Serine phosphorylation of Stat5 proteins in lymphocytes stimulated with IL-2
Hai-Hui Xue1,
Donald W. Fink, Jr3,
Xiaolong Zhang2,5,
Jun Qin2,6,
Christoph W. Turck4 and
Warren J. Leonard1
Laboratories of 1 Molecular Immunology and 2 Biophysical Chemistry, National Heart, Lung and Blood Institute, NIH, Bethesda, MD 20892-1674, USA 3 Laboratory of Stem Cell Biology/Neurotrophic Factors, US-FDA/CBER, 1401 Rockville Pike/Suite 200N, Rockville, MD 20825-1448, USA 4 Howard Hughes Medical Institute and Department of Medicine, University of California San Francisco, San Francisco, CA 94143-0724, USA 5 Present address: Department of Structure Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA 6 Present address: Departments of Biochemistry and Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Correspondence to: W. J. Leonard; E-mail: wjl{at}helix.nih.gov
Transmission editor: T. Hirano
 |
Abstract
|
---|
Tyrosine phosphorylation regulates cytokine-induced dimerization of STAT proteins. Serine phosphorylation has also been found to occur in a number of STAT proteins, including Stat1, Sat3, Stat4, Stat5a, Stat5b and Stat6, and was shown to be important for maximal transcriptional activation mediated by Stat1, Stat3 and Stat4, but not for Stat5a or Stat5b. As these latter proteins were studied in transiently transfected COS-7 cells stimulated with prolactin, we sought to further investigate the significance of their serine phosphorylation in a more physiologically based system in response to IL-2. Both Stat5a and Stat5b were rapidly phosphorylated on serine in response to IL-2 and the phosphorylation site in Stat5a was mapped to Ser780, which is not conserved in Stat5b. In vitro studies with reporter constructs, and experiments in which wild-type and mutant Stat5a retroviruses were used to transduce Stat5a-deficient splenocytes revealed that the serine mutant constructs were not diminished in their ability to mediate IL-2 signaling and if anything exhibited augmented proliferative capability. Thus, in contrast to the apparent importance of serine phosphorylation for transcriptional activation by Stat1, Stat3 and Stat4 in response to IFN, IL-6 and IL-12 respectively, serine phosphorylation of Stat5a does not enhance Stat5a-mediated signaling in response to IL-2.
Keywords: cytokine, mass spectrometry, retroviral transduction, signal transduction, T lymphocyte
 |
Introduction
|
---|
Cytokines and IFN signal through a number of signaling pathways, including the JakSTAT pathway (1,2). Following the binding of these molecules to their receptors, Janus family tyrosine kinases (Jak kinases) are activated, resulting in the tyrosine phosphorylation of tyrosine residues on receptors, some of which can serve as docking sites for the binding of STAT proteins, which bind to the phosphorylated receptor via their SH2 domains (1,2). The STAT proteins in turn are phosphorylated on a conserved tyrosine. When the STAT proteins dissociate from the receptor, they then can dimerize, translocate to the nucleus, bind regulatory sequences in target genes and act as transcription factors (1,2). Tyrosine phosphorylation of the STAT proteins is essential for their homo- or heterodimerization by an SH2 domain-mediated mechanism in which the SH2 domain of each STAT protein monomer interacts with the phosphorylated tyrosine in the other monomer. A C-terminal region of STAT proteins is required for transactivation (1,2).
In addition to Jak kinase-mediated tyrosine phosphorylation, it has become evident that a number of STAT proteins can be phosphorylated on serine [reviewed in detail in (3)]. Stat1 and Stat3 were the first STAT noted to contain phosphoserine based on phosphoamino acid analysis (35). Subsequently, Stat4, Stat5a and Stat5b were also noted to contain phosphoserine (3,610). The initial studies on Stat1 and Stat3 revealed a conserved residue, Ser727, as the phosphorylation site (5). This residue is within the transactivation domain and was identified as biologically important, as its mutation to alanine in the context of either Stat1 or Stat3 resulted in a partial decrease in cytokine-induced transcriptional activation (11,12). This was shown for IFN-
in the case of Stat1 (13), and for IFN-
and IL-6 in the case of Stat3 (14). Similar effects have been shown for mutation of Ser721 of Stat4, the homologous residue (15).
In contrast to the apparent importance of these serine residues in Stat1, Stat3 and Stat4, the situation for Stat5 proteins is somewhat different and is perhaps analogous to Stat6 where multiple serine residues in the transactivation domain are phosphorylated in response to IL-4, but the functional significance of this is not clear (16,17). In the context of prolactin signaling, it was found that Ser725 of murine Stat5a was constitutively phosphorylated in COS-7 cells and Nb2 lymphocytes, whereas phosphorylation of Ser730 of Stat5b was potently induced by prolactin (8,9). An independent study revealed that Ser779 in murine Stat5a was constitutively phosphorylated in multiple tissues, and that mutagenesis of Ser725 and Ser779 did not affect prolactin-induced activation of a ß-casein reporter construct, but, interestingly, a Ser725 mutant exhibited sustained DNA binding activity in transiently transfected COS-7 cells (10). Another study revealed that phosphorylation of Ser725 and Ser779 of murine Stat5a cooperatively suppressed prolactin-stimulated transcription from the ß-casein promoter in COS-7 and MCF-7 mammary cells (18). Consistent with this observation, mutation of Ser779, alone or in combination with Ser725, of Stat5a and mutation of Ser730 of Stat5b led to an increase in growth hormone-induced ß-casein promoter activity (19). In contrast, these mutations caused decreased activity of a reporter construct containing four copies of a Stat5-binding site derived from the promoter of the rat ntcp gene, suggesting that serine phosphorylation of Stat5 proteins may modulate the expression of target genes based on the promoter context (19). We have now investigated serine phosphorylation of Stat5a and Stat5b in response to IL-2. We identified serine phosphorylation of both proteins by phosphoamino acid analysis, but both mass spectrometry and radioactive sequencing of H332PO4-labeled cells only revealed a serine-phosphorylated site (Ser780) in human Stat5a. Mutagenesis of Stat5a at positions 725 and 780 and reconstitution experiments in murine splenocytes showed that the mutants were at least as active as wild-type Stat5a, underscoring the difference in the importance of serine phosphorylation of Stat5a versus Stat1/Stat3.
 |
Methods
|
---|
Metabolic labeling and immunoprecipitation of Stat5a phosphoproteins
YT cells (1.5 x 107/well) were cultured overnight in RPMI 1640 media supplemented with 2% FBS and labeled with H332PO4 (1 mCi/ml, 85009120 Ci/mmol; NEN, Boston, MA) for 4 h at 37°C in phosphate-free RPMI 1640 containing 2% dialyzed FBS. 32P-labeled cells were incubated in the absence or presence of IL-2 (100 U/ml,
2 nM) for 15 min at 37°C. Cells were then collected, washed and lysed in 1 ml of lysis buffer (50 mM TrisHCl, pH 7.4 containing 1% Triton X-100, 10% glycerol, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylethylsulfonyl fluoride and 10 µg/ml leupeptin). Insoluble material was pelleted by centrifugation, lysates pre-cleared with 25 µl Protein Aagarose (Pierce, Rockford, IL) for 60 min at 4°C, and then immunoprecipitated overnight at 4°C with antibodies to Stat5a and Stat5b. Immune complexes were collected by incubation with 25 µl of Protein Aagarose for 60 min at 4°C, washed 5 times with 1 ml ice cold washing buffer [50 mM Tris, pH 8.5; 1% (v/v) Triton X-100, 1% deoxycholate, 0.1% SDS, 150 mM NaCl and 0.2% sodium azide], and once each with 1 ml of ice-cold PBS and H2O. Washed immune complexes were suspended in 25 µl of SDS sample buffer and heated for 7.5 min at 90°C. Released immunoprecipitated proteins were resolved by SDSPAGE through 8% Trisglycine gels (Invitrogen, Carlsbad, CA). Electrophoresed phosphoproteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) and the Stat5 proteins identified by autoradiography using XAR film (Kodak, Rochester, NY).
Phosphoamino acid analysis of immunoprecipitated, phosphorylated protein
Phosphorylated Stat5 protein bands located by autoradiography were excised from the Immobilon-P membranes and subjected to hydrolysis in 150 µl of 6 N HCl at 100°C for 90 min, thereby liberating 32P-labeled phosphoamino acids from the membrane. The hydrolysates were evaporated to dryness and reconstituted in 12 µl of electrophoresis buffer [7.8% acetic acid and 2.5% formic acid (v/v), pH 1.9] containing unlabeled phosphoamino acid standards (Sigma, St Louis, MO). Samples were spotted onto 20 x 20 cm DEAE cellulose-coated glass TLC plates (Merck/EM Science, Gibbstown, NJ) and 32P-labeled phosphoamino acids resolved by two-dimensional horizontal electrophoresis according to an established procedure (20) using the HTLE-7000 peptide mapping system (CBS Scientific, Del Mar, CA). Briefly, sample-spotted TLC plates were pre-wet with pH 1.9 electrophoresis buffer and electrophoresed in the first dimension for 20 min at 1500 V. After air-drying, TLC plates were pre-moistened with pH 3.5 electrophoresis buffer [5% acetic acid, 0.5% pyridine (v/v), 0.5 mM EDTA, pH 3.5], rotated 90° and electrophoresed in the second dimension for 18 min at 1350 V. The locations of resolved phosphoamino acid standards were visualized by ninhydrin staining and co-migrating radiolabeled phosphoamino acids were subsequently detected by autoradiography.
Mapping of protein phosphorylation sites
Final purification of radiolabeled Stat5a and Stat5b was achieved by immunoprecipitation and SDSPAGE. Gels were fixed, dried and exposed to X-ray film to visualize the radiolabeled proteins. In-gel digestion of the proteins with trypsin (Roche Diagnostics, Indianapolis, IN) was performed as described (21). The resultant peptides were separated using reversed-phase HPLC on a microbore C8 column (Vydac, Hesperia, CA) and an aliquot of each collected fraction subjected to scintillation counting. Individual radioactive peptides were subjected to covalent Edman degradation on a Sequelon AA membrane (Perseptive Biosystems, Cambridge, MA) with a protein sequencer, model 492 (Applied Biosystems, Foster City, CA). The ATZ-amino acids were extracted from the membrane with neat trifluoroacetic acid and scintillation counted. Radioactive profiles for each sequencing run were compared to theoretical peptide sequences derived from Stat5a and Stat5b respectively. For confirmation of the phosphorylation sites, synthetic peptides were prepared corresponding to the possible Stat5a tryptic phosphopeptides. These peptides were mixed with the radioactive tryptic digest and detected by UV absorption using reversed-phase HPLC.
Mass spectrometry for the identification of phosphorylation sites
The procedure for identifying phosphorylation sites was previously described (22). Briefly, matrix-assisted laser desorption/ionization mass spectrometry (MALDI/TOF) with delayed extraction (Voyager-DE; Perseptive Biosystems, Framingham, MA) was used for the identification of phosphopeptides. Electrospray ion trap mass spectrometry (LCQ; Finnigan, San Jose, CA) coupled on-line with a microbore HPLC (Magic 2002, Auburn, CA) were used for the identification of the phosphorylation site. A monitor C18 column (5 µm particle diameter, 150 Å pore size, 0.2 x 50 mm dimension) was used for the on-line liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis.
Constructs and mutagenesis
Human Stat5a and murine Stat5a were subcloned into the XhoI site of pGFP-RV, a retroviral vector (23). Mutations of Ser726 and/or Ser780 in human Stat5a and Ser725 and/or Ser779 in murine Stat5a were introduced in their respective original vectors using the Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, CA), the sequences of the mutant constructs were confirmed, and the inserts were then subcloned into the pGFP-RV vector.
Retroviral transduction of splenocytes from Stat5a/ mice
The mice used in this study were F2 or F3 animals derived from Stat5a heterozygous crosses of mixed background (129 SvEv x NIH Black Swiss SvCv x BALB/c) animals [(24) kindly provided by Dr Lothar Hennighausen]. All experiments were performed under protocols approved by the National Institutes of Health Animal Use and Care Committees and followed the National Institutes of Health Guidelines Using Animals in Intramural Research. Splenic cells were isolated from 8- to 12-week-old Stat5a/ mice, and suspended in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, antibiotics and 50 µM 2-mercaptoethanol (complete RPMI) containing 10 mM HEPES (pH 7.0). Lymphocytes were isolated using Histopaque 1.119 (Sigma) and T cells were then purified by panning on dishes coated with goat anti-mouse IgG (100 µg/ml in PBS for 1 h at room temperature). Splenic T cells were resuspended in complete RPMI containing 1 µg/ml anti-CD28 (BD PharMingen, San Diego, CA) and 1 nM recombinant human IL-2 (Hoffmann La Roche, Nutley, NJ), and were activated for 24 h by culturing in dishes precoated with anti-CD3 (1 µg/ml, 37°C for 1.5 h).
Retroviruses harboring wild-type human or murine Stat5a or their serine mutants were packaged in 293T cells by co-transfection of Stat5a-expressing pGFP-RV and pCLeco, the retroviral packaging vector (25). For each infection, 2 ml of filtered retroviral supernatant was supplemented with 4 µg/ml polybrene and added to 1 x 106 splenic T cells. The cells were centrifuged at 2500 r.p.m. at 30°C for 45 min. The supernatant was removed and the cells were cultured in full RPMI supplemented with 0.5 µg/ml anti-CD3, 1 µg/ml anti-CD28 and 1 nM IL-2. The retroviral infection was repeated 24 h later, and the cells were maintained in full RPMI containing 1 nM IL-2 for 710 days before flow cytometric analysis and proliferation assays.
Retrovirus-infected splenic T cells were stained and analyzed on a FACSort (Becton Dickinson, San Jose, CA) using CellQuest software as previously described (26). Phycoerythrin (PE)-conjugated anti-IL-2R
(PC61) and rat IgG1 (a
isotype-matched control antibody; BD PharMingen) were used for direct staining.
Proliferation assays
After three washes with PBS, retrovirus-infected splenic T cells (2x105 cells/well) were cultured in complete RPMI 1640 with or without 1 nM IL-2 for 24 h in 96-well plates and pulsed with 1 µCi of [3H]thymidine (6.7 mCi/mmol; NEN) for the last 12 h of culture. Radioactivity incorporated into cells was collected on printed glass fiber filters with a cell harvester and was counted using a Trilux 1450 micro-ß-counter (Wallac, Turku, Finland).
 |
Results and discussion
|
---|
To evaluate IL-2-induced phosphorylation of Stat5 proteins, two equal aliquots (15 x 106 cells each) of YT cells were labeled with H332PO4 and then either not stimulated or stimulated with IL-2. Cells were then lysed and lysates immunoprecipitated with antisera to Stat5a or Stat5b. By one-dimensional analysis, IL-2 resulted in an increase in the overall level of phosphorylation of each Stat5 protein (Fig. 1). The amino acid specificity of this phosphorylation was determined by a two-dimensional electrophoretic phosphoamino acid analysis (Fig. 2). In the absence of IL-2, basal serine phosphorylation was detected for both Stat5a and Stat5b. Stimulation with IL-2 increased serine phosphorylation (2.5-, 3.2- and 3.7-fold for Stat5a and 1.7-, 3.7- and 8.9-fold for Stat5b in three independent experiments) and induced robust tyrosine phosphorylation. Threonine phosphorylation was not detected in either untreated or IL-2-treated samples. IL-2-induced increases in serine and tyrosine phosphorylation were also observed in NK3.3 cells (data not shown). IL-2-stimulated phosphorylation of Stat5a and Stat5b was also evaluated in human peripheral blood lymphocytes. IL-2 uniformly induced serine and tyrosine phosphorylation of Stat5a; in the case of Stat5b, while induction of tyrosine phosphorylation was seen in all three donors examined, induction of serine phosphorylation was more variable (data not shown). The reason for the difference in sensitivity of Stat5b serine versus tyrosine phosphorylation is unknown.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 1. IL-2 induced an overall increase in phosphorylation of Stat5 proteins. YT cells were labeled with H332PO4 as described in Methods and then cultured with (lanes 3, 4, 7 and 8) or without (lanes 1, 2, 5 and 6) IL-2 for 15 min. The cells were lysed and immunoprecipitated with antisera to Stat5a or Stat5b. The proteins were resolved by SDSPAGE, transferred to Immobilon-P membranes and autoradiographed. Shown is one representative experiment with each condition performed in duplicate.
|
|

View larger version (111K):
[in this window]
[in a new window]
|
Fig. 2. Phosphoamino acid analysis of Stat5a and Stat5b. Stat5a (A and B) and Stat5b (C and D) were analyzed from 15 x 106 YT cells either not treated (A and C) or stimulated with IL-2 (B and D). Phosphorylated Stat5 proteins were excised from membranes as shown in Fig. 1 and subjected to acid hydrolysis. The phosphoamino acids thus released were developed on DEAE cellulose-coated plates and autoradiographed. pSer and pTyr stand for phosphorylated serine and phosphorylated tyrosine respectively.
|
|
To determine the specific sites of phosphorylation, YT cells were labeled with H332PO4, Stat5a and Stat5b were immunoprecipitated, and radiolabeled bands corresponding to Stat5a and Stat5b were excised. In-gel digestion of the proteins was performed with trypsin, peptides separated by HPLC and then subjected to Edman degradation. Radioactivity contained in each fraction was counted as detailed in Methods. For Stat5a, both before (data not shown) and after IL-2 stimulation (Fig. 3A), a radioactive peak was detected in fractions 3435. An additional peak of radioactivity was identified in fraction 29 only after IL-2 stimulation (Fig. 3A). Sequencing of each of these peaks showed that the 32P-labeled residue was in cycle 5 of the fraction 29 peptide (Fig. 3B) and cycle 2 of the fraction 3435 peptide (Fig. 3C). Examination of the various possible tryptic peptide sequences derived from Stat5a with a tyrosine residue in position (sequencing cycle) 5 (Fig. 3B) allowed us to unequivocally identify the radioactive peptide in fraction 29 (Fig. 3A) as A690VDGpYVKPQIK700. This peptide includes the known Y694 tyrosine phosphorylation site of Stat5a. Based on the elution profile from the HPLC column and the fact that it carries a radioactive amino acid in sequencing cycle 2 (Fig. 3C), we hypothesized that fractions 3435 (Fig. 3A) corresponded to the phosphopeptide L779pSPPAGLFTSAR790, which includes Ser780, a serine residue that is not conserved in Stat5b. Analogous to fraction 29 for Stat5a, a strongly phosphorylated peptide was detected in fraction 26 of Stat5b (data not shown). This site corresponds to A695VDGpYVK701, which includes the known Stat5b Y699 tyrosine phosphorylation residue (data not shown). Unexpectedly, however, we could not identify a second radioactive peptide for Stat5b, even though two-dimensional phosphoamino acid analysis had revealed serine phosphorylation. This finding might be explained by the inability to elute the phosphorylated peptide from the gel.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3. Reversed-phase HPLC of the tryptic digest of radiolabeled Stat5a (A) and radioactive Edman degradation of the radioactive peptides eluting in fractions 29 (B) and 3435 (C). In (A), the tryptic digest peptide mixture was fractionated on a reversed-phase C8 column and 10% of each 1-min fraction was counted. In (B) and (C), the radioactive peptides were bonded covalently to the sequencing membrane and subjected to Edman degradation. The membrane extracts from each sequencing cycle were counted.
|
|
In order to confirm these findings, we used mass spectrometry to analyze in vivo phosphorylation of Stat5 in YT cells using an experimental procedure described previously (22). Two phosphopeptides were identified upon treatment of the trypsin digest with a calf intestine phosphatase from an in-gel trypsin digestion of the Stat5a protein. Figure 3(A) shows a peak corresponding to a phosphorylated peptide. The phosphorylated peptides were sequenced by LC/MS/MS to identify the phosphorylation sites as Ser780 (see legend for explanation) and Y699 (data not shown) respectively. Analogous to the radioactive phosphorylated peptide analysis, for Stat5b, mass spectrometry analysis only revealed the Y699 peptide, but no serine-phosphorylated peptide (data not shown).
Having identified Ser780 as the site of serine phosphorylation in human Stat5a, we next investigated whether either this residue or Ser726, the human homologue of murine Ser725, which Yamashita et al. identified as a Stat5a phosphorylation site (9), or both were required for Stat5a-dependent transcription in response to IL-2. As an assay system, we used retrovirus-mediated transduction of splenocytes from Stat5a/ mice. IL-2-induced proliferation and IL-2R
expression were then evaluated. As splenocytes from Stat5a/ mice are known to exhibit diminished IL-2-induced IL-2R
expression (27), we tested the ability of retrovirus-transduced Stat5a to rescue this phenotype. IL-2R
expression in GFP-RV vector-transduced Stat5a/ splenocytes was similar to that observed in non-infected cells (Fig. 5A), while the cells infected by a retrovirus harboring wild-type Stat5a showed greatly elevated IL-2R
expression (Fig. 5B). The higher percentage of green fluorescent protein (GFP)+ cells in the Stat5-transduced group can be attributed to a higher proliferation rate of those cells in which Stat5 expression is restored (discussed below). Transduction with human Stat5aS726A, S780A or S726A/S780A mutants all potently restored IL-2R
expression in Stat5a/ splenocytes (Fig. 5C, D and E, and Table 1). Interestingly, in most experiments, the mutants were if anything more effective than wild-type Stat5a (Table 1). Proliferation assays were also performed 710 days after retrovirus infection when GFP+ cells comprised >70% of the viable cells. As shown in Table 2, transduction of wild-type Stat5a or the S726A, S780A or S726A/S780A mutants of human Stat5a each yielded reasonably similar levels. At the highest concentrations of IL-2, the double mutant yielded slightly higher levels of proliferation. Similar results for IL-2R
expression (Table 1) and proliferation (Table 2) were obtained with wild-type murine Stat5a, with S725A or S779A mutants, or with S725A/S779A double mutants. Thus, mutation of serine residues in Stat5a did not diminish Stat5a function. If anything, particularly for the double mutant, both basal and maximal functional activities were slightly higher for the serine mutants, although the basis for and significance of this finding remains unclear. It will be interesting to determine if there are interacting protein(s), whose interaction is affected by serine phosphorylation of Stat5, that directly or indirectly affect proliferation. There tended to be increased proliferation in Stat5a (wild-type or serine mutants)-transduced splenocytes compared with those transduced with pRV alone. However, because the cells are pre-activated, this could reflect a Stat5-dependent effect of endogenous IL-2 that persisted even after resting the cells.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 5. Rescue of IL-2R expression of splenocytes from Stat5a/ mice by retroviral transduction of wild-type Stat5 and its serine mutants. Splenocytes were isolated from Stat5a/ mice. The cells were pre-activated and, as described in Methods, infected with retrovirus expressing GFP alone (A), or with retrovirus expressing human wild-type Stat5a (B), hStat5a Ser726Ala (C), hStat5a Ser780Ala (D) or hStat5a Ser726/780Ala (E) in addition to GFP. After subsequent expansion for 710 days in the presence of 1 nM IL-2, the cells were analyzed for expression of IL-2R . Values shown in the upper right quadrant represent the percentage of GFP+ cells among activated splenocytes and those in the upper left quadrant represent the percentage of GFP cells. The mean fluorescent intensities of IL-2R PE in the upper right quadrant for (A)(E) are 200, 1533, 1938, 1693 and 1877 respectively. For (B)(E), in which the splenocytes were infected with Stat5a-expressing retroviruses, the level of IL-2R GFP+ was elevated as compared with the respective GFP population. Representative data from four independent experiments are shown.
|
|
This study represents the first report of the serine phosphorylation of human Stat5a and Stat5b, and the first to specifically investigate Stat5 phosphorylation sites in response to IL-2. As noted above, in studies focusing on prolactin and growth hormone, Ser779 and Ser725 were separately identified as sites of phosphorylation for Stat5a, while Ser730 was found to be a phosphorylation site for Stat5b (810,19). As noted above, initial studies showed no functional deficit in reporter assays when these serines were mutated to alanines (9,10), while more recent studies suggest different regulatory effects depending on the promoter context (18,19). Because these functional studies were all performed using transient transfection and in view of the important functional roles for serine phosphorylation reported for Stat1, Stat3 and Stat4 (4,5,15), we sought to further investigate the role of serine phosphorylation for Stat5 proteins in the context of IL-2 and to use a more physiologically based assay system. Although both Stat5a and Stat5b appeared to be phosphorylated on serine, we could only map a site in Stat5a as Ser780 (the human homologue of Ser779). It is conceivable that sites in Stat5b and other site(s) in Stat5a could be phosphorylated, but that they could not be identified either by the experimental approaches used. The failure to identify such sites might result, for example, from the phosphorylation of multiple sites, with each individual site being phosphorylated at too low a stoichiometry to allow detection.
To investigate the functional significance of serine phosphorylation of Stat5a, we mutated each of the sites identified in this study as well as those found by others in previous studies. The effect of site-directed mutagenesis was evaluated using reporter assays in cell lines (data not shown) and by reconstitution of primary splenocytes from Stat5a/ mice, where we evaluated cellular proliferation in response to IL-2. No defects were found in any of the assays. These data indicate that, at least for the activities examined, serine phosphorylation of Stat5a is not essential for IL-2 signaling. In fact, unexpectedly, in five of six experiments, there was somewhat augmented CD25 expression in splenocytes reconstituted with mutated Stat5a. This is consistent with the observations that mutation of Ser725 and/or Ser779 to Ala in murine Stat5a showed prolonged prolactin-induced DNA binding activity (10,18) and delayed dephosphorylation of Tyr694 (10). In addition, mutation of Ser730 in Stat5b caused increased Stat5 DNA-binding activity and an increase in growth hormone-induced ß-casein promoter activity (19). Collectively, these results point to a possible role of serine phosphorylation in limiting the duration of Stat5 signals, although the possible significance of this finding and mechanism require additional investigation. Nevertheless, based on reconstitution experiments in normal murine splenocytes, we can conclude that the functional significance of serine phosphorylation of Stat5 proteins is distinctive from its role in Stat1, Stat3 and Stat4 signaling, where it has been shown to serve a positive regulatory function. It is interesting that while Stat1, Stat3 and Stat4 share a ProMetSerPro motif, the Stat5a Ser725 residue is in a ProSerPro motif, whereas the Ser780 peptide is closer to the C-terminus and contains an ArgLeuSerPro sequence. The fact that these motifs are different from those present in Stat1, Stat3 and Stat4 is consistent with a potentially different function, as well as with the potential utilization of different serine/threonine kinases. Evolutionarily, it has been suggested that STAT proteins can be divided into two classes, B-type STAT and A-type STAT, with Stat1, Stat2, Stat3 and Stat4 falling into the former category, and Stat5a, Stat5b and Stat6 falling into the latter (28,29). Consistent with this categorization, none of these latter A-type STAT proteins contain the ProMetSerPro motif nor has serine phosphorylation been shown to be essential for their transactivation function or DNA binding. Thus, the importance of serine phosphorylation for different STAT proteins may correlate with evolutionary considerations.
 |
Acknowledgements
|
---|
We thank Dr Jian-Xin Lin for valuable discussions, critical comments and other contributions to the study, and Ms Maria Berg for assist ance in growing YT cells. We thank Dr Kenneth M. Murphy and Dr Inder M. Verma for providing GFP-RV and the pCL packaging vector respectively.
 |
Abbreviations
|
---|
GFPgreen fluorescent protein
LC/MS/MSliquid chromatography/mass spectrometry/mass spectrometry
MALDI/TOFmatrix-assisted laser desorption/ionization mass spectrometry
PEphycoerythrin

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4. Serine phosphorylation of Stat5a in vivo. (A) The portion of the MALDI/TOF mass spectra before (top panel) and after (bottom panel) treatment with CIP showing the disappearance of the phosphorylated peptide encompassing the sequence, 770RPMDSLDSRLSPP AGLFTSAR790 (calculated m/z [mass to charge ratio, with z typically equal to 1] of 2355.6 average; see solid circle with dashed arrow). Each peak corresponds to a tryptic peptide of the indicated mol. wt. The height of each peak shows the relative rather than absolute abundance. As expected, the dephosphorylated ion at m/z 2275.21 increases in relative abundance after treatment with CIP (solid circle). There appears to be comparable amounts of phosphorylated and unphosphorylated protein originally present, although MALDI is imprecise in such quantitation. Note that trypsin inefficiently cleaves after Arg770 due to the adjacent Pro771 and inefficiently cleaves after Arg778, presumably because of the phosphorylation of Ser780. (B) The LC/MS/MS (often referred to as liquid chromatograph/mass spectrometry2) spectrum of the phosphopeptide identified in (A) at 2355.6 + 2 H (hydrogen ions) = 2357.6. When this is divided by the full charge of +3, one gets 786 (3+ charged). A window of ±2 Da was used to select the ion. Ser780 was identified as the site of phosphorylation. The ions marked with signify that 98 a.m.u. corresponding to H3PO4 has been lost. The b and y nomenclature (30) used corresponds to the typical cleavage points of the peptide. The b ions correspond to the amido CN cleavage points (between the carbonyl and the adjacent -amino group at each peptide bond) with the charge being retained by the N-terminal fragment; similarly, the y ions correspond to the same cleavage with the charge retained by the C-terminal fragment, plus 2 H atoms that have migrated to the N-terminal Nitrogen atom of each resulting peptide fragment. Thus, the y ions correspond to sequential N-terminal deletions of 1 or more amino acids. Specifically, for example, b7 comprises amino acids 770776, whereas y7 comprises residues 784790. The yn+10 ion refers to the y10 ion that carries n positive charges; therefore, its mass will be the (y10 mass + [n 1] H)/n [the charge]; thus y2+10 has a mass of (y10 + 1H)/2. The b7, y10 and y2+10 ions have masses that indicate that they do not contain phosphate, restricting the phosphate site to Ser777 or Ser780. Ions at y2+14 (770.6), and y 2+14 and b11, b2+11, and b 11 confirm this. The ion at 1086 for y 11 allows assignment of the phosphate to Ser780.
|
|
 |
References
|
---|
- Leonard, W. J. and OShea, J. J. 1998. Jaks and STATs: biological implications. Annu. Rev. Immunol.16:293.[ISI][Medline]
- Stark, G., Kerr, I. M., Williams, B. R. G., Silverman, R. H. and Schreiber, R. D. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67:227.[ISI][Medline]
- Decker, T. and Kovarik, P. 2000. Serine phosphorylation of STATs. Oncogene 19:2628.[ISI][Medline]
- Zhang, X., Blenis, J., Li, H. C., Schindler, C. and Chen-Kiang, S. 1995. Requirement of serine phosphorylation for formation of STATpromoter complexes. Science 267:1990.[ISI][Medline]
- Wen, Z., Zhong, Z. and Darnell, J. E. 1995. Protein maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241.[ISI][Medline]
- Cho, S. S., Bacon, C. M., Sudarshan, C., Rees, R. C., Finbloom, D., Pine, R. and OShea, J. J. 1996. Activation of STAT4 by IL-12 and IFN-alpha: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation. J. Immunol. 157:4781.[Abstract]
- Beadling, C., Ng, J., Babbage, J. W. and Cantrell, D. A. 1996. Interleukin-2 activation of STAT5 requires the convergent action of tyrosine kinases and a serine/threonine kinase pathway distinct from the Raf1/ERK2 MAP kinase pathway. EMBO J. 15:1902.[Abstract]
- Kirken, R. A., Malabarba, M. G., Xu, J., Liu, X., Farrar, W. L., Hennighausen, L., Larner, A. C., Grimley, P. M. and Rui, H. 1997. Prolactin stimulates serine/tyrosine phosphorylation and formation of heterocomplexes of multiple Stat5 isoforms in Nb2 lymphocytes. J. Biol. Chem. 272:14098.[Abstract/Free Full Text]
- Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A. and Rui, H. 1998. Differential control of the phosphorylation state of proline-juxtaposed serine residues Ser725 of Stat5a and Ser730 of Stat5b in prolactin-sensitive cells. J. Biol. Chem. 273:30218.[Abstract/Free Full Text]
- Beuvink, I., Hess, D., Flotow, H., Hofsteenge, J., Groner, B. and Hynes, N. E. 2000. Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity. J. Biol. Chem. 275. 10247.
- Kovarik, P., Stoiber, D., Novy, M. and Decker, T. 1998. Stat1 combines signals derived from IFN-gamma and LPS receptors during macrophage activation. EMBO J. 17:3660.[Abstract/Free Full Text]
- Ng, J. and Cantrell, D. 1997. STAT3 is a serine kinase target in T lymphocytes. Interleukin 2 and T cell antigen receptor signals converge upon serine 727. J. Biol. Chem. 272:24542.[Abstract/Free Full Text]
- Eilers, A., Georgellis, D., Lose, B., Schindler, C., Ziemiecki, A., Harpur, A. G., Wilks, A. F. and Decker, T. 1995. Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages. Mol. Cell. Biol. 15:3579.[Abstract]
- Schuringa, J. J., Jonk, L. J., Dokter, W. H., Vellenga, E. and Kruijer, W. 2000. Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components. Biochem. J. 347:89.[ISI][Medline]
- Visconti, R., Gadina, M., Chiariello, M., Chen, E. H., Stancato, L. F., Gutkind, J. S. and OShea, J. J. 2000. Importance of the MKK6/p38 pathway for interleukin-12-induced STAT4 serine phosphorylation and transcriptional activity. Blood 96:1844.[Abstract/Free Full Text]
- Pesu, M., Takaluoma, K., Aittomaki, S., Lagerstedt, A., Saksela, K., Kovanen, P. E. and Silvennoinen, O. 2000. Interleukin-4-induced transcriptional activation by stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of stat6. Blood 95:494.[Abstract/Free Full Text]
- Wick, K. R. and Berton, M. T. 2000. IL-4 induces serine phosphorylation of the STAT6 transactivation domain in B lymphocytes. Mol. Immunol. 37:641.[ISI][Medline]
- Yamashita, H., Nevalainen, M. T., Xu, J., LeBaron, M. J., Wagner, K. U., Erwin, R. A., Harmon, J. M., Hennighausen, L., Kirken, R. A. and Rui, H. 2001. Role of serine phosphorylation of Stat5a in prolactin-stimulated beta-casein gene expression. Mol. Cell. Endocrinol. 183:151.[ISI][Medline]
- Park, S. H., Yamashita, H., Rui, H. and Waxman, D. J. 2001. Serine phosphorylation of GH-activated signal transducer and activator of transcription 5a (STAT5a) and STAT5b: impact on STAT5 transcriptional activity. Mol. Endocrinol. 15:2157.[Abstract/Free Full Text]
- Kamps, M. P. and Sefton, B. M. 1989. Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. Anal. Biochem. 176:22.[ISI][Medline]
- Hellman, U., Wernstedt, C., Gonez, J. and Heldin, C. H. 1995. Improvement of an ingel digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 224:451.[ISI][Medline]
- Zhang, X. L., Herring, C. J., Romano, P. R., Szczepanowska, J., Brezeska, H., Hinnebusch, A. G. and Qin, J. 1998. Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis. Anal. Chem. 70:2050.[ISI][Medline]
- Ouyang, W., Ranganath, S. H., Weindel, K., Bhattacharya, D., Murphy, T. L., Sha, W. C. and Murphy, K. M. 1998. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism. Immunity 9:745.[ISI][Medline]
- Liu, X., Robinson, G. W., Wagner, K. U., Garrett, L., Wynshaw-Boris, A. and Hennighausen, L. 1997. Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev. 11:179.[Abstract]
- Naviaux, R. K., Costanzi, E., Haas, M. and Verma, I. M. 1996. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 70:5701.[Abstract]
- Cao, X., Shores, E. W., Hu-Li, J., Anver, M. R., Kdelsall, B. L., Russell, S. M., Drago, J., Noguchi, M., Grinberg, A., Bloom, E. T., Paul, W. E., Kats, S. I., Love, P. E. and Leonard, W. J. 1995. Defective lymphoid development in mice lacking expression of the common cytokine receptor
chain. Immunity 2:223.[ISI][Medline]
- Nakajima, H., Liu, X. W., Wynshaw-Boris, A., Rosenthal, L. A., Imada, K., Finbloom, D. D., Hennighausen, L. and Leonard, W. J. 1997. An indirect effect of Stat5a in IL-2-induced proliferation: a critical role for Stat5a in IL-2-mediated IL-2 receptor alpha chain induction. Immunity 7:691.[ISI][Medline]
- Barillas-Mury, C., Han, Y. S., Seeley, D. and Kafatos, F. C. 1999. Anopheles gambiae Ag-STAT, a new insect member of the STAT family, is activated in response to bacterial infection. EMBO J. 18:959.[Abstract/Free Full Text]
- Copeland, N. G., Gilbert, D. J., Schindler, C., Zhong, Z., Wen, Z., Darnell, J. E., Jr, Mui, A. L. F., Miyajima, A., Quelle, F. W., Ihle, J. N. and Jenkins, N. A. 1995. Distribution of the mammalian Stat gene family in mouse chromosomes. Genomics 29:225.[ISI][Medline]
- Biemann, K. 1990. Nomenclature for peptide Fragment Ions (Positive Ions). In McCloskey, J. A., ed., Methods in Enzymol. 193, appendix 5, p. 886. Academic Press, Orlando, FL.