGF (0.933 ± 0.089); Y699/725/740F EGF (0.745 ± 0.113). For Src++ cells: wtSTAT5b EGF (0.889 ± 0.133); Y725/740/743F EGF (0.838 ± 0.081); Y699/725/743F EGF (0.541 ± 0.081); Y699/725/740F EGF (0.307 ± 0.051). Student’s t test was used to determine statistical significance between SYF and Src cells. *, P = 0.031; {diamondsuit}, P = 0.030.

 
Specificity of the Anti-Phospho-Y740/743 Antibody
Although transient transfection of various tyrosine mutants demonstrated that EGF stimulation induced Y740 and Y743 phosphorylation, the ability of endogenous STAT5b to be phosphorylated on Y740 and Y743 had not been examined. Thus, development of an anti-phospho-Y740/743-specific antibody was undertaken. The specificity of the antibody was tested in two different cell lines. MEF5–/– cells were transfected with various STAT5b constructs and treated with or without EGF. In response to EGF stimulation, the total anti-phosphotyrosine antibody recognized the wtSTAT5b, Y699/725F, and Y740/743F constructs, whereas the Y699/725/740/743F was not recognized (Fig. 7AGo). The anti-phospho-Y740/743 antibody only recognized the EGF-treated Y699/725F construct and not the Y740/743F construct, suggesting that this antibody is specific for phosphorylated Y740 and Y743. Because it had previously been noted that Y740 and Y743 were more readily phosphorylated in the SYF cells (Fig. 6Go), the specificity of this antibody was also examined in these cells. Similar to the MEF5–/– cells, SYF cells were transfected with various STAT5b constructs and treated with or without EGF. As predicted, EGF stimulation induced tyrosine phosphorylation of all of the constructs except the Y699/725/740/743F mutant (Fig. 7BGo). Consistent with its specificity, the anti-phospho-Y740/743 antibody recognized the Y699/725F mutant and not the Y740/743F mutant. Further characterization demonstrated that the anti-phospho-Y740/743 antibody recognized phosphorylated Y740 and Y740 individually (data not shown). In contrast to the MEF5–/– cells in which Y740 and Y743 phosphorylation was not detected for wtSTAT5b (Fig. 7AGo), the wtSTAT5b expressed in the SYF cells demonstrated an EGF-induced phosphorylation at Y740 and Y743 (Fig. 7BGo). Together, the results support a role for the c-Src tyrosine kinase inhibiting phosphorylation of Y740 and Y743.


Figure 7
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  		Fig. 7. Specificity of the Phospho-Y740/743 Antibody

A, MEF5–/– cells were transfected with various STAT5b constructs. Cells were treated with medium alone (–) or plus 100 ng/ml EGF (+) for 15 min at 37 C and lysates prepared. Immunoprecipitations were performed using antibodies directed specifically against STAT5b and analyzed by immunoblotting with antibodies directed toward phospho-Y740/743 (top), phosphotyrosine (middle), or STAT5b (bottom). B, SYF were transfected with various STAT5b and treated as described above. Nickel-NTA magnetic agarose beads were used to isolate the His-tagged proteins. Proteins were analyzed as described in A.

 
Endogenous STAT5b Phosphorylation
Previous studies (Fig. 3CGo) demonstrated that exogenously expressed STAT5b could be phosphorylated at Y699, Y725, Y740, and Y743 in SKBr3 cells. Using the newly developed and specific anti-phospho-Y740/743 antibody, the ability of Y740 and Y743 to be phosphorylated on endogenous STAT5b was investigated. Because these cells overexpress both the EGFR (a positive regulator of Y740/743 phosphorylation) and c-Src (a negative regulator of Y740/743 phosphorylation), it was important to determine whether these sites were phosphorylated endogenously. STAT5b immunoprecipitations were performed from SKBr3 cells lysates stimulated from 1 to 60 min with EGF (Fig. 8Go). EGF-induced tyrosine phosphorylation was detected as early as 1 min, with tyrosine phosphorylation being greatly decreased by 60 min. In contrast, Y740/743 phosphorylation was not detected at any of these times. Other human breast cancer cell lines that overexpress both the EGFR and c-Src, including MDA-MB-486, MDA-MD-231, and BT-20, were also examined, but endogenous STAT5b was not detectably phosphorylated on Y740 and Y743 in either control or EGF treated (data not shown). The anti-phospho-Y740/743 blot shown in Fig. 8Go was stripped and reprobed with the anti-phospho-Y699 antibody. Figure 8Go, bottom panel, demonstrated that phosphorylation of Y699 was similar to that seen with total tyrosine phosphorylation, with detection at 1 min of EGF stimulation and decreased by 60 min. Together, these data suggest that although the Y699/725F mutant exogenously expressed in SKBr3 cells (Fig. 3BGo) was phosphorylated upon EGF stimulation, Y740 and Y743 are not endogenously phosphorylated (Fig. 8Go) in these cells under the same conditions.


Figure 8
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  		Fig. 8. Y740 and Y743 Are Not Phosphorylated Endogenously

After serum starvation, SKBr3 cells were treated with 100 ng/ml EGF for various times. Endogenous STAT5b was immunoprecipitated using STAT5b-specific antibodies. The immunoprecipitation was split equally among three gels. Immunoprecipitants were analyzed by immunoblotting with anti-STAT5b antibody (top), anti-phosphotyrosine (second blot), and anti-phospho-Y740/743 (third blot). The anti-phospho-Y740/743 blot was then stripped and reprobed with anti-phospho-Y694/699 (STAT5a/b) (bottom).

 
To determine whether the c-Src tyrosine kinase was potentially suppressing phosphorylation of Y740 and Y743 in the SKBr3 cells, the cells were treated with a Src inhibitor. Figure 9Go shows the tyrosine phosphorylation of STAT5b from lysates that were pretreated with increasing concentrations of the Src family kinase inhibitor PP2. Whereas 0.1 µM PP2 treatment did not alter EGF-induced tyrosine phosphorylation, SKBr3 cells treated with 1.0 µM PP2 followed by EGF stimulation resulted in detectable phosphorylation of Y740 and Y743. Note that the 10 µM PP2 treatment abrogated detectable STAT5b tyrosine phosphorylation, probably due to the ability of higher concentrations of PP2 to inhibit the EGFR kinase activity. Together, these data suggest that phosphorylation of Y740 and Y743 does not occur on endogenous STAT5b in c-Src-overexpressing breast cancer cells potentially due to c-Src kinase activity. However, inhibition of the c-Src kinase activity allows for detectable Y740 and Y743 phosphorylation upon EGF stimulation of SKBr3 cells.


Figure 9
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  		Fig. 9. Inactivation of Src Enhances Endogenous STAT5b Phosphorylation on Y740/743

Serum-starved SKBr3 cells were treated with increasing concentrations of PP2 followed by with (+) or without (–) EGF stimulation. STAT5b immunoprecipitations were performed and analyzed by immunoblotting with anti-phospho-Y740/743 (top), anti-phosphotyrosine (middle), or anti-STAT5b (bottom).

 
Together, our data support a model whereby 1) both c-Src and EGFR tyrosine kinases mediate STAT5b phosphorylation on sites other than Y699, 2) Y699 phosphorylation is required for EGF-induced DNA synthesis, 3) the presence of Y725 enhances EGF-mediated STAT5b activity, 4) Y740 and Y743 are involved in the inhibition of basal STAT5b activity, and 5) overexpression of the c-Src tyrosine kinase inhibits phosphorylation of Y740 and Y743.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Understanding the molecular mechanisms of the signaling pathways that are involved in breast cancer proliferation is important to developing targeted therapies to inhibit its growth. Given the precedence for cancer cells to become resistant to therapies over time, it is necessary to target multiple points along these signaling pathways. The STAT5a/b proteins are overexpressed and activated in a number of cancers including squamous cell cancer of the head and neck, and lung, prostate, and breast cancers (25, 26, 33, 34). We have shown that STAT5b is the major STAT5 protein expressed in breast cancer cells, whereas STAT5a is either expressed at relatively low levels, or not at all (Fig. 2AGo). Additionally, several publications (24, 25, 26) have illustrated that STAT5b, not STAT5a, has a pro-proliferative effect in several cancers. STAT5b has been shown here, as well as by others, to be the predominantly expressed form in breast cancer cell lines (24, 29). Although both STAT5a and -5b are translocated to the nucleus upon prolactin treatment, STAT5b, but not STAT5a, translocates to nucleus in the presence of constitutive active c-Src (35). Xenograft models of head and neck carcinomas have demonstrated that inhibition of STAT5b, but not STAT5a, by antisense oligonucleotides repressed tumor growth and hindered expression of STAT5 regulated genes cyclin D1 and Bcl-xL (25, 26). Reintroduction of STAT5a into the BT-20 breast cancer line inhibited invasion and migration (24). Transfection of STAT5a into SKBr3 cells inhibited EGF-induced DNA synthesis, whereas STAT5b increased DNA synthesis (Fig. 2CGo). The data presented here lend further support to published studies demonstrating that STAT5b, but not STAT5a, mediates a pro-proliferative signal. One explanation for the differential effects of these two highly conserved transcriptional factors is their ability to form N-terminally mediated tetramers (36, 37). STAT5a more readily forms tetramers than STAT5b, and this enables STAT5a to bind consensus and nonconsensus STAT5 binding sites (37).

Previously published studies (27) demonstrate an essential role of STAT5b activation in breast cancer models in which the EGFR and c-Src are co-overexpressed. In a separate study using mouse and human cell lines engineered to overexpress the EGFR and c-Src, we showed that in addition to Y699, EGF induces the phosphorylation of three tyrosines in the transactivation domain of STAT5b (Y725, Y740, and Y743). Furthermore, mutation of these tyrosines increases STAT5b transcriptional activity (15). The goal of the studies presented in this manuscript was to understand the potential biological implications and molecular mechanism by which these tyrosines modulate STAT5b activity in an EGFR/c-Src co-overexpressing breast cancer cell model.

The human breast cancer cell line SKBr3, which overexpresses the EGFR, HER2, and c-Src tyrosine kinases, was used as a model. Our studies demonstrate that in addition to the known activating tyrosine phosphorylation site (Y699), three additional tyrosines (Y725, Y740, and Y743) were phosphorylated in response to EGF treatment of SKBr3 cells (Fig. 3CGo). Importantly, no further basal or EGF-induced phosphorylation was seen in this cell context (Fig. 3BGo). Therefore, in the context of a breast cancer cell that co-overexpresses HER family members and c-Src tyrosine kinase, all four tyrosines are likely to play an important physiological role in STAT5b function.

Our studies provide support for the role of the overexpressed tyrosine kinases found in breast cancer cells in tyrosine phosphorylation of STAT5b. The c-Src tyrosine kinase is overexpressed in approximately 70% of breast cancers, whereas HER2 is in 25–30%, and EGFR from 16–48% (1, 38). In the absence of c-Src kinase activity, the EGFR kinase mediated EGF-induced phosphorylation of all four tyrosines (Y699, Y725, Y740, and Y743). We found that the c-Src kinase mediated phosphorylation of the positive regulatory tyrosine sites in STAT5b, Y699 and Y725, but not the negative regulatory sites, Y740 and Y743 (data not shown). On the contrary, c-Src kinase activity inhibits the phosphorylation of Y740 and Y743. Inhibiting Y740 and Y743 phosphorylation, in addition to mediating phosphorylation of Y699 and Y725, is potentially a mechanism by which c-Src increases STAT5b activity in breast cancer cells.

Our results have shown that mutation of Y740 and/or 743 affect STAT5b in numerous ways: changes in electrophoretic mobility, enhanced basal Y699 phosphorylation, increased basal transcriptional activity, and increased basal DNA synthesis. Because Y740 and Y743 are not phosphorylated endogenously in breast cancer cell lines, it is unlikely that the increased activity of the Y740/743F mutant is due to the inability of the phenylalanine mutant to be phosphorylated. With the use of antibodies directed toward the N terminus or C terminus of STAT5b, Kabotyanksi et al. (14) demonstrated that the C terminus of STAT5b undergoes different conformations upon activation by prolactin vs. c-Src. To examine whether the mutation of Y740 and Y743 altered the conformation of the C terminus, similar studies comparing wtSTAT5b and the Y740/743F mutant were performed using N-terminal and C-terminal antibodies, but no differences were observed (data not shown). However, our results are consistent with a model whereby an induced conformational change of STAT5b mediates the increased basal activity of the Y740/743F mutant.

To understand how mutation of Y740 and Y743 could affect STAT5b in such ways, we propose a model as depicted in Fig. 10Go. This model is based on the enhanced basal activity and altered electrophoretic mobility seen with the Y740/743F mutant. In this model, the STAT5b protein is depicted in different conformations depending on the status of the tyrosine sites. Upon activation of the EGFR/HER2/c-Src signaling complex, either by EGF binding and/or overexpression of the kinases, STAT5b localizes to the membrane and through binding to the complex changes conformation such that the EGFR/c-Src complex can phosphorylate the activating site, Y699. Whereas c-Src kinase can additionally phosphorylate Y725, the EGFR kinase can mediate the phosphorylation of Y699, Y725, Y740, and Y743. However, in the presence of c-Src kinase activity, Y740 and Y743 phosphorylation will be reduced or not detected. Conformation I represents wtSTAT5b, shown in a folded closed conformation as it would appear in an inactivated state. Mutation of Y740 and Y743 opens the STAT5b protein such that it can more effectively be phosphorylated by c-Src or EGFR (Conformation II). This conformation would be similar to that present in EGF-stimulated cells. Thus, mutation of Y740 and Y743 generates a STAT5b that is similar to activated STAT5b.


Figure 10
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  		Fig. 10. STAT5b Signaling Model in SKBr3 Cells

In the EGFR/HER2 and c-Src-overexpressing breast cancer cell line SKBr3, EGF stimulation mediates STAT5b tyrosine phosphorylation. Activation of the EGFR/c-Src signaling complex leads to conformationally open STAT5b such that these kinases can then mediate STAT5b tyrosine phosphorylation. Conformation I depicts STAT5b in a closed inactive form. Conformation II illustrates that mutation of Y740 and Y743 creates a conformationally open STAT5b protein.

 
Due to the location of Y740 and Y743 within the transactivation domain, there are a number of plausible mechanisms by which these sites could mediate an inhibitory effect on STAT5b activity. The carboxyl terminus of STAT5b is involved in interaction with other proteins including coactivators p300/CBP, p100, and centrosomal P4.1-associated protein (CPAP) (39, 40, 41, 42). Alternatively, the signal-transducing adaptor protein 2 (STAP-2) physically associates with STAT5b and negatively modulates STAT5b activity by hindering tyrosine phosphorylation (43). Future studies will include investigating the potential role of binding partners in STAT5b function.

The transactivation domain of STAT5a/b is also known to be important for STAT5a/b inactivation (44, 45). Therefore, an alternative mechanism for the inhibitory role of Y740 and Y743 is that their mutation could disrupt a negative regulatory pathway of STAT5b. The inability of an inhibiting factor, such as a phosphatase, to bind to the Y740/743F STAT5b mutant could result in increased and prolonged Y699 phosphorylation. SHP-2 binds to and dephosphorylates STAT5a, and SHP-2-deficient cells have prolonged Y694 phosphorylation (46). If the Y740 and Y743 region provides a binding site for a phosphatase, then mutation of these tyrosines would inhibit this binding and impair the dephosphorylation of Y699 and/or Y725. Given that Y699 phosphorylation is required for STAT5b activation and Y725 is shown here to be a positive regulator of STAT5b activity, prolonged phosphorylation at these sites would result in an increase in STAT5b activity. We are currently pursuing studies to identify potential binding partners of STAT5b that require the presence and/or phosphorylation of Y740 and Y743.

In summary, the studies presented here demonstrated the ability of Y725, Y740, and Y743 to biologically and mechanistically modulate STAT5b activity in an EGFR/HER2/c-Src-overexpressing breast cancer cell line. Whereas Y699 and Y725 are positive regulators of STAT5b activity, Y740 and Y743 are negative regulators. Understanding the molecular mechanisms of the signaling pathways that are involved in breast cancer proliferation is important in developing targeted therapies to inhibit its growth. We propose that targeting the STAT5b transcriptional activation domain will prove useful for this strategy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Lines and Transient Transfections
The human breast cancer cell lines, SKBr3, MCF7, MDA-MB-231, MDA-MB-468, T47D, BT-20, BT-549, were obtained from American Type Culture Collection (Manassas, VA). Cells were passaged twice per week and maintained in DMEM plus 10% fetal calf serum. MEFs (MEF5–/–) from STAT5a/5b knockout mice (provided by Dr. J. Ihle, St. Jude Children’s Hospital, Memphis, TN) and SYF MEFs (deficient for Src, Yes, and Fyn) and Src++ MEFs (provided by Dr. T. Parsons, University of Virginia, Charlottesville, VA) were passaged twice per week and maintained in DMEM plus 10% fetal calf serum. Cells were transiently transfected with HA-tagged or His-tagged STAT5b constructs (15) using LipofectAMINE Plus per manufacturer’s directions (Invitrogen, Gaithersburg, MD).

Reagents
Recombinant human EGF (rhEGF) was obtained from Invitrogen (Gaithersburg, MD). The polyclonal STAT5b-specific antibody was developed in our laboratory, as previously described (15). Polyclonal STAT5a and monoclonal anti-phosphotyrosine antibody (PY-99) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-HA antibody (16B12) and anti-His antibody were purchased from Covance (Princeton, NJ). The polyclonal STAT5a/5b antibody directed against the SH2 domain was obtained from Zymed (Carslbad, CA). Monoclonal anti-phospho-STAT5a/5b (Y694/Y699) antibody was obtained from Upstate Biotechnology (Lake Placid, NY). The protease cocktail inhibitor and the Src inhibitor PP2 were from Calbiochem (San Diego, CA). The acrylamide was from Bio-Rad (Hercules, CA), and prestained molecular weight standards and all tissue culture reagents were from Invitrogen. Except where noted, other reagents were of either reagent or molecular biological grade from Sigma (St. Louis, MO).

Treatment of Cells
Cells were preincubated overnight in DMEM containing 0.1% BSA. After preincubation, cells were treated either with medium alone (control) or 100 ng/ml rhEGF at 37 C for 15 min. After incubation, cells were washed twice in PBS. For c-Src inhibition, cells were preincubated as described above, and then treated with various concentrations of PP2 (Calbiochem) at 37 C for 30 min.

Isolation of His-Tagged Proteins
Cells were lysed in 1% Triton X-100, 10 mM imidazole, 50 mM NaH2PO4/300 mM NaCl (pH 8.0). Protease inhibitor cocktail I (Calbiochem) and phosphatase inhibitor (sodium orthovanadate) were added to the lysis buffer just before use. The His-tagged expressed proteins were isolated using nickel-nitrilotriacetic acid (NTA)-agarose magnetic beads (Qiagen, Valencia, CA), which were extensively washed with 20 mM imidazole before proteins were eluted with 250 mM imidazole. Eluates were mixed 1:1 with 2x Laemmli buffer and analyzed as described in Immunoblotting.

Immunoprecipitations
Cells were treated as described above and then lysed in 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% deoxycholate, 50 mM Tris (pH 7.4), containing protease and phosphatase inhibitors. Lysates were incubated with the indicated antibody overnight at 4°C, and protein A-agarose (Santa Cruz) was added for an additional 1 h at 4 C. Agarose pellets were washed three times in the detergent lysis buffer, and the bound proteins were removed by boiling in 1x Laemmli buffer.

Immunoblotting
Eluted proteins isolated as described above were separated on a 7.5% polyacrylamide gel and electrophoretically transferred to nitrocellulose (Pall Corporation, Pensacola, FL). Blocking buffers were made in TBS-T [0.15 M NaCl, 0.1% Tween 20, 50 mM Tris (pH 8.0)] and contained 3% BSA for the anti-phosphotyrosine antibody or 5% nonfat dry milk for all other antibodies. Secondary antibodies were either donkey anti-rabbit or sheep anti-mouse conjugated to horseradish peroxidase, and antibody binding was detected using the enhanced chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ). Blots were stripped in buffer [2% sodium dodecyl sulfate, 0.1 M 2-mercaptoethanol, 62.5 mM Tris (pH 6.8)] at 70 C for 80 min and rinsed extensively in TBS-T before being reprobed.

Generation of Specific Anti-Phospho-Y740/743-STAT5b Antibodies
The phosphopeptide CPQAH(pY)NM(pY)PQNP, corresponding to amino acid residues 735–747, was used as an immunogen in rabbits (Biosource/Quality Controlled Biochemicals, Hopkinton, MA). The antiserum was precleared over a column of unphosphorylated peptide, followed by absorption over the two mono-phosphopeptide columns. The final affinity purification over the dual-phosphopeptide column was eluted with a low pH glycine buffer. For immunoblotting, blots were blocked in 3% BSA/TBS-T for 1 h at 25 C followed by incubation overnight at 4 C with the antibody at a concentration of 2 µg/ml.

Luciferase Assay
SKBr3 cells were transiently transfected with Spi 2.1-containing luciferase reporter plasmid, specific for STAT5 as used in previous studies (15). Twenty-four hours post transfection, cells were treated for 16 h, either control (medium) or 100 ng/ml EGF. Lysates were prepared and luciferase activity was measured. The luciferase values (arbitrary units), as measured by a Berthold luminometer, were normalized to total protein.

DNA Synthesis Assay
SKBr3 cells were transiently transfected using Lipofectamine PLUS (Invitrogen) with HA-tagged or His-tagged STAT5b constructs according to manufacturer’s recommendations (15). After transfection, cells were serum starved for 24 h, and then incubated with 100 µM BrdU with or without 100 ng/ml EGF treatment for an additional 8 h. Cells were fixed in 4% paraformaldehyde for 20 min at 25 C, and then permeablized with 0.1% Triton X-100 for 5 min at 25 C followed by 2 N HCl for 1 h at 37 C. After neutralization with 0.1 M borate buffer for 5 min at 25 C, cells were blocked in 20% goat serum/PBS for 30 min at 37 C, and then incubated with mouse monoclonal anti-HA or anti-His antibody (Covance) for 1 h at 37 C followed by antimouse fluorescein isothiocyanate (Molecular Probes, Eugene, OR) for 1 h at 37 C. Cells were then incubated with anti-BrdU-Alexa Fluor 594 (Molecular Probes) for 1 h at 37 C. Expression of the HA- or His-tagged STAT5b construct and BrdU incorporation into DNA were visualized using a Leica DM RBE Fluorescence microscope (model RS232C).


   ACKNOWLEDGMENTS
 
We appreciate the expert technical assistance of Kristen K. Laughlin and Elise M. Branch. We thank Drs. Julie Boerner, Allison Belsches-Jablonski, Stuart Frank, and Andrew Catling for insightful comments and Dr. Sarah J. Parsons’s research group for fruitful discussions.


   FOOTNOTES
 
This research was supported by National Institutes of Health/National Cancer Institute Grants RO1-CA085462 and 5T32CA001909 and Susan G. Komen Foundation BCTR0503476.

The authors have nothing to disclose.

First Published Online June 13, 2006

Abbreviations: BrdU, Bromodeoxyuridine; dn, dominant negative; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MEF, mouse embryo fibroblast; NTA, nitrilotriacetic acid; rh, recombinant human; STAT, signal transducer and activator of transcription; wt, wild type.

Received for publication October 20, 2005. Accepted for publication June 7, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Silva CM 2004 Role of STATs as downstream signal transducers in Src family kinase-mediated tumorigenesis. Oncogene 23:8017–8023[CrossRef][Medline]
  2. Levy DE, Darnell JEJ 2002 STATs: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3:651–662[CrossRef][Medline]
  3. Horvath CM 2000 STAT proteins and transcriptional responses to extracellular signals. Trends Biochem Sci 25:496–502[CrossRef][Medline]
  4. Bromberg J 2000 Signal transducers and activators of transcription as regulators of growth, apoptosis and breast development. Breast Cancer Res 2:86–90[CrossRef][Medline]
  5. Liu X, Robinson G, Wagner K 1997 Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev 11:179–186[Abstract/Free Full Text]
  6. Teglund S, McKay C, Schuetz E, van Deursen J, Stravopodis D, Wang D, Brown M, Bodner S, Grosveld G, Ihle JN 1998 Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell 93:841–850[CrossRef][Medline]
  7. Udy GB, Towers RP, Snell RG, Wilkins RJ, Park W, Ram PA, Waxman DJ, Davey HW 1997 Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc Natl Acad Sci USA 94:7239–7244[Abstract/Free Full Text]
  8. Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L 1995 Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. Proc Natl Acad Sci USA 92:8831–8835[Abstract/Free Full Text]
  9. Moriggl R, Gouilleux-Gruart V, Jahne R, Berchtold S, Gartmann C, Liu X, Hennighausen L, Sotiropoulos A, Groner B, Gouilleux F 1996 Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype. Mol Cell Biol 16:5691–5700[Abstract]
  10. Wang D, Stravopodis D, Teglund S, Kitazawa J, Ihle JN 1996 Naturally occurring dominant negative variants of Stat5. Mol Cell Biol 16:6141–6148[Abstract]
  11. Lin JX, Mietz J, Modi WS, John S, Leonard WJ 1996 Cloning of human Stat5B. Reconstitution of interleukin-2-induced Stat5A and Stat5B DNA binding activity in COS-7 cells. J Biol Chem 271:10738–10744[Abstract/Free Full Text]
  12. Gouilleux F, Wakao H, Mundt M, Groner B 1994 Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J 13:4361–4369[Medline]
  13. Olayioye M, Beuvink I, Horsch K, Daly JM, Hynes N 1999 ErbB receptor-induced activation of Stat transcription factors is mediated by Src tyrsoine kinases. J Biol Chem 274:17209–17218[Abstract/Free Full Text]
  14. Kabotyanski EB, Rosen JM, Kazansky AV 2003 Signal transduction pathways regulated by prolactin and Src result in different conformations of activated Stat5b. J Biol Chem 278:17218–17227[Abstract/Free Full Text]
  15. Kloth MT, Catling AD, Silva CM 2002 Novel activation of STAT5b in response to epidermal growth factor. J Biol Chem 277:8693–8701[Abstract/Free Full Text]
  16. Calo V, Migliavacca M, Bazan V, Macaluso M, Buscemi M, Gebbia N, Russo A 2003 STAT proteins: from normal control of cellular events to tumorigenesis. J Cell Physiol 197:157–168[CrossRef][Medline]
  17. de Groot R, Raaijamkers J, Lammers J, Jove R, Koenderman L 1999 STAT5 activation by BCR-Abl contributes to transformation of K562 leukemia cells. Blood 94:1108–1112[Abstract/Free Full Text]
  18. Bowman T, Garcia R, Turkson J, Jove R 2000 STATs in oncogenesis. Oncogene 19:2474–2488[CrossRef][Medline]
  19. Sternberg DW, Gilliland DG 2004 The role of signal transducer and activator of transcription factors in leukemogenesis. J Clin Oncol 22:361–371[Abstract/Free Full Text]
  20. Yu H, Jove R 2004 The STATs of cancer—new molecular targets come of age. Nat Rev Cancer 4:97–105[Medline]
  21. Darnell Jr JE 2002 Transcription factors as targets for cancer therapy. Nat Rev Cancer 2:740–749[CrossRef][Medline]
  22. Gesbert F, Griffin JD 2000 Bcr/Abl activates transcription of the Bcl-X gene through STAT5. Blood 96:2269–2276[Abstract/Free Full Text]
  23. Buettner R, Mora LB, Jove R 2002 Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clin Cancer Res 8:945–954[Abstract/Free Full Text]
  24. Sultan AS, Xie J, LeBaron M, Ealley E, Nevalainen MT, Rui H 2005 STAT5 promotes homotypic adhesion and inhibits invasive characteristics of human breast cancer cells. Oncogene 24:746–760[CrossRef][Medline]
  25. Xi S, Zhang Q, Dyer KF, Lerner EC, Smithgall T, Gooding WE, Kamens J, Grandis JR 2003 Src kinases mediate STAT growth pathways in squamous cell carcinoma of the head and neck. J Biol Chem 278:31574–31583[Abstract/Free Full Text]
  26. Xi S, Zhang Q, Gooding WE, Smithgall TE, Grandis JR 2003 Constitutive activation of Stat5b contributes to carcinogenesis in vivo. Cancer Res 63:6763–6771[Abstract/Free Full Text]
  27. Kloth MT, Laughlin KK, Biscardi JS, Boerner JL, Parsons SJ, Silva CM 2003 STAT5b, a mediator of synergism between c-Src and the epidermal growth factor receptor. J Biol Chem 278:1671–1679[Abstract/Free Full Text]
  28. Liu X, Gallego MI, Smith GH, Robinson GW, Hennighausen L 1998 Functional rescue of STAT5a-null mammary tissue through the activation of compensating signals including STAT5b. Cell Growth Differ 9:795–803[Abstract]
  29. Yamashita H, Iwase H, Toyama T, Fujii Y 2003 Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells. Oncogene 22:1638–1652[CrossRef][Medline]
  30. Gebert CA, Park S, Waxman DJ 1997 Regulation of signal transducer and activator of transcription (STAT) 5b activation by the temporal pattern of growth hormone stimulation. Mol Endocrinol 11:400–414[Abstract/Free Full Text]
  31. Smit LS, Vanderkuur JA, Stimage A, Han Y, Luo G, Yu-Lee L, Schwartz J, Carter-Su C 1997 Growth hormone-induced tyrosyl phosphorylation and deoxyribonucleic acid binding activity of Stat5A and Stat5B. Endocrinology 138:3426–3434[Abstract/Free Full Text]
  32. Klinghoffer RA, Sachsenmaier C, Cooper JA, Soriano P 1999 Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J 18:2459–2471[CrossRef][Medline]
  33. Sordella R, Bell DW, Haber DA, Settleman J 2004 Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 305:1163–1167[Abstract/Free Full Text]
  34. Haura EB, Turkson J, Jove R 2005 Mechanisms of disease: insights into the emerging role of STATs in cancer. Nat Clin Pract 2:315–324
  35. Kazansky AV, Kabotyanski EB, Wyszomierski SL, Mancini MA, Rosen JM 1999 Differential effects of prolactin and src/abl kinases on the nuclear translocation of STAT5B and STAT5A. J Biol Chem 274:22484–22492[Abstract/Free Full Text]
  36. John S, Vinkemeier U, Soldaini E, Darnell JEJ, Leonard WJ 1999 The significance of tetramerization in promoter recruitment by Stat5. Mol Cell Biol 19:1910–1918[Abstract/Free Full Text]
  37. Soldaini E, John S, Moro S, Bollenbacher J, Schindler U, Leonard WJ 2000 DNA binding site selection of dimeric and tetrameric Stat5 proteins reveals a large repertoire of divergent tetrameric Stat5a binding sites. Mol Cell Biol 20:389–401[Abstract/Free Full Text]
  38. Lin N, Winer EP 2004 Small molecule tyrosine kinase inhibitors. Breast Cancer Res 6:204–210[CrossRef][Medline]
  39. Pfitzner E, Jahne R, Wissler M, Stoecklin E, Groner B 1998 p300/CREB-binding protein enhances the prolactin-mediated transcriptional induction through direct interaction with the transactivation domain of Stat5, but does not participate in the Stat5-mediated suppression of the glucocorticoid response. Mol Endocrinol 12:1582–1593[Abstract/Free Full Text]
  40. Shuai K 2000 Modulation of STAT signaling by STAT-interacting proteins. Oncogene 19:2638–2644[CrossRef][Medline]
  41. Peng B, Sutherland K, Sum E, Olayioye M, Wittlin S, Tang T, Lindeman G, Visvader J 2002 CPAP is a novel Stat5-interacting cofactor that augments Stat5-mediated transcriptional activity. Mol Endocrinol 16:2019–2033[Abstract/Free Full Text]
  42. Paukku K, Silvennoinen O, Yang J 2003 Tudor and nuclease-like domains containing protein p100 function as coactivators for signal transducer and activator of transcription 5. Mol Endocrinol 17:1805–1814[Abstract/Free Full Text]
  43. Sekine Y, Yamamoto T, Yumioka T, Sugiyama K, Tsuji S, Oritani K, Shimoda K, Minoguchi M, Yoshimura A, Matsuda T 2005 Physical and functional interactions between STAP-2/BKS and STAT5. J Biol Chem 280:8188–8196[Abstract/Free Full Text]
  44. Chen Y, Dai X, Haas AL, Wen R, Wang D 2006 Proteasome-dependent down-regulation of activated STAT5a in the nucleus. Blood 108:566–574[Abstract/Free Full Text]
  45. Wang CS, Donoghue DJ, Wang D 2000 A small amphipathic {alpha}-helical region is required for transcriptional activities and proteasome-dependent turnover of the tyrosine-phosphorylated Stat5. EMBO J 19:392–399[CrossRef][Medline]
  46. Chen Y, Wen R, Yang S, Schuman J, Zhang EE, Taolin Y, Feng GS, Wang D 2003 Identification of Shp-2 as a Stat5a phosphatase. J Biol Chem 278:16520–16527[Abstract/Free Full Text]



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