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Division of Cell and Molecular Biology, Department of Biology, Boston University (S.-H.P., D.J.W.), Boston, Massachusetts 02215; and Department of Pathology, Uniformed Services University of the Health Sciences (H.Y., H.R.), Bethesda, Maryland 20814
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In addition to this JAK-catalyzed tyrosine phosphorylation reaction, STAT proteins may undergo serine phosphorylation in a manner that can be cell type and stimulus dependent (4, 5, 6). In the case of STAT1, -3, and -4, serine phosphorylation is directed at a conserved PMSP motif located about 20–30 amino acids C-terminal of the conserved tyrosine phosphorylation site. This phosphorylation can be stimulated by cytokine treatment and may be catalyzed by a downstream kinase, e.g. a cytokine- or growth factor-activated MAPK. Basal serine phosphorylation of STAT proteins has also been observed (for a review, see Ref. 7). Mutation of the conserved PMSP serine phosphorylation site (e.g. STAT1-Ser727, STAT3-Ser727, or STAT4-Ser721) decreases cytokine-induced transcriptional activity, supporting the hypothesis that serine phosphorylation is required for maximal transcriptional activity of these three STATs and may modulate cytokine responses (4, 8, 9), at least on some promoters (10). Serine phosphorylation can also negatively regulate cytokine-induced tyrosine phosphorylation in the case of STAT3 (11). STAT nuclear translocation and DNA-binding activity are generally not affected by STAT serine phosphorylation, suggesting that serine phosphorylation modulates STAT’s intrinsic transcriptional potential, which is mediated by a C-terminal trans-activation domain just downstream of the serine phosphorylation site.
Serine phosphorylation of STAT5a and the closely related (>90% identical) STAT5b (12) has been observed in cells and tissues stimulated with STAT5-activating ligands such as GH (13, 14, 15), PRL (16, 17), and IL-2 (18). In PRL-stimulated cells, both STAT5 forms are phosphorylated on a conserved serine residue (STAT5a-Ser725 and STAT5b-Ser730) located within a PSP sequence, which corresponds in location to the PSMP serine phosphorylation sequence of STAT1, -3, and -4 (19). STAT5a is additionally phosphorylated at a second site, recently identified as serine 779 (20, 21). STAT5 serine phosphorylation may in part be mediated by the MAPK cascade, as suggested by binding interactions between STAT5a and the MAPKs ERK1 and ERK2 (21) and by the inhibition of constitutive, but not PRL-inducible, STAT5a serine 725 phosphorylation in Nb2 lymphocytes by the MAPK pathway inhibitor PD98059 (19). Functional studies of the effects of serine phosphorylation on STAT5’s transcriptional activity have not provided a consistent picture. In the case of IL-2-activated STAT5 (STAT5a and/or STAT5b), cytokine-stimulated reporter gene activity is abolished in cells treated with the serine kinase inhibitor H7, which blocks STAT5 serine phosphorylation (18). Similarly, GH-activated STAT5a activity is blocked by inhibition of MAPK activity (22), which may play a role in STAT5a serine phosphorylation (21). In contrast, no difference in PRL-stimulated STAT5 reporter gene activity was seen when cells were transfected with serine to alanine mutant forms of STAT5b (Ser730 to Ala) or STAT5a (Ser725 mutated to Ala, Ser779 to Ala, or the Ser725,779Ala double mutant) compared with the corresponding wild-type STAT5 forms (19, 20). Delayed tyrosine dephosphorylation was, however, reported for STAT5a-S725A in cells stimulated with PRL (20). These findings raise the possibility that the consequences of STAT5 serine phosphorylation may vary with the activating hormone or cytokine, or perhaps with the target gene used to evaluate the impact of STAT5 serine phosphorylation. These and related issues are investigated in the present study, where we evaluate the functional consequences of STAT5 serine phosphorylation in GH-stimulated cells using site- specific mutants of STAT5a and STAT5b. Our findings reveal that GH induces the same pattern of STAT5 serine phosphorylation as that previously reported for PRL. Moreover, we report that serine phosphorylation can modulate the transcriptional activity of both STAT5a and STAT5b in a promoter-dependent manner.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Initial experiments were carried out using COS-1 cells transfected with
GH receptor and either STAT5b or the site-specific mutant STAT5b-S730A
and then stimulated with GH. Western blotting with anti-STAT5b antibody
revealed multiple protein bands, which were previously identified as
differentially phosphorylated forms of STAT5b (Fig. 1A
). STAT5b band 0 (Fig. 1A) migrates as
a doublet of bands, neither of which appears to be phosphorylated, as
shown previously by phosphatase treatment experiments, whereas STAT5b
band 1a corresponds to serine-phosphorylated STAT5b (14).
This latter conclusion is supported by the absence of STAT5b band 1a in
unstimulated cells transfected with STAT5b-S730A (Fig. 1A, lane 3
vs. lane 1). STAT5b band 2, previously identified as STAT5b
phosphorylated on both tyrosine and serine, is the major GH-induced
phosphorylated form of wild-type STAT5b (lane 2). In contrast,
STAT5b-S730A was converted to a doublet of proteins after GH treatment
(Fig. 1A, lane 4 vs. lane 3). Both bands of the doublet were
phosphorylated on tyrosine, as shown by immunoprecipitation with
anti-STAT5b antibody followed by antiphosphotyrosine 4G10 Western
blotting (data not shown; also see below). The lower band corresponds
in mobility to STAT5b phosphorylated on tyrosine alone, i.e.
STAT5b band 1, whereas the upper band of the doublet remains
unidentified (band X). The STAT5b doublet dominates in
STAT5b-S730A-transfected cells treated with GH and is not further
converted to the doubly phosphorylated STAT5b band 2, presumably
because of the block in the secondary serine phosphorylation on residue
730 (see below). The precise relationship between STAT5b-S730A bands 1
and band X is uncertain. The two proteins may correspond to the
tyrosine-phosphorylated counterparts of the two marginally resolved
STAT5b protein forms seen in unstimulated cells (both designated band
0; Fig. 1A, lanes 1 and 3).
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, lanes 5–9). Thus, STAT5b was basally
phosphorylated on Ser730 (band 1a; Fig. 1, middle panel, lane 6), and GH stimulated the
formation of STAT5b phosphorylated on both Ser730
and Tyr699 (band 2; Fig. 1, lane 7). STAT5b
Ser730 phosphorylation was blocked in cells
transfected with STAT5b-S730A (Fig. 1, lanes 8 and 9, middle
panel), whereas GH stimulated Tyr699
phosphorylation of STAT5b-S730A to form a band that migrated just below
band 2 (band 1/band X; Fig. 1, upper panel, lane 9). [Of
note, STAT5b bands 0, 1 and X are poorly resolved in the right
panel of Fig. 1A (c.f. lanes 1 and
4).]
We next investigated whether GH induces phosphorylation of
STAT5b on Ser730, e.g. via a
GH-stimulated serine kinase, and whether
phospho-Ser730-STAT5b serves as a substrate for
the GH-stimulated tyrosine phosphorylation reaction. These studies were
carried out using the GH-responsive liver cell line CWSV-1, where all
the components required for GH-induced STAT5b tyrosine and serine
phosphorylation are expressed endogenously (14, 24).
CWSV-1 cells were stimulated with GH for varying periods of time, and
cell extracts were prepared and analyzed by immunoprecipitation with
anti-STAT5b antibody, followed by sequential probing with the
antibodies shown in Fig. 1B. As reported previously (14),
STAT5b is basally phosphorylated on serine in CWSV-1 cells
(c.f. presence of STAT5b band 1a in unstimulated cells; Fig. 1B, lane 1, lower panel). At least a portion of this
phosphorylation is on Ser730, as indicated by the
reactivity of band 1a with
phospho-Ser730-specific anti-STAT5 antibody
(anti-pS730; upper panel). Moreover, GH-induced tyrosine
phosphorylation of Ser730-phosphorylated STAT5b
was readily detectable, as revealed by the appearance of the
pS730-reactive STAT5b, band 2 (lane 2). The GH-stimulated increase in
total pS730-STAT5b normalized to total STAT5b
immunoreactivity (lane 2 vs. lane 1, lower
portion of Fig. 1B) suggests that STAT5b
Ser730 can be phosphorylated by a
GH-stimulated serine kinase in addition to the basal
Ser730 kinase activity. Both the basal and the
GH-stimulated STAT5b Ser730 kinase activity are
partially inhibited by the serine kinase inhibitor H7 (Fig. 1B, lanes 7
and 8 vs. lanes 1 and 2). This conclusion is supported by
densitometric analysis of pS730-STAT5b immunoreactivity normalized to
total STAT5b protein (lower portion of Fig. 1B). Moreover,
the specific phospho-Ser730 content of STAT5b is
seen to decline back to the basal level from its peak 30 min after GH
stimulation.
We cannot determine from the above data whether the
diphosphorylated STAT5b (band 2) is preferentially formed by
GH-stimulated tyrosine phosphorylation of preexisting
phospho-Ser730-STAT5b (band 1a) or by tyrosine
phosphorylation of STAT5b band 0, followed by a secondary,
GH-stimulated Ser730 phosphorylation of a STAT5b
band 1 intermediate. Support for the latter possibility is provided
by Western blot analysis of GH-stimulated CWSV-1 extracts using
antibody specific for STAT5b phosphotyrosine 699. Figure 1C shows that
GH stimulates the transient formation of pY699-STAT5b at 5 min,
followed by conversion to band 2 in an apparent serine phosphorylation
reaction (lanes 3 and 4 vs. lane 2). In cells treated with
H7, this secondary conversion to STAT5b band 2 is partially blocked.
This finding supports the partial inhibition of GH-stimulated S730
phosphorylation shown in Fig. 1B (note the persistence in Fig. 1C of
the pY699-STAT5b-immunoreactive doublet, even at 40–80 min; lanes
11–13 vs. single band at 20 min in lane 15 in the absence
of H7). Densitometric analysis of the
phospho-Tyr699 signals normalized to total STAT5b
protein verified that H7 treatment slows down the decay in STAT5b
signaling, as shown previously (24).
Transcriptional Activity of Site-Specific STAT5 Serine Mutants
To ascertain the functional significance of GH-stimulated STAT5
serine phosphorylation, serine to alanine mutations were introduced at
the conserved PSP serine phosphorylation site of both STAT5 forms
(STAT5a Ser725 and STAT5b
Ser730). The effects of these site-specific
mutations on GH-stimulated, STAT5-dependent gene transcription were
evaluated in transfection experiments using a luciferase reporter gene
driven by four copies of a STAT5-binding site derived from the promoter
of the rat ntcp gene (25). These studies were
carried out in the liver cell line HepG2, which has low endogenous GH
receptor and STAT5, but serves as a useful model for STAT reporter gene
studies and for expression of GH-regulated liver promoters (25, 26). Wild-type STAT5a and wild-type STAT5b
trans-activated ntcp promoter activity in these
cells by 32- to 40-fold after GH stimulation (Fig. 2A). STAT5a-Y694F and STAT5b-Y699F, which
are mutated at the established STAT5 tyrosine phosphorylation site,
were inactive, consistent with the absolute requirement of STAT5
tyrosine phosphorylation for activation of gene transcription. By
contrast, mutation of the PSP serine phosphorylation site had a more
subtle effect on STAT5-dependent transcriptional activity. No
significant change in ntcp promoter activity was seen with
STAT5a-S725A compared with wild-type STAT5a, whereas a substantial
(
50%) reduction in activity was seen with the corresponding
STAT5b-S730A mutant (Fig. 2A). This effect was observed in each of four
independent HepG2 transfection experiments and was confirmed in a
second cell model, transfected COS-1 cells (Table 1).
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). By
contrast, mutation of the STAT5 tyrosine phosphorylation site abolished
STAT5 DNA-binding activity, as anticipated (Fig. 2B, lanes 4 and 10).
We conclude that phosphorylation of STAT5b at
Ser730 is not required for DNA-binding activity,
but is required to achieve full activation of ntcp promoter
activity, as demonstrated in both HepG2 and COS-1 cells. In the case of
STAT5a, phosphorylation of the corresponding
Ser725 is not required for maximal
transcriptional activity.
Role of STAT5a Ser779 Phosphorylation
In addition to Ser725 phosphorylation,
STAT5a can be constitutively phosphorylated at a second site,
identified as Ser779 (20, 21). To
investigate the significance of Ser779
phosphorylation, we prepared and then assayed the transcriptional
activity of STAT5a expression plasmids containing a serine to alanine
mutation at position 779 alone, or serine to alanine mutations at
positions 725 and 779 in combination. Figure 3A shows that GH-stimulated
ntcp promoter activity was significantly lower in the case
of the double serine mutant STAT5a construct in both HepG2 and COS-1
cells. Moreover, the fold stimulation of promoter activity was
substantially decreased (32- to 38-fold for wild-type STAT5a
vs. 12- to 15-fold for STAT5a-S725,779A; Table 1), despite
the expression of the STAT5a mutant protein at a level at least as
great as wild-type STAT5a (Fig. 3B, lanes 7–10 vs. lanes 3
and 4). A less dramatic decrease in transcriptional activity was seen
in the case of STAT5a-S779A (Table 1), indicating that phosphorylation
of STAT5a on Ser779 in the context of
phosphorylation on Ser725 is required for full
STAT5a transcriptional activity. Phospho-STAT5a analysis confirmed that
mutation of the STAT5a serine phosphorylation sites did not alter
tyrosine phosphorylation of STAT5a at Tyr699
(Fig. 3B, middle panel, lanes 8, 10, and 12 vs.
lane 4). Moreover, mutation of Ser779 did not
decrease phosphorylation of Ser725 (Fig. 3B, upper panel, lanes 9 and 10 vs. lanes 3 and
4).
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(left panel) shows that PRL
activation of STAT5a or STAT5b led to a substantial increase in
ntcp reporter activity. This gene activation was reduced in
cells transfected with the serine- mutated STAT5 forms,
STAT5a-S725,779A and STAT5b-S730A, just as it was in GH-stimulated
cells.
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(right panel) shows that ß-
casein promoter activity is strongly activated in COS-1 cells
transfected with PRL receptor together with either STAT5a or STAT5b and
stimulated overnight with PRL. However, in sharp contrast to the
reduced transcriptional activity obtained in parallel experiments with
the ntcp promoter-based luciferase reporter (left
panel), ß-casein promoter transcriptional
activity was increased to an approximately 2-fold higher level in cells
transfected with either STAT5a-S725,779A or STAT5b-S730A compared with
the corresponding wild-type STAT5 proteins. A similar stimulatory
effect of the STAT5 serine mutations on ß-casein promoter
activity was observed in GH receptor-transfected COS-1 cells stimulated
with GH (data not shown; also see below).
The differential effect of STAT5 serine mutation on ntcp vs.
ß-casein promoter activity shown in Fig. 4A suggests that
the effects of a mutational block in STAT5 serine phosphorylation may
be influenced by promoter context. To further investigate this finding,
we examined the effects of the STAT5b S730A mutation on reporter gene
activity in cells stimulated with GH for times ranging from 3–22 h. As
shown in Fig. 4B, mutation of Ser730 to alanine
led to a decrease in GH-stimulated ntcp reporter activity
assayed at each time point. By contrast, this same mutation increased
ß-casein promoter activity when assayed 22 h after GH
addition, as seen in Fig. 4A in the case of PRL stimulation, but had no
significant impact when the cells were assayed 3, 5, or 8 h after
GH addition (Fig. 4C). Analysis of extracts prepared from GH-stimulated
cells revealed a higher level of Tyr699
phosphorylation in the STAT5b-S730A-transfected cells (Fig. 5A, upper panel, lanes 8–14
vs. lanes 1–7). Correspondingly, a higher DNA-binding
activity was obtained at all time points examined (Fig. 5B). This
increased activity of STAT5b-S730A largely reflects a higher level of
STAT5b-S730A protein expression (Fig. 5A, lower panel, lanes
8–12 vs. lanes 1–5). Consequently, total cellular STAT5
DNA-binding activity is higher in the case of the STAT5b mutant (Fig. 5B), and as a result there is a differential in the DNA-binding
activity found in STAT5b-transfected cells vs.
STAT5b-S730A-transfected cells that is maximal 22 h after GH
stimulation (Fig. 5C). This finding provides an explanation for the
increased ß-casein promoter activity of STAT5b-S730A at
this time point (c.f. Fig. 4C).
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). Table 2 shows,
however, that H7 treatment of COS-1 cells transfected with wild-type
STAT5b leads to a complete inhibition of GH-stimulated,
STAT5b-dependent transcription of the ntcp-luciferase
reporter gene, in contrast to the 50% activity decrease seen with the
STAT5b-S730A mutant. Moreover, H7 treatment led to a similar inhibition
of the transcriptional activity of STAT5b-S730A, demonstrating that the
inhibitory action of H7 is unrelated to its effects on STAT5b serine
phosphorylation. Complete inhibition of STAT5b and
STAT5b-S730A-stimulated transcription was also observed using the pZZ1
reporter (Table 2). Finally, a similar inhibitory effect of H7 on
STAT5b-S730A transcriptional activity was obtained in cells stimulated
with GH for a shorter time period (3 h; c.f. 6 h GH
stimulation in Fig. 5A; data not shown).
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) or DNA-binding activity (data not shown).
Moreover, H7 treatment did not impair nuclear translocation of STAT5b
protein in its tyrosine-phosphorylated form, as revealed by
immunofluorescence microscopy using anti-STAT5b and anti-pY699-STAT5b
antibodies (Fig. 6). We conclude that H7
inhibits STAT5b transcriptional activity at a stage that is downstream
of the STAT5b nuclear translocation and DNA-binding steps.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-activated STAT1
interacts with the nuclear factor minichromosome maintenance-5 in a
phospho-Ser727-dependent manner (9).
Finally, changes in the trans-activation potential of STAT5
can also be achieved by mutations elsewhere in the C-terminal region
(e.g. increased activity of STAT5a-T757V)
(32). The increased DNA-binding activity of STAT5b-S730A compared with wild-type STAT5b appears to be due at least in part to increased expression and/or stability of the Ser730-mutated STAT5 protein. This raises the possibility that phosphorylation of Ser730 in the wild-type protein may enhance the turnover of STAT5b. Although the apparent increase in signaling of STAT5b-S730A at longer times of GH stimulation could additionally involve a decrease in the rate of STAT5b tyrosine dephosphorylation, as was suggested earlier for STAT5a mutated at serine 725 and/or 779 (20), this has not been established. Independent of the mechanism underlying this effect, the increased cellular DNA-binding activity of STAT5b-S730A compared with wild-type STAT5b appears to account for the enhanced ß-casein reporter activity seen at longer times after GH stimulation. Nevertheless, ntcp reporter activity was decreased under these same conditions, highlighting the intrinsic differences in the effects of Ser730 mutation on transcription of the two STAT5-responsive reporter genes, discussed above.
Of the two STAT5a serine phosphorylation sites, residues 725 and 779,
Ser779 appears to be more important for maximal
ntcp promoter activity. This is supported by the decrease in
ntcp reporter activity in cells transfected with
STAT5a-S779A, but not in cells transfected with STAT5a-S725A. This
decrease cannot be explained by an effect of the
Ser779 mutation on the phosphorylation of STAT5a
on Tyr694 or Ser725 (Fig. 3B). Both STAT5a serine residues are likely to be important, however,
as suggested by the more substantial decrease in ntcp
reporter activity displayed by the double mutant, STAT5a-S725,779A.
Similarly, mutation of Ser779, alone or in
combination with Ser725, led to an increase in
ß-casein promoter activity, a response that was also seen
with STAT5b-S730A. In contrast to STAT5b Ser730,
whose phosphorylation is inducible, STAT5a Ser725
and Ser779 can both be constitutively
phosphorylated in a variety of cells and tissues, including developing
mammary gland in the case of Ser779 (19, 20). It is unclear, however, whether both serine residues can be
simultaneously phosphorylated in a given STAT5a molecule, leaving open
the possibility that phosphorylation of Ser725
may inhibit, and thereby help regulate, Ser779
phosphorylation and the resultant
phospho-Ser779-dependent transcriptional
responses.
The stimulatory effects of the STAT5a-Ser779 and STAT5b-Ser730 mutations on GH-induced ß-casein promoter activity seen in the present study contrast with the absence of a clear effect of these mutations in previous studies in PRL-stimulated cells (19, 20). This discrepancy does not reflect differences in the stimulating hormone, as we were able to duplicate our findings in experiments in which STAT5a and STAT5b were activated via the PRL receptor. Rather, it may relate to differences in the time-dependent effects of the Ser730 mutation on ß-casein promoter activity documented in the present study. Also of note, the two previous studies were carried out in COS-7 cells under conditions where only a 2- to 3-fold stimulation of ß-casein promoter activity was achieved, which may limit or mask the modulatory effects of mutating the STAT5 serine phosphorylation sites. In contrast, the present studies were carried out in COS-1 cells under conditions where a 20- to 40-fold activation of the ß-casein promoter was routinely achieved. Further study will be required to clarify this point.
The serine kinase inhibitor H7, which inhibits GH-stimulated STAT5b serine phosphorylation, strongly inhibited STAT5b-dependent reporter gene activity. A strong inhibitory action of H7 was also seen with STAT5b-S730A, independent of whether activity was assayed with the ntcp or ß-casein promoter, indicating that the transcriptional inhibition effected by H7 is unrelated to the resultant changes in STAT5b Ser730 phosphorylation. Mechanistic studies revealed that H7 does not interfere with GH-stimulated STAT5b tyrosine phosphorylation, nuclear translocation, or DNA-binding activity, strongly suggesting that H7 exerts a specific inhibitory action at the level of STAT5 trans-activation. This effect may thus be distinct from the prolonged signaling by the GH receptor-JAK2 complex after H7 treatment that we have previously described in GH-stimulated liver cells (24). Conceivably, H7 may inhibit STAT5-dependent transcription by altering the phosphorylation of a STAT5b-interacting coactivator that is required for the STAT transcriptional response. Alternatively, the inhibition by H7 of STAT5 transcriptional activity may be mechanistically linked to the prolonged signaling by GH receptor-JAK2 to STAT5b by way of a block in STAT5-stimulated transcription of feedback inhibitory regulators of GH receptor-JAK2 signaling, such as SOCS/CIS proteins (33, 34). Further investigation is needed to address this issue.
The kinase(s) that catalyze the constitutive phosphorylation of STAT5a on Ser725 and Ser779 and the signaling pathways that lead to the inducible phosphorylation of STAT5b on Ser730 in response to GH or PRL stimulation remain to be identified. Inhibitor studies suggest a role for a MAPK-like activity in the constitutive phosphorylation of STAT5a on Ser725, but not for the PRL-inducible phosphorylation of STAT5b on the corresponding Ser730 (19). Interestingly, in cells in which the constitutive phosphorylation of STAT5a Ser725 is blocked by the MAPK kinase inhibitor PD98059, PRL can induce phosphorylation at that site (19), demonstrating that Ser725 is intrinsically responsive to PRL stimulation, in a manner that is analogous to the inducible phosphorylation of Ser730 in the case of STAT5b. The additional site of STAT5a phosphorylation, at residue Ser779, is within the COOH-terminal 20 amino acids of STAT5a, where the two STAT5 proteins are highly divergent in sequence. This residue is thus absent from STAT5b. In vitro phosphorylation of STAT5a by MAPK is strongly inhibited by mutation of Ser779, as is the interaction of STAT5a with the MAPK ERK1 and ERK2 (21), suggesting a role for MAPK in this phosphorylation reaction as well. In other studies, carried out in a different cell model, phosphorylation of STAT5a at Ser779 was not blocked by inhibitors of MAPK or PI3K (20). Interestingly, Ser779 occurs within a sequence (RLSPPA) that corresponds to a consensus motif for phosphorylation by PKA, but not by nine other serine protein kinases, as revealed by computer analysis using the web-based PhosphoBase program (35). Accordingly, further investigation of the role of PKA/cAMP-dependent signaling pathways in the phosphorylation of STAT5a at this COOH-terminal site may be warranted.
STAT5a and STAT5b play distinct physiological roles in mediating hormonal responses to PRL (STAT5a) and GH (STAT5b) in the mammary gland and liver, respectively (36, 37). Although this differential endocrine function may largely reflect the distinct tissue distributions of the two STAT5 forms, there is increasing evidence that the biological properties of STAT5a and STAT5b, although very similar, are distinguishable in several important ways. STAT5a and STAT5b not only display biochemical differences in apparent DNA binding specificity (38, 39) and propensity to bind to DNA as tetramers (STAT5a > STAT5b) (29, 40), but they exhibit potentially important differences in their regulation by serine phosphorylation. Thus, the phosphorylation of STAT5a vs. STAT5b on Ser725/730 is not only subject to differential regulation (constitutive phosphorylation of STAT5a vs. inducible phosphorylation of STAT5b), but leads to modulatory effects on gene transcription only in the case of STAT5b. In the case of STAT5a, such a modulatory effect requires phosphorylation on Ser779, a residue unique to this STAT5 form.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture and Transfections
COS-1 and HepG2 cells were maintained in DMEM containing 10%
FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin. For transient
transfections, cells were seeded in 24-well plates at a density of
1.3 x 105 HepG2 cells/well or 5 x
104 COS-1 cells/well. Fugene 6 transfection
reagent (Roche Molecular Biochemicals, Indianapolis, IN)
was used as described in the manufacturer’s protocol, at a ratio of
1.3:1 of Fugene 6/DNA (vol/wt). Each well received a total of 600 ng
DNA, including 150–200 ng luciferase reporter plasmid, 50 ng GH
receptor, and 100–200 ng STAT5 expression plasmid. pRL-tk-Luc plasmid
(Renilla luciferase; 50 ng DNA) was included as an internal
control for transfection efficiency. Twenty-four hours after
transfection, the cells were treated with rat GH (200 ng/ml) or rat PRL
(10 nM) for an additional 18–24 h unless
specified otherwise. H7 (200 µM) was included
as indicated. Total cell extracts were prepared using 1x lysis buffer
(Promega Corp., Madison, WI) for measuring luciferase
activities. For Western blot and EMSA analysis, total cell lysates were
centrifuged for 30 min at 15,000 x g. Firefly and
Renilla luciferase activities were measured using a Dual
Reporter Assay System (Promega Corp.) and a Monolight 2010
luminometer (Analytical Luminescence Laboratory, San Diego, CA). Data
shown in the individual figures are relative values based on normalized
luciferase activity (i.e. firefly/Renilla
luciferase activities; mean ± SD for three
replicates).
Growth and passage of CWSV-1 cells was carried out as described previously (14). For serine phosphorylation studies, CWSV-1 cells were stimulated with GH at 200 ng/ml in the presence or absence of H7 (200 µM) for varying periods of time. Total cell extracts were prepared in lysis buffer containing 20 mM HEPES (pH 7.9); 1% Triton X-100; 1 mM each of EDTA, EGTA, Na3VO4, Na2P2O7, and dithiothreitol; 0.5 mM phenylmethylsulfonylfluoride; and 1 µg/ml each of pepstatin, antipain, and leupeptin. Total cell extracts were passed through a 27-gauge needle seven times, adjusted to 150 mM NaCl, and centrifuged at 15,000 x g for 30 min at 4 C. Protein concentrations were determined using the Dc detergent protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).
EMSA Analysis
Total cell extracts (5 µg) were assayed for STAT5 DNA-binding
activity using a ß-casein STAT5 response element probe
(14). Gels were exposed to PhosphorImager plates
overnight, followed by quantitation of radioactivity intensity using a
Molecular Dynamics, Inc. PhosphorImager and ImageQuant
software (Sunnyvale, CA).
Western Blotting and Immunoprecipitation
Total cell extracts (20–30 µg) were electrophoresed on 7.5%
Laemmli SDS gels, electrotransferred to nitrocellulose membranes, and
then probed with anti-STAT5b antibodies (catalogue no. sc-835,
Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Blocking
and probing conditions were described previously (41).
Probing with anti-pY699-STAT5b antibody (Cell Signaling Technology,
Beverly, MA) was performed with a 1-h incubation of the blot at room
temperature in TST buffer [10 mM Tris-HCl (pH 7.6), 0.1%
Tween 20, and 100 mM NaCl] containing 5% nonfat dry milk,
followed by incubation with anti-pY699 antibody (1:1000 dilution) in
5% BSA-TST buffer overnight at 4 C. Washings were carried out as
specified by the manufacturer. For STAT5 immunoprecipitation, CWSV-1
cells grown on 100-mm dishes were solubilized in 1 ml lysis buffer [10
mM Tris-HCl (pH 7.6), 5 mM EDTA, 50
mM NaCl, 30 mM
Na2P2O7
50 mM NaF, 1 mM
Na3VO4, 1% Triton X-100,
and 1 mM phenylmethylsulfonylfluoride] in the presence of
phosphatase inhibitors (14). Clarified total cell extracts
were incubated for 2 h on ice with 2 µl polyclonal rabbit
anti-STAT5b antiserum (antibody raised against a peptide corresponding
to amino acid residues 776–786 of mouse STAT5b was obtained from Dr.
L. Hennighausen, NIH) (42). Immune complexes were captured
with protein A-Sepharose beads (Pharmacia Biotech,
Piscataway, NJ), electrophoresed on 7.5% Laemmli-SDS gels, and then
transferred onto nitrocellulose membranes (Millipore Corp., Bedford, MA). Membranes were blocked for 1 h at 37 C
with 5% nonfat dry milk in TST buffer. Incubations with site-specific
antiphosphoserine STAT5 antibody (anti-pS730) (19) were
carried out for 16 h at a dilution of 1:5000 in the cold-room.
Anti-pS730-STAT5 antibody was raised against the phosphopeptide
DQAP[pS]PAVC, corresponding to amino acid residues 726–734 of human
STAT5b (19). Antibody binding was visualized on x-ray film
by enhanced chemiluminescence using the ECL kit from Amersham Pharmacia Biotech (Arlington Heights, IL; anti-STAT5b and
anti-pS730-STAT5b) or the SuperSignal ECL kit from Pierce Chemical Co. (Rockford, IL; anti- pY699-STAT5b).
Immunofluorescence Studies
CWSV-1 cells were seeded at about 60% confluence onto four-well
chamber slides (catalog no. 62409-294, VWR Scientific Products, Boston,
MA) in RPCD medium (14) containing 3% FBS and
allowed to adhere overnight. The medium was then replaced with
serum-free RPCD medium. The following day, the cells were pretreated
with H7 (200 µM) for 1 h as indicated, then treated
with GH (200 ng/ml) and H7 for 30 min. Cells were rinsed with ice-cold
PBS and fixed with 100% MeOH for 20 min at -20 C. Fixed cells were
blocked with 3% charcoal-stripped calf serum in PBS for 1 h at
room temperature and then incubated with anti-STAT5b antibody (1:500
dilution; Santa Cruz Biotechnology, Inc.) in blocking
solution overnight at room temperature. For anti-pY699-STAT5b
immunostaining, fixed cells were blocked with 5.5% charcoal-stripped
calf serum in TBST buffer [50 mM Tris-HCl (pH 7.4), 150
mM NaCl, and 0.1% Triton X-100] for 1 h at room
temperature and then incubated for 24 h at 4 C with
anti-pY699-STAT5b antibody (1:500 dilution; Cell Signaling Technology,
Beverly, MA) in TBS buffer [50 mM Tris-HCl (pH 7.4) and
150 mM NaCl] containing 3% BSA. The samples were then
washed (three times, 5 min/wash) with PBS containing 3% calf serum for
anti-STAT5b and with TBST for anti-pY699-STAT5b antibody. Cells were
then incubated for 1 h at 37 C with fluorescein
isothiocyanate-conjugated goat antirabbit IgG antibody (1 µg/ml;
Molecular Probes, Inc., Eugene, OR). Cells were
counterstained with 50 ng/ml propidium iodide (Sigma) to
localize nuclei. For confocal analysis, immunofluorescent cells were
scanned with an BX-50 confocal laser scanning microscope (Olympus Corp., New Hyde Park, NY) equipped with a x60 objective
(Carl Zeiss, New York, NY).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by NIH Grant DK-33765 (to D.J.W.).
Abbreviations: anti-pS730 antibody, Antibody specific for phosphoserine 730 of STAT5b and phosphoserine 725 of STAT5a; anti-pY699 antibody, antibody specific for tyrosine 699 or 694 of STAT5b and STAT5a, respectively; JAK, Janus kinase; PMSP motif, Pro-Met-Ser-Pro; PSP, Pro-Ser-Pro; STAT, signal transducer and activator of transcription; TST buffer, 10 mM Tris-HCl (pH 7.6), 5 mM EDTA, 50 mM NaCl, 30 mM Na2P2O7 50 mM NaF, 1 mM Na3VO4, 1% Triton X-100, and 1 mM phenylmethylsulfonylfluoride.
Site-specific STAT5 mutants are designated using the single letter amino acid code; thus, STAT5b-S730A corresponds to STAT5b with Ser730 mutated to alanine.
Received for publication May 9, 2001. Accepted for publication August 28, 2001.
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