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J. Biol. Chem., Vol. 280, Issue 25, 24175-24180, June 24, 2005

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ERBB4/HER4 Potentiates STAT5A Transcriptional Activity by Regulating Novel STAT5A Serine Phosphorylation Events^*

Diane E. Clark, Christopher C. Williams, Tamika T. Duplessis, Kimberly L. Moring, Amy R. Notwick¶, Weiwen Long||, William S. Lane**, Iwan Beuvink, Nancy E. Hynes, and Frank E. Jones

From the Departments of Biochemistry, ^¶Pathology, and ^||Structural and Cellular Biology, Tulane University Health Sciences Center, Tulane Cancer Center, New Orleans, Louisiana 70112-2669, ^**Harvard Microchemistry and Proteomics Analysis Facility, Harvard University, Cambridge, Massachusetts 02138, and the Friedrich Miescher Institute, P. O. Box 2543, CH-4002 Basel, Switzerland

Received for publication, December 14, 2004 , and in revised form, March 28, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The epidermal growth factor receptor family member ERBB4 is required for mammary gland development and lactation. ERBB4 activities in the breast are mediated through the signal transducer and activator of transcription (STAT) family member STAT5A, and ERBB4 directly activates STAT5A, in part, through phosphorylation of STAT5A at the regulatory Tyr-694. Here we show that STAT5A regulation by ERBB4 is also mediated through STAT5A serine phosphorylation. Using a reverse-phase high performance liquid chromatography tandem mass spectrometry analysis of proteolytically digested STAT5A coexpressed with ERBB4, we identified STAT5A serine phosphorylations at the previously described Ser-779 and at the novel Ser-127/Ser-128. Immunohistochemistry of wild-type and ERBB4-null mammary glands at late pregnancy showed that ERBB4 expression was required for STAT5A phosphorylation at Ser-779. Independent serine-to-alanine residue substitutions in full-length STAT5A revealed that although STAT5A Ser-779 phosphorylation was dispensable for phosphorylation of STAT5A at Tyr-694 and subsequent DNA binding, Ser-779 was required to stabilize an interaction with ERBB4 and mediate ERBB4-induced STAT5A stimulation of gene expression. STAT5A Ser-127/Ser-128, on the other hand, was required for ERBB4-induced phosphorylation of Tyr-694, whereas Ser-779 and as yet unidentified tyrosine residues were phosphorylated in the absence of Ser-127/Ser-128. In addition, STAT5A S127A/S128A remained associated with ERBB4 but failed to bind DNA or activate transcription in response to ERBB4 coexpression. Our studies demonstrate that phosphorylation of STAT5A at Ser-127/Ser-128 and Ser-779 are obligatory events regulating ERBB4-mediated activation of STAT5A.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
STAT5A, initially identified as a mammary differentiation factor, is a member of the signal transducer and activator of transcription (STAT)¹ family of transcription factors. STAT5A transcriptional activity is regulated through signals initiated at the cell surface by both cytokine and growth factor receptors, leading to the phosphorylation, nuclear translocation, and binding to -interferon activation sites in the promoters of target genes. In the mammary gland, STAT5A regulates multiple pregnancy-induced developmental events including specification of ductal epithelium to a secretory phenotype, epithelial terminal differentiation, and activation of milk gene expression during lactation (1-5).

STAT5A activity is regulated by phosphorylation events at specific residues through the actions of receptor tyrosine kinases, cytokine receptors, and non-receptor tyrosine kinases (6, 7). Phosphorylation of the regulatory Tyr-694 is indispensable for STAT5A transcriptional activity and has been used as a biochemical indicator of STAT5A transcriptional activation (8). In addition, constitutive serine phosphorylation of STAT5A at positions 725 and 779 has recently been reported, and convincing evidence indicates that phosphorylation of STAT5A at Ser-779 occurs within the mouse mammary gland during multiple developmental stages, including lactation (9, 10). Although a conserved residue substitution at STAT5A Ser-725 failed to impact prolactin (Prl)-induced -casein promoter activity (9, 10), in one study a similar residue substitution at STAT5A Ser-779 suppressed Prl-induced STAT5A transcriptional activity (10). The lack of a dramatic influence of STAT5A serine phosphorylation on Prl-induced STAT5A transactivation raises the possibility that STAT5A serine phosphorylation modulates the activity of an alternative mammary gland signaling network.

The ERBB/epidermal growth factor receptor family of receptor tyrosine kinases play critical roles in normal development (11) and neoplastic transformation of the breast (12). Substantial in vivo evidence indicates that ERBB4, the final member of this receptor family to be discovered, is the essential mediator of STAT5A activation during epithelial differentiation at late pregnancy and lactation at parturition (4, 5, 13). Indeed, STAT5A phosphorylation at the regulatory Tyr-694 was abolished and the expression of STAT5A regulated milk genes was dramatically impaired in mice harboring mammary gland-specific deletions of both ERBB4 alleles (4, 5). In tissue culture models, we have shown that ERBB4 physically interacts with and induces Tyr-694 phosphorylation of STAT5A in a STAT5A Src homology 2 domain-dependent manner (13). Furthermore, ligand-activated and proteolytically processed ERBB4 regulates STAT5A stimulation of the -casein promoter by functioning as a STAT5A nuclear chaperone (14, 15). Accordingly, we have demonstrated by chromatin immunoprecipitation assay that ERBB4 binds with STAT5A at the endogenous -casein promoter in the T47D breast cancer cell line (15). In addition to the unique function of ERBB4 as a STAT5A nuclear chaperone, ERBB4 induces phosphorylation of novel STAT5A tyrosine residues in addition to Tyr-694 (13). The critical role of ERBB4 as a unique regulator of STAT5A in breast cancer cell lines and the mouse mammary gland raises the possibility that ERBB4 is an important functional regulator of STAT5A serine phosphorylation. Indeed, phosphorylation of STAT5A at Ser-779 peaks at late pregnancy and early parturition (9, 10), coincident with the essential contribution of ERBB4 to STAT5A activation (4, 5).

In this study, we investigated the role of STAT5A serine phosphorylation on ERBB4-induced STAT5A stimulation of gene expression. Phosphopeptide mapping of STAT5A revealed that ectopic ERBB4 expression is associated with STAT5A phosphorylation at Ser-127/Ser-128, as well as at Ser-779. To determine the functional significance of these phosphorylation events, we incorporated independent base substitutions within STAT5A at Ser-127/Ser-128 and Ser-779 and characterized the influence of these mutations on ERBB4 regulation of STAT5A biological activity. In contrast to previous descriptions where STAT5A serine phosphorylation failed to influence Prl-regulated STAT5A signaling, serine phosphorylation of STAT5A had a significant impact on ERBB4 regulation of STAT5A activity. Moreover, our experiments identified distinct biological functions for STAT5A phosphorylation at Ser-127/Ser-128 and Ser-779 and underscore the role of ERBB4 as a regulator of unique STAT5A signaling mechanisms.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
ERBB4 cDNA—The human ERBB4 cDNA used in these experiments has been sequenced in its entirety (16) and represents the JM-a isoform (17). This isoform retains both tumor necrosis factor -converting enzyme (TACE) and -secretase recognition sequences and is therefore an ERBB4 isoform that undergoes complete proteolytic processing at the cell surface following ligand stimulation.

Tissue Culture and Transfections—HEK 293 cells were obtained from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 2 mM L-glutamine. The dramatic growth-inhibitory effect of ectopic ERBB4 expression (18) precluded analysis of ERBB4 and STAT5A in many common mammalian cell lines. We therefore performed all functional assays in the MCF-7 human breast cancer cell line MCF-7B, which was modified to stably overexpress BCL-2 (19) and is resistant to ERBB4-induced cell killing.² MCF-7B cells were cultured in minimum essential medium containing 10% fetal bovine serum, 1 mM sodium pyruvate, and 2 mM L-glutamine.

Transfections were performed on 2 x 10⁵ cells in 35-mm tissue culture dishes with 1 µg of each transfected DNA by using FuGENE 6 transfection reagent (Roche Applied Science) as described by the manufacturer. In each experiment, transfected cells were harvested at 48 h post-transfection.

Plasmid Constructs—With the exception of mouse STAT5A harboring a residue substitution of S779A (9), the human ERBB4 and mouse STAT5A expression plasmids have been described elsewhere (13). The plasmid pEF-STAT5AS127/128A harboring alanine residue substitutions at serines 127 and 128 was generated in an NruI/BamHI fragment of pEF-STAT5A by PCR-mediated, site-directed mutagenesis using the upstream primer 5'-GCTGCAGAAGAAGG-3' (nucleotides 241-254; GenBank^TM accession number BC008998 [GenBank] ), the downstream primer 5'-GGGCAAACTGAGCT-3' (nucleotides 602-589), and the mutation incorporating oligonucleotide primer pairs 5'-AATTGCGCAGCCCCTGCTGGTGTCCTGGTT-3' (nucleotides 413-442) and 5'-AGCAGGGGCTGCGCAATTGTTGGCTTCGCG-3' (nucleotides 430-401). The STAT5AS127/128A mutagenic primers were designed to generate a unique FspI site. The mutations were verified by sequencing.

Phosphopeptide Mapping of STAT5A—HEK 293 cells were cotransfected with mouse STAT5A and human ERBB4 expression vectors. At 48 h post-transfection, STAT5A was immunoprecipitated from cell lysates and separated by PAGE as described elsewhere (13). The resolving gel was stained with Gel Code Blue (Pierce), and the 95-kDa STAT5A band was excised and stored at -70 °C until use. The STAT5A sequence was evaluated using an in-house program, Enzyme Optimizer, for a dual enzyme strategy that would optimize for coverage of phosphorylated residues. The program considers factors that influence recovery and detection of a predicted peptide rather than simple protein coverage. The gel slices were subjected to in-gel tryptic and chymotryptic digestions after reduction and carboxyamidomethylation. The resultant digests were pooled just prior to liquid chromatography tandem mass spectrometry (MS/MS) injection. Phosphorylated peptide sequences were determined using a 75-µm reverse phase microcolumn (New Objective, Woburn MA) terminating in a custom nanoelectrospray source directly coupled to an LCQ DECA XP Plus quadrupole ion trap mass spectrometer (Thermo Electron, San Jose CA). Flow was nominally 200 nl/min. The ion trap repetitively surveyed the range m/z 395-1600, executing data-dependent MS/MS for peptide sequence information on the four most abundant ions in each survey scan. MS/MS spectra were acquired with a relative collision energy of 30% and an isolation width of 2.5 Da, and recurring ions were dynamically excluded. After data base correlation with the algorithm SEQUEST (32), phosphorylated peptides were confirmed by manual, de novo interpretation of the MS/MS spectra using the FuzzyIons program (20). When needed, a second targeted ion MS/MS experiment was conducted to increase detection sensitivity and spectrum quality. In this run, the predicted precursor mass-to-charge ratio (m/z) of the phosphopeptide was subjected to MS/MS for the entire chromatographic run, capturing the peptide of interest at the moment of its chromatographic elution.

STAT5A Phosphoserine779 Immunohistochemistry—Generation of paraffin-embedded mouse mammary glands at 18 days post-coitus from normal control mice (ErbB4^+/+WAP-Cre) and mice with conditional deletions of both ERBB4 alleles (ErbB4^Flox/FloxWAP-Cre) has been described elsewhere (4). Immunohistochemical detection of the STAT5A nuclear antigen phosphorylated at Ser-779 was performed exactly as described previously (13) with the following modifications. STAT5A Ser(P)-779-specific antiserum (9) was diluted 1:100 and incubated on the tissue sections in the presence of 1 µM unphosphorylated peptide (9); the biotinylated goat anti-rabbit (Vector Labs) secondary antibody was diluted to 15 µg/ml. The diaminobenzidine tetrahydrochloride (DAB) substrate was prepared fresh before use (4). Sections were lightly counterstained in hematoxylin (Polysciences) according to the manufacturer's instructions, dehydrated in ethanol, cleared in xylene, and coverslipped with Permount (Fisher).

Western Blot Analysis—Immunoprecipitations from total cell lysates were separated on a 7.5% polyacrylamide gel, transferred to Hybond ECL (Amersham Biosciences) membrane, and analyzed by Western blot as described previously (13). Primary antibodies were anti-ERBB4 (Santa Cruz Biotechnology), anti-STAT5A (Santa Cruz Biotechnology), anti-P-Stat5 (Santa Cruz Biotechnology), and anti-phosphotyrosine (Santa Cruz Biotechnology). The STAT5A Ser(P)-779-specific antiserum was diluted 1:5000 and incubated in the presence of 2 µg/ml unphosphorylated peptide (9).

Luciferase Reporter Assay—Luciferase assays were performed exactly as described elsewhere (15). Each sample was performed in duplicate, and the entire experiment was performed at least three times. Statistically significant differences between data sets were determined using the paired Student's t test.

Electrophoretic Mobility Shift Assay—NIH3T3 cells were transfected with ERBB4, STAT5A, STAT5AS127/128A, or STAT5AS779A expression plasmids, and whole cell extracts were prepared at 48 h post-transfection as described elsewhere (9). STAT5A DNA binding activity was assayed by incubating 20 µg of whole cell lysate with gel shift binding buffer (50 mM Tris, pH 7.5, 5 mM MgCl_2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 2% glycerol, and 250 µg/ml poly(dI-dC) (Promega) for 10 min prior to the addition of a ³²P-5'-end-labeled oligonucleotide probe corresponding to the STAT5A binding sequence derived from the bovine -casein promoter (5'-AGATTTCTAGGAATTCAATCC-3') and further incubation at room temperature for 30 min. Lysates were run on a native 5% polyacrylamide gel for 1 h, dried, and exposed to x-ray film.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
STAT5A Ectopically Coexpressed with ERBB4 Is Phosphorylated at Serines 127/128 and 779—When ectopically coexpressed with ERBB4, STAT5A is phosphorylated at the regulatory Tyr-694 (13, 21) in addition to as yet unidentified tyrosine residues (13). Recent evidence demonstrating that STAT5A is constitutively phosphorylated at serine residues raises the possibility that coexpressed ERBB4 and STAT5A may result in serine phosphorylation of STAT5A. We therefore performed a phosphoserine peptide analysis of STAT5A coexpressed with ERBB4 by microcapillary reverse-phase high performance liquid chromatography nanoelectospray MS/MS. This analysis revealed two unambiguous STAT5A serine phosphorylation events at Ser-127/Ser-128 and Ser-779 (Table I, Supplemental Fig. S1 (available in the on-line version of this article), and Fig. 2A). Interestingly, activation of STAT5A through Prl or growth hormone failed to result in phosphorylation of STAT5A at Ser-127/Ser-128 (9, 10, 22), suggesting that this phosphorylation event is unique to ERBB4 activation of STAT5A. Similar to our previous results (9), constitutive phosphorylation of STAT5A at Ser-725 (23) was not detected in our analysis of STAT5A phosphoserine residues. In summary, our data suggest that STAT5A regulation by ERBB4 may be partially mediated by a pathway involving serine kinases in addition to regulation by tyrosine phosphorylation.

STAT5A Serine Phosphorylation Is Necessary for ERBB4-Induced STAT5A Transcriptional Activity—Most recently, we have demonstrated that the ectopic coexpression of STAT5A with ERBB4 results in transcriptional activation of a reporter gene fused to a -casein promoter harboring STAT5A DNA binding sites (15). Here, by ectopically coexpressing ERBB4 with STAT5A harboring independent serine-to-alanine substitutions at Ser-127/Ser-128 and Ser-779 (STAT5AS127/128A and STAT5AS779A, respectively), we determined the contribution of STAT5A serine phosphorylation to ERBB4 regulation of STAT5A transactivation in a -casein promoter luciferase reporter assay. In concordance with our previous report (15), independent expression of STAT5A or ERBB4 failed to stimulate -casein promoter activity above vector control levels (Fig. 2C). Luciferase reporter activity was increased 8-fold, however, when STAT5A was coexpressed with ERBB4 (Fig. 2C). Despite equivalent levels of STAT5A and ERBB4 expression (Fig. 2B), -casein promoter activity was abolished when ERBB4 was coexpressed with STAT5AS127/128A or STAT5AS779A (Fig. 2C). Independent mutation of STAT5A Ser-127 or Ser-128 resulted in a STAT5A protein transcriptionally stimulated by ERBB4 (data not shown), suggesting that phosphorylation of one serine residue compensates for the loss of phosphorylation of the other site. Taken together, our results indicate that phosphorylation of STAT5A serine residues Ser-127/Ser-128 and Ser-779 is essential for ERBB4-induced STAT5A stimulation of the -casein promoter.

View larger version (133K): [in this window] [in a new window]   FIG. 1. Phosphorylation of STAT5A at Ser-779 in mammary epithelium requires ERBB4 expression. Wild-type (ErbB4+/+WAP-Cre) (A) and ERBB4-null (ErbB4Flox/FloxWAP-Cre) (B) mammary glands isolated from mice at 18 days pregnancy were fixed in 4% paraformaldehyde and embedded in paraffin, and 6-µm sections were stained by immunohistochemistry using an antibody directed against STAT5A phosphorylated at Ser-779. Arrowheads in panel A indicate nuclear accumulation of STAT5A phosphorylated at Ser-779 within the mammary secretory epithelium.

View larger version (28K): [in this window] [in a new window]   FIG. 2. ERBB4-induced STAT5A transcriptional activity requires STAT5A phosphorylation at Ser-127/Ser-128 (S127/128) and Ser-779 (S779). A, schematic of mouse STAT5A polypeptide indicating coiled-coil, DNA-binding, linker, and transactivation domains. The STAT5A Ser-127/Ser-128 and Ser-779 residues are located at the amino terminus/coiled-coil junction and transactivation domain, respectively. Y694, Tyr-694; SH2, Src homology 2. B, Western blot analysis of ERBB4 and STAT5A immunoprecipitations from MCF-7B cell lysates prepared from transfections with the indicated cDNAs. C, MCF-7B cells were co-transfected with the bovine -casein promoter fused to luciferase and plasmids expressing the indicated cDNAs. Cell lysates were prepared at 48 h post-transfection, and luciferase activity was determined using standard methods. Results are reported as the fold increase in luciferase activity relative to -casein promoter fused to luciferase and co-transfected with empty vector controls (mean ± S.E. of at least three experiments). Asterisks indicate ERBB4/STAT5A stimulation of the -casein promoter significantly greater than each of the other treatments as determined by paired Student's t test (p < 0.001).

View larger version (63K): [in this window] [in a new window]   FIG. 3. ERBB4-induced STAT5A Tyr-694 phosphorylation requires Ser-127/Ser-128, whereas STAT5A Ser-779 phosphorylation stabilizes STAT5A interaction with ERBB4. HEK 293 cells were transfected with the indicated expression plasmids, and cell lysates were prepared at 48 h post-transfection. ERBB4 (left sections) or STAT5A (right sections) was immunoprecipitated (IP) from 1 mg of total lysate and analyzed by Western blot for ERBB4 (IB:ERBB4), STAT5A (IB:STAT5A), tyrosine phosphorylation (IB:P-Tyr), STAT5A phosphorylated at Tyr-694 (IB:P-STAT5A), and STAT5A phosphorylated at Ser-779 (IB:P-Ser-779).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (49K): [in this window] [in a new window]   FIG. 4. STAT5A Ser-127/Ser-128 phosphorylation is required for ERBB4-induced STAT5A DNA-binding. NIH3T3 cells were transfected with the indicated expression plasmids, whole cell lysates were prepared at 48 h post-transfection, and 20 µg of lysate was incubated with a radiolabeled oligonucleotide corresponding to the STAT5A DNA-binding site of the bovine -casein promoter. STAT5A DNA-binding activity was determined by an electrophoretic mobility shift assay.

View larger version (24K): [in this window] [in a new window]   FIG. 5. A model for the role of STAT5A serine phosphorylation during ERBB4-induced STAT5A activation. Step 1, coexpression of ERBB4 and STAT5A results in a stable interaction between ERBB4 and STAT5A (13, 15) mediated by the STAT5A Src homology 2 domain (SH2) and stabilized by phosphorylation (P) of STAT5A at Ser-779 (S779). Step 2, although STAT5A lacking Ser-127/Ser-128 (S127/128) retains phosphorylation of Ser-779 and ERBB4-specific unidentified tyrosine residues (P-Y?), phosphorylation of STAT5A at Ser-127/Ser-128 is required for Tyr-694 phosphorylation (P-Y694) and subsequent DNA-binding of STAT5A. Step 3, DNA-bound STAT5A requires phosphorylation of Ser-779 for ERBB4-induced STAT5A stimulation of gene expression. GAS, interferon-activating site.

At this point the kinase(s) regulating STAT5A serine phosphorylation in response to ERBB4 remains to be determined. One likely candidate is the serine/threonine kinase p21-activated kinase 1, which is regulated by members of the epidermal growth factor receptor family through the ERBB4 ligand heregulin (25). Significantly, transgenic expression of a dominant negative form of p21-activated kinase 1 in the developing mammary gland results in lobuloalveolar and lactational defects (26) coincident with identical phenotypes observed in ERBB4-null mammary glands (4, 5). Moreover, both ERBB4 and p21-activated kinase 1 are required for late pregnancy phosphorylation of STAT5A at Ser-779, and this phosphorylation event is required for maximal stimulation of the -casein promoter induced by ERBB4 or p21-activated kinase 1 (26). Collectively, these results support a developmentally important signaling pathway involving coupled ERBB4 and Pak1 regulation of STAT5A in the mammary gland at late pregnancy.

Phosphorylation of STAT5A at Tyr-694 and subsequent DNA binding was thought to be sufficient to stimulate gene expression; however, our observations suggest that serine phosphorylation plays an important regulatory role. For example, the STAT5AS779A mutant was phosphorylated at Tyr-694 and bound DNA in response to ERBB4 coexpression, but this mutant failed to activate gene expression. We also show that STAT5A phosphorylation at Tyr-694 and transactivation activity requires Ser-127/Ser-128. STAT5AS779A retains phosphorylation at Tyr-694, implying that STAT5AS779A is also phosphorylated at Ser-127/Ser-128, excluding impaired Ser-127/Ser-128 phosphorylation as an explanation for the lack of STAT5AS779A transactivation activity. Thus, it seems likely that STAT5A Ser-779 phosphorylation is required to recruit transcription coactivators critical for ERBB4-induced STAT5A stimulation of gene expression. For example, ERBB4 itself harbors transactivation activity (15, 27) and binds with STAT5A at the endogenous -casein promoter (15). Therefore, the lack of potent STAT5AS779A transactivation activity may be a result of the suppressed interaction with ERBB4 observed in our experiments. We are currently investigating the contribution of STAT5A serine phosphorylation to the recruitment of ERBB4 and other transcriptional coactivators at STAT5A target promoters.

In summary, our findings provide the first description of STAT5A serine phosphorylation events critical for STAT5A function. Furthermore, we have identified a novel STAT5A serine phosphorylation event at Ser-127/Ser-128 critical for ERBB4-induced STAT5A phosphorylation at the regulatory Tyr-694 and for STAT5A-stimulated gene expression. In addition, the previously described STAT5A phosphorylation at Ser-779, which had minimal impact on PrlR regulation of STAT5A, is regulated by ERBB4 expression in mammary glands at late pregnancy and is essential for ERBB4 regulation of STAT5A transactivation activity. Thus, the ERBB4/STAT5A signaling axis appears to be mechanistically similar to STAT1, STAT3, and STAT4, which are functionally regulated by serine phosphorylation events (28-31).

	FOOTNOTES

 
^* This work was supported by National Cancer Institute/National Institutes of Health Grants RO1CA95783 and RO1CA96717 (to F. E. J.), National Cancer Institute/National Institutes of Health Research Supplement for Underrepresented Minorities (to C. C. W.), and funds generously supplied through the National Cancer Coalition and the Tulane Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at www.jbc.org) contains Supplementary Fig. S1, featuring a mass spectrometry analysis of STAT5A phosphopeptides.

These authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Biochemistry, Tulane University Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112-2669. Tel.: 504-988-6585; Fax: 504-988-1687; E-mail, fjones{at}tulane.edu.

¹ The abbreviations used are: STAT, signal transducer and activator of transcription; MS/MS, tandem mass spectrometry; Prl, prolactin; PrlR, prolactin receptor; STAT5AS127/128A, STAT5A serines 127/128 mutated to alanine.

² W. Long, G. A. Vidal, A. Naresh, W. C. Wimley, L. Marrero, C. I. Sartor, S. Tovey, T. G. Cooke, J. M. S. Bartlett, and F. E. Jones, unpublished observations.

	ACKNOWLEDGMENTS

 
We are grateful to Lacey Sullivan for excellent laboratory management and to additional members of the Jones laboratory for critical discussions.

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