From the Hormel Institute, University of Minnesota, Austin, Minnesota 55912
Received for publication, October 9, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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Phosphorylation at Ser727 is
known to be required for complete activation of STAT3 by diverse
stimuli including UV irradiation, but the kinase(s) responsible for
phosphorylating STAT3 (Ser727) is still not well discerned.
In the present study, we observed that activation of ATM is required
for a UVA-stimulated increase in Ser727 phosphorylation of
STAT3 as well as in activation and phosphorylation of p90 ribosomal
protein S6 kinases (RSKs). Moreover, UVA-stimulated activation of
upstream kinases, such as c-Jun N-terminal kinases (JNKs) and ERKs,
involved in mediating phosphorylation of RSKs and STAT3 was defective
or delayed in ATM-deficient cells. Furthermore, we provide evidence
that RSK2-deficient cells were defective for UV-induced
Ser727 phosphorylation of STAT3, and the defect was
restored after ectopic expression of transfected full-length RSK2.
In vitro experiments showed that active RSK2 and JNK1
induce the phosphorylation of STAT3 precipitates from
immunoprecipitation but not from glutathione S-transferase
(GST) pull-down. Interestingly, the GST fusion STAT3 proteins mixed
together with STAT3 immunoprecipitates can be phosphorylated by JNK.
However, the in vitro phosphorylation of STAT3 was reduced by the GST-STAT3 Signal transducer and activator of transcription 3 (STAT3; also
called STAT3 Based on the fact that Ser727 is located within a potential
mitogen-activated protein kinase (MAPK) consensus motif of the
C-terminal transcriptional domain in STAT3 (9, 11), MAPKs, including extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases
(JNKs) and p38 kinase, were hypothesized to catalyze phosphorylation of
STAT3 (Ser727). Indeed, earlier investigations showed that
MAPKs were likely to be associated with induction of STAT3
(Ser727) phosphorylation (22-25), but direct evidence was
not provided in these studies. Later, some studies (26-28) further
showed that MAPKs had an indirect effect on induction of
Ser727 phosphorylation involved in STAT3 signaling
activation. However, JNKs were recently reported by us and others to
directly induce STAT3 (Ser727) phosphorylation (14, 29). In
addition, studies also demonstrated that Ser727
phosphorylation of STAT3 may be mediated by H7-sensitive kinase (18),
protein kinase C (30), mammalian target of rapamycin (12), MAPK kinase
(e.g. MEK1/SEK1) (28, 31), or MEK1 kinase (MEKK1) (32).
Overall, these findings suggest that in addition to a cell type- and
stimulus-dependent induction of STAT3 (Ser727)
phosphorylation, MAPKs may also modulate Ser727
phosphorylation indirectly via a serine-threonine kinase of MAPK upstream (e.g. MEK1/SEK1, Rac, or MEKK1) or downstream
(e.g. mitogen- and stress-activated protein kinase-1 (MSK1)
or p90 ribosomal protein S6 kinases (RSKs)).
Our recent work showed that MAPKs were required in mediating STAT3
(Ser727) phosphorylation via MSK1 in the cellular response
to UVA (320-400 nm), and in vitro kinase assays indicated
that MSK1 might be a potential kinase involved in phosphorylation of
STAT3 (Ser727) (29). However, the UVA-stimulated
phosphorylation of STAT3 (Ser727) was not completely
abolished in cell lines expressing a kinase-dead mutant of MSK1,
suggesting the involvement of other downstream kinases (e.g.
RSKs) of MAPKs in the phosphorylation process. Furthermore, RSKs and
STAT3, as well as their upstream MAPKs, were reported to be
concomitantly activated and to play a role in controlling cell
development, growth, and survival (33), suggesting that a certain
association existed between MAPKs/RSKs and STAT3. Here, we find that
RSKs are also required for phosphorylation of STAT3 (Ser727) following exposure of cells to UVA or UVC
(200-290 nm). Moreover, UVA-stimulated but not UVC-stimulated
activation and phosphorylation (Ser380) of RSKs as well as
STAT3 (Ser727) phosphorylation are modulated by activation
of ATM (ataxia telangiectasia, mutated) kinase. Taken together, our results suggest that
RSKs may be potential upstream kinases for modulating phosphorylation of STAT3, and the process may be regulated by ATM. Interestingly, further in vitro kinase assays provide evidence that active
RSKs or JNK1 only in the presence of a downstream kinase or cofactor may induce phosphorylation at the C-terminal Ser727 and/or
non-Ser727 residues of STAT3 Amplification of the Wild-type STAT3 Construction and Mutagenesis of GST Fusion STAT3
Vectors--
The STAT3 GST Fusion STAT3 Protein Expression and Pull-down--
The
pGEX-5X-C plasmids encoding the wild-type full-length STAT3 Cell Lines and Cell Culture--
GM09621 is a human lymphoblast
line containing wild-type RKS2 genes and therefore was used as the
internal control cells; GM03317 is a lymphoblast line from a patient
with Coffin-Lowry syndrome that is linked to RSK2 mutation
(RSK2 UV Sources and Cell Treatments--
To diminish the basal level
of protein phosphorylation or protein kinase activity, the experimental
cell lines were starved for 36-48 h in the above mentioned RPMI 1640 containing 0.5% FBS for lymphoblast lines or in serum-free Dulbecco's
modified Eagle's medium for AT fibroblast lines prior to treatments
with UVA or UVC irradiation. Nonirradiated cell samples were used as
negative controls. For the detailed description of UVA or UVC sources, see our previous reports (38, 40).
Transfection of RSK2 STAT3-mediated Luciferase Activity Assays--
The above
mentioned pGEX-5X-C vectors expressing wild type STAT3 Western Blot Analysis of Protein Phosphorylation--
Equal
numbers of experimental cells (1 × 106 to 1.5 × 106) were cultured for 12-24 h in 100-mm dishes. The cells
were starved for 48 h and then irradiated with UVA or UVC. At the
indicated times after irradiation, the cells were harvested and washed
once with ice-cold phosphate-buffered saline. Then the cell samples were disrupted in 200 µl of radioimmune precipitation buffer (1× phosphate-buffered saline, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, and the inhibitors added before use, 10 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 100 µM Na3VO4). The cell lysates were
clarified by microcentrifuging at 13,000 rpm, and the supernatant
fractions were saved. The samples containing equal amounts of proteins
(modified Lowry's method; Sigma) in an equal volume of radioimmune
precipitation buffer were diluted with 3× SDS sample buffer (187.5 mM Tris-HCl (pH 6.8), 6% (w/v) SDS, 30% (v/v) glycerol,
150 mM dithiothreitol, and 0.3% (w/v) bromphenol blue).
Then samples were subjected to separation by 8% SDS-PAGE followed by
Western blot analysis according to the methods reported previously (29,
38-40). Antibodies against a specifically phosphorylated RSK
(Ser380), STAT3 (Ser727), ERKs, JNKs, or p38
kinase, as well as against total STAT3, STAT1, ERKs, JNKs, p38 kinase,
GST, or In Vitro Assay for STAT3 Phosphorylation by Protein
Kinases--
After starvation for 48 h in Eagle's minimum
essential medium and 0.1% FBS, JB6 Cl 41 cells were disrupted
and then subjected to immunoprecipitation with a STAT3 antibody
according to the procedure reported previously (29, 39). The STAT3
proteins from immunoprecipitation or GST pull-down or the mixture of
both were incubated at 30 °C for 1 h with active RSK1, RSK2,
ERK2, or JNK1 (Upstate Biotechnology) in kinase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
1 mM EGTA, 1 mM dithiothreitol, and 0.01% (v/v) Brij 35) containing 0.5 mM ATP or 10 µCi of
[ Assay for RSK1 and RSK2 Activity--
After treatments of the
experimental cells with UVA or UVC, the lysates were subjected to
immunoprecipitation with a RSK1 or RSK2 antibody, followed by RSK1 or
RSK2 activity assays that were performed according to the methods
recommended by Upstate Biotechnology or as reported previously (39). A
peptide, AKRRRLSSLRA, in the S6 kinase assay kit (Upstate
Biotechnology) was used as a substrate for the RSK1 immune complexes,
whereas another peptide KKRNRTLTK served as a substrate for RSK2
(Upstate Biotechnology). After the reactions, the radioactive phosphate
incorporated into the indicated substrate peptide in the samples was
quantified with a MAPK Activity Assay--
ERKs, JNKs, and p38 kinase activities
were measured by immune complex kinase assays (39). In brief, the
lysates from UV-irradiated cells were subjected to immunoprecipitation
with an antibody to ERKs, JNKs, or p38 kinase. Then the
immunoprecipitates of ERKs, JNKs, or p38 kinase were incubated for 20 min at 30 °C, respectively, with Elk1, c-Jun, or ATF2 fusion protein
as substrates (Cell Signaling). The radioactive phosphate incorporated
into the reactive substrate was quantified as described above.
Statistical Analysis--
Significant differences between
stimulated activity of RSK1, RSK2, ERKs, JNKs, or p38 kinase and their
corresponding controls, as well as STAT3-mediated luciferase
activities, were determined using the Student's t test.
ATM Kinase Activation Is Involved in Regulating UVA-stimulated
Phosphorylation of STAT3 and STAT1--
ATM kinase is known to be a
member of the phosphatidylinositol 3-kinase-related protein kinase
family and plays a vital role in controlling cell development, growth,
and survival (reviewed in Refs. 43-45). Our recent studies indicated
that ATM kinase is activated by irradiation with UVA but not UVC, and
the activation is involved in UVA-induced signaling (42). Here, our
data showed that UVA-induced Ser727 phosphorylation of
STAT3 as well as STAT1 (Fig.
1A) was differentially reduced
in ATM-deficient cells (ATM UVA-induced Activation and Phosphorylation of MAPKs Are Modulated
by ATM Activation--
Our previous studies have indicated that MAPKs
are upstream kinases involved in UVA-stimulated signaling activation of
STAT3 (29). ATM kinase has been shown to be located upstream of
multiple signal transduction pathways (reviewed in Ref. 44). However, whether ATM kinase has an effect on activation and phosphorylation of
MAPKs is unclear. Here, our further experiments revealed that UVA-stimulated phosphorylation and activation of JNKs (Fig.
2, A and
C) were markedly prevented in ATM-deficient
cells compared with ATM-expressing control cells. Moreover, UVA-induced
phosphorylation and activity of ERKs (Fig. 2, A and
D) and p38 kinase (Fig. 2, A and E)
were diminished and/or delayed in ATM-deficient cells. Conversely, ATM
deficiency had no inhibitory effect on UVC-stimulated phosphorylation
of ERKs, JNKs, or p38 kinase (Fig. 2B), and no change in
total levels of ERKs, JNKs, or p38 kinase (Fig. 2, A and
B) was observed in the two cell lines. Overall, these data suggest an involvement of ATM kinase in the regulation of the MAPK
cascades contributing to activation of STAT3 signaling by UVA. But how
the activated ATM kinase mediates phosphorylation and activation of
MAPKs remains to be further investigated.
UVA-induced but Not UVC-induced Activation and Phosphorylation of
RSKs Are Mediated by ATM Kinase Activity--
Our recently reported
data indicated that activation of JNKs and ERKs are required in
UVA-induced phosphorylation and activation of RSKs (39). Further, the
UVA activation of JNKs and ERKs was initiated by ATM kinase as
described above. However, whether ATM plays a role in regulating RSKs
activation is as yet unknown. To evaluate the role of ATM, we assayed
stimulated levels of phosphorylation (Ser380) of RSKs as
well as activity of RSK1 or RSK2 in two UV-irradiated AT cell lines.
The results showed that UVA-induced phosphorylation of RSKs
(Ser380) was markedly prevented (Fig.
3A) in
ATM-deficient cells compared with control cells, but the
phosphorylation by UVC was not different between the two cell lines
(Fig. 3B). In addition, no change of total levels of RSK1 or
RSK2 expression (Fig. 3, A and B) was detected in
the two cell lines. The phosphorylation of RSKs (residue 380) may
indirectly reflect total levels of RSK activity, based on the fact that
the specific-phospho-RSK (Ser380) antibody is known to
cross-react with the homologous sequence of RSK1, RSK2, and RSK3 (39)
(see the Cell Signaling site on the World Wide Web at
www.cellsignal.com). Furthermore, both RSK1 and RSK2 activity
stimulated by UVA (Fig. 3, C and E), but not UVC
(Fig. 3, D and E), were significantly
(p < 0.05) diminished in ATM-deficient cells
(ATM RSK2 Is Required in UV-stimulated Phosphorylation of STAT3 but not
STAT1--
As described above, phosphorylation and activation of
STAT3, RSKs, and MAPKs were triggered by UVA-activated ATM kinase
(Figs. 1-3). The UVA-stimulated MAPKs were shown to be upstream
kinases responsible for the intracellular phosphorylation or activation of STAT3 (29) and RSKs (39). However, phosphorylation of the STAT3
immunoprecipitates was induced directly by active JNKs only, and not
ERKs or p38 kinase in vitro (29), although MAPKs are postulated to be a potential kinase for catalyzing phosphorylation of
STAT3 (Ser727) (9, 11). These observations, therefore,
suggest that a downstream effector such as RSK may be involved in
mediation of ATM-initiated phosphorylation of STAT3. To examine the
suggestion, we used two cell lines expressing normal human wild-type
RSK2 or a mutant RSK2 gene
(RSK2
In addition, the RSK2 Phosphorylation of STAT3 (Ser727) Is Restored by
Ectopic Expression of Full-length RSK2 in RSK2 Phosphorylation of the STAT3 Immunoprecipitates Is Induced
Indirectly by RSKs or JNK1 in Vitro--
Our recent report (29)
indicated that Ser727 phosphorylation of STAT3
immunoprecipitates (IP-STAT3) from JB6 Cl 41 cell lysates was induced
in vitro by active MSK1 as well as JNKs but not by ERKs or
p38 kinase. Further studies showed an involvement of ERKs/p38 kinase-mediated MSK1 in the intracellular phosphorylation of STAT3 (29). Here, we performed further in vitro kinase assays
followed by Western blotting with a phospho-Ser727 antibody
(Fig. 6, A and
F) or with radioisotope
[
To further identify a kinase directly responsible for catalyzing
phosphorylation of STAT3 (Ser727), we established a GST
fusion STAT3 protein expression system to pull down a wild-type
full-length STAT3 Dependence of STAT3-mediated Luciferase Activity on
RSK2--
Here, to examine the functional significance of STAT3 serine
phosphorylation by related kinases, we used a STAT-responsive luciferase Cy1luc reporter containing the CyRE region of the
human VIP gene (12). Two motifs within the CyRE of
the Cy1luc reporter have been determined to bind transcriptional
complexes that are made up of STAT dimers and AP-1 (12). On the
other hand,
Furthermore, we performed co-transfection of wild-type RSK2 cells with
The most information regarding regulation of STAT3 or STAT1
signaling is concentrated heavily on tyrosine phosphorylation by Janus
kinases or other tyrosine kinases, leading to the idea that tyrosine
phosphorylation is indispensable for the activation process of STAT3 or
STAT1 (reviewed in Refs. 1-3). This idea, however, is challenged by
recent findings revealing the existence of tyrosine
phosphorylation-independent activation mechanisms of STAT3 or STAT1
(17-21). In fact, serine phosphorylation (Ser727) was
shown to be essential for activation of STAT3 (9-16) or STAT1 (46,
47), in particular, following stimulation of cells with diverse
stresses, including UVA (29) or UVC (14, 46) irradiation. These
findings thus demonstrate that multiple signaling pathways may regulate
STAT3 or STAT1 signaling activation by Ser727
phosphorylation. However, the upstream kinases responsible for phosphorylating Ser727 in STAT3 or STAT1 are not fully
identified. MAPKs, including ERKs, JNKs, and p38 kinase, were
postulated to phosphorylate STAT3 or STAT1 (Ser727) (9-12)
and were shown to be involved in the induction of Ser727
phosphorylation (22-25), but no direct evidence was presented. Recently, we reported that MSK1 is a potential upstream kinase required
for the Ser727 phosphorylation contributing to UVA-induced
STAT3 signaling activation (29). However, the UVA-induced
Ser727 phosphorylation of STAT3 was not totally prevented
by blocking MSK1 activity by pretreatment with H89, a potential
inhibitor of MSK1, or expression of a MSK1 kinase-dead mutant (29),
suggesting that the intracellular phosphorylation process may also
occur through a MSK1-independent pathway (e.g. RSK).
RSKs are another downstream serine-threonine kinase family of ERKs and
PDK1 and are involved in the signaling regulation of some transcription
factors to execute a variety of cellular programs, such as embryonic
development, cell growth, proliferation, and differentiation (39, 48)
(reviewed in Ref. 49). Indeed, these cellular processes also require
functional regulation of STAT3 (reviewed in Ref. 50) or STAT1 (51) in
addition to MAPKs (e.g. ERK2) (52). Furthermore, STAT3 has
been shown to be an oncogene (53) and is activated concomitant with
MAPKs and RSKs following stimulation of some oncogenes by UVA or UVC
(29, 33, 39) (reviewed in Refs. 7, 8, 49, and 54). Therefore, these
findings suggest that MAPK-mediated RSKs may be involved in the
regulation of the STAT3 signaling pathway. To explore the relationship,
we here used a cell line expressing a mutant RSK2 gene
(RSK2 ATM has been shown to be a protein kinase of the phosphatidylinositol
3-kinase family and to be involved in multiple signal transduction
pathways that regulate diverse cellular functions (reviewed in Refs.
43-45). Recently, we reported that ATM kinase was activated by UVA,
but not UVC, and the activation was required for UVA activation of p53
and JNK signaling pathways leading to apoptosis (42). In the present
study, we provide evidence showing that UVA-stimulated, but not
UVC-stimulated, activity of RSK1 and RSK2 as well as phosphorylation of
RSKs (Ser380), STAT3 (Ser727), and STAT1
(Ser727), were differentially prevented in ATM-deficient
cells compared with ATM-expressing control cells. Furthermore, the
defective kinase activation of ATM caused a reduction and/or delay in
UVA-stimulated phosphorylation and activity of ERKs, JNKs, and p38
kinase but had no effect on the response to UVC. But basal protein
expression of the above mentioned signal molecules, including RSK1,
RSK2, STAT1, STAT3, ERKs, JNKs, and p38 kinase, was not influenced by deficiency of ATM kinase. Moreover, RSKs have been shown to be activated and phosphorylated by active PDK1 (54), ERKs, and JNKs but
not p38 kinase (39). However, phosphorylation (Ser727) of
STAT3 immunoprecipitates was induced by active JNKs but not ERKs or p38
kinase, and the UVA-stimulated intracellular phosphorylation of STAT3
was not mediated by the phosphatidylinositol 3-kinase/PDK1 pathway
(29). Overall, these findings suggest a model for UVA-induced STAT3
phosphorylation (Ser727) (Fig.
8). The model indicates that RSK2 is a
potential upstream kinase required for STAT3 phosphorylation
(Ser727), and ATM kinase may play a regulatory role in
UVA-induced phosphorylation of STAT3 through a pathway involving ERKs,
JNKs, and RSK2. In addition, the regulation of ATM kinase may also
occur via an ERK/p38 kinase-dependent MSK1 pathway.
However, whether ATM kinase plays a direct role in the phosphorylation
of RSK2 or STAT3 will be further determined. On the other hand, our
studies also suggest that UVC-stimulated STAT3 phosphorylation
(Ser727) may be triggered by an ATM-independent JNK/RSK
pathway.
protein, a dominant negative form of STAT3. Taken
together, our results demonstrate that the STAT3 phosphorylation at
Ser727 is triggered by active RSK2 or JNK1 in the presence
of a downstream kinase or a cofactor, and thereby the intracellular
phosphorylation process is stimulated through a signaling pathway
involving ATM, MAPKs, RSK2, and an as yet unidentified kinase or
cofactor. Additionally, RSK2-mediated phosphorylation of STAT3
(Ser727) was further determined to be required for basal
and UVA-stimulated STAT3 transcriptional activities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)1 was shown
to be highly homologous to STAT1 and was first identified as an
interleukin-6-activated transcription factor that transduces signals to the nucleus and activates expression of many targeted genes
(reviewed in Refs. 1-3). STAT3 has since been shown to play several
important roles in controlling diverse cellular processes such as
growth, development (reviewed in Refs. 3-6), cell transformation, and
oncogenesis (reviewed in Refs. 7 and 8). In these processes, STAT3
signaling is activated by cytokines and growth factors, and the
activation is well documented to occur through Tyr705
phosphorylation by Janus kinase family members or receptor or nonreceptor tyrosine kinases (1, 4, 6). Further, maximal activation of
STAT3 signaling was demonstrated to require modulation of
Ser727 phosphorylation as well (9-12) (reviewed in Ref.
13). Interestingly, STAT3 signaling was also activated by various
stresses, including UV, hyperosmolarity, and osmotic shock (14-16),
and the activation was triggered by Ser727 phosphorylation
but independent of Tyr705 phosphorylation (17-21). These
findings, therefore, demonstrate that STAT3 is a converging point for
numerous intracellular signaling pathways and that phosphorylation at
Ser727 is essential for STAT3 signaling activation.
However, the kinases responsible for phosphorylating STAT3
(Ser727) are not yet well elucidated. Hence, identification
of the STAT3 kinases will help in understanding the mechanism of STAT3 activation.
, and the phosphorylation is
diminished by STAT3
, a dominant-negative variant of STAT3
(34).
In addition, a role of RSKs and ATM in regulation of STAT1
phosphorylation is also investigated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and STAT3
cDNA--
The cDNA fragments of the STAT3
and STAT3
including ORF (2310 and 2166 bp, respectively) were amplified by the
PCR with primers 5'-CGT GTC GAC TGG CCC AAT GGA ATC AGC
T-3' (SalI site underlined) and 5'-TCG ACG CGT
CGT TCT CAG CTC CTC ACA TG-3' (MluI site underlined) and
cloned into pACT vector (Promega, Madison, WI) via SalI and
MluI restriction sites. The PCR-amplified sequences of
STAT3
and STAT3
were verified by comparison of restriction fragment length and DNA sequence analysis. The primer synthesis and DNA
sequencing were performed by Sigma.
encoding a fragment digested with
SalI/EcoRV from the pACT was cloned into
SalI/SmaI sites of the pUC19 vector. To introduce
the point mutation in the position of the 705(Y705F) or the 727(S727A)
residue, a pair of sense and antisense primers for STAT3
Y705F (5'-CG
CTG CCC CAT TCC TGA AGA CCA AG-3' and 5'-CT TGG TCT TCA
GGA ATG GGG CAG CG-3') and another pair of the primers for
STAT3
S727A (5'-C CTG CCG ATG GCC CCC CGC-3' and 5'-GCG
GGG GGC CAT CGG CAG G-3') (underlined nucleotides indicate
the codon coding a mutated amino acid) were synthesized, respectively
(Sigma). Subsequently, the STAT3
Y705F or STAT3
S727A nucleotide
substitution was accomplished using a QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA) and then confirmed by DNA
sequence analysis (Sigma). Further, the STAT3
Y705F and STAT3
S727A
were ligated into the SalI/MluI sites of the
pGEX-5X-C modified multicloning sites from the pGEX-5X-1 vector. In
addition, wild type STAT3-
and STAT3-
were directly introduced
into the SalI/MluI sites of the pGEX-5X-C vector.
The above-mentioned restriction endonucleases and ligases were
purchased from New England BioLabs, Inc. (Beverly, MA).
or
STAT3
as well as the STAT3
point mutants (STAT3
Y705A or
STAT3
S727A) as glutathione S-transferase (GST) fusion
protein sources were used to transform a DH5
-competent
Escherichia coli strain (Invitrogen). After induction of
protein expression with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside (Sigma) for 4 h, the bacteria were resuspended in a lysis buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1% (v/v) Triton X-100,
10 µg/ml aprotinin and leupeptin, and 100 µM
Na3VO4) and then were further disrupted by the
addition of 0.1 volume of 10% mg/ml lysozyme (Sigma) and subsequent
sonication. Following centrifugation at 10,000 × g for
20 min, the supernatant fraction containing the induced proteins was
incubated with a 50% slurry of pretreated glutathione-Sepharose 4B
(Amersham Biosciences). After washing twice with the above-mentioned
lysis buffer and an additional two times with kinase buffer (described
below), the beads were subjected to SDS-polyacrylamide gel
electrophoresis followed by Western blotting with a GST or STAT3
antibody to determine expression of the GST fusion STAT3 proteins. In
subsequent in vitro kinase reactions, eluates of the
beads with 20 mM reduced GSH (Roche Molecular Biochemicals)
were used as enzymatic substrates. Control experiments were performed
with GST-Sepharose beads generated by expression of GST alone, using
the empty pGEX-5X-C vector.
) (35). The two cell lines were bought
from the NIGMS Human Genetic Cell Repository, Coriell Institute for
Medical Research (Camden, NJ) and were cultured in RPMI 1640 medium
(Invitrogen) supplemented with 15% heat-inactivated FBS (Gemini
Bio-Products, Inc., Calabasas, CA), 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin in a 37 °C humidified atmosphere of 5%
CO2. Human ataxia telangiectasia (AT) fibroblast lines
stably transfected with an empty mammalian expression vector, pEBS7
(Atm
), or a construct, pEBS7-YZ5, containing
recombinant full-length ATM (Atm+) (a generous
gift from Dr. Yosef Shiloh) (36, 37) was maintained in Dulbecco's
modified Eagle's medium (Invitrogen) containing 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml of hygromycin B (Sigma) for
selection. In addition, mouse epidermal tumor promotion-sensitive JB6
Cl 41 cells were grown in 5% FBS with Eagle's minimum essential
medium (BioWhittaker, Inc., Walkersville, MD) (29, 38, 39).
Cells--
An RSK2 cell line,
GM03317, from a Coffin-Lowry syndrome patient was exponentially grown
in the RPMI 1640 medium containing 15% FBS and then was transfected
with an empty vector pcDNA3.1(+) (3.0 µg) (Invitrogen) or
co-transfected with 0.3 µg of the empty vector and 2.7 µg of a
construct expressing a wild-type full-length RSK2 enzyme (a generous
gift from Dr. Morten Frodin (41)) according to the modified protocol
recommended by Qiagen Inc. (Valencia, CA). Briefly, 2 × 106 cells were washed twice with phosphate-buffered saline,
resuspended in 1 ml of fresh RPMI 1640 containing 1%
phosphate-buffered saline, and then seeded into each well of a six-well
plate. Three micrograms of the plasmid DNA were mixed in 96 µl of
serum/antibiotic-free RPMI 1640 medium, and then 15 ml of SuperFect
Reagent was added to the DNA solution. After incubation at 4 °C for
10 min, the transfection complexes gently mixed with 400 µl of RPMI
1640 containing 1% FBS and antibiotics were dropped into the prewashed
cells. After incubation for 3-5 h, some cells were observed to die.
Then the transfected cells were grown for 3 days in the normal complete medium containing 15% FBS and subsequently were selected in a 250 µg/ml G418 RPMI medium.
and STAT3
and point-mutated STAT3 were amplified by PCR with the STAT3 upstream
and downstream primers: 5'-CCC AAG CTT GGG ATG GCC CAA TGG
AAT CAG CTA-3' (HindIII site underlined) and 5'-TCC
CCC CGG GGG ACG TTC TCA GCT CCT CAC ATG GG-3'
(SmaI site underlined). These PCR products were digested
with HindIII/SmaI and then subcloned into the
HindIII/EcoRV sites of the pcDNA3.1(+) vector
(Invitrogen). Cy1luc (VH3) vector, a STAT-responsive luciferase reporter containing the 180-base pair cytokine response element (CyRE)
region of the human VIP gene (12), was truncated out with KpnI/PstI (187 bp). Then the truncated
Cylluc reporter was reacted with Klenow and dNTP and ligated with a T4
ligase, and it is designated here as
Cy1luc, an internal
control. Within the CyRE, two motifs have been identified to bind the
transcriptional complexes made up of STAT dimers and AP-1 (12).
Thus, the truncated
Cy1luc still contains a partial STAT binding
motif. The above mentioned restriction endonucleases and ligases were
purchased from New England BioLabs, Inc. (Beverly, MA). Subsequently,
these STAT3 vectors, Cy1luc, or
Cy1luc reporters were or were
not transfected or co-transfected into wild-type cells or
RSK2-deficient cells according to the procedure as described above. The
transfected cells were cultured for 24 h in normal media and then
starved for 24 h with serum-free media. The cells were harvested
4 h after irradiation with UVA (80 kJ/m2) and
destroyed in 100 µl of lysis buffer containing 0.1 M
potassium phosphate, pH 7.8, 1% Triton X-100, 1 mM
dithiothreitol, 2 mM EDTA. The STAT3-mediated luciferase
activity in the supernatant fraction of samples was measured by
Luminoskan Ascent (Labsystems Inc. Franklin, MA). Results of the
luciferase activity are expressed in relative luminescence units.
-actin were from Cell Signaling, Inc. (Beverly, MA).
Antibodies to a specific phospho-STAT1 (Ser727) and total
RSK1 or RSK2 were purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY). The total RSK3 antibody (C-20) was from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). The levels of total protein were
used as internal controls to verify basal level of protein expression
or equal amounts of protein loading. In addition, some Western blots
were analyzed, and areas were calculated using the ImageQuant Microsoft system.
-32P]ATP. Subsequently, phosphorylation of STAT3 at
Ser727 and total STAT3 proteins were detected by Western
blot analysis with specific antibodies (29) or by autoradiography
(42).
-scintillation counter. Then the stimulated
activity of RSK1 or RSK2 was normalized to the nonirradiated control
value after subtraction of background and is expressed as -fold change compared with controls.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) compared with
control cells ectopically expressing wild-type full-length ATM kinase
(ATM+). On the other hand, UVC-stimulated
Ser727 phosphorylation of STAT3 and STAT1 (Fig.
1B) were not significantly changed in either ATM-deficient
or ATM-expressing cells. Importantly, the deficiency of ATM kinase did
not influence total levels of STAT3 or STAT1 expression (Fig. 1,
A and B). These results suggest that ATM
activation may be involved in the regulation of phosphorylation (Ser727) of STAT3 or STAT1 in the cellular response to UVA
but not UVC. In addition, the partial reduction in the UVA-stimulated
phosphorylation of STAT3 or STAT1 (Fig. 1A) indicates a
requirement for activation of other signaling pathways involved in the
process.
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Fig. 1.
Diminishment of UVA-stimulated
phosphorylation of STAT3 (Ser727) by ATM deficiency.
ATM-expressing (ATM+) and ATM-deficient
(ATM ) cells were harvested at the indicated times
following irradiation with UVA (80 kJ/m2) (A) or
UVC (60 J/m2) (B). Then, the cell lysates were
subjected to Western blot analysis to detect phosphorylation of STAT3
or STAT1 at Ser727, as well as total levels of STAT3 or
STAT1. In addition, the intensity of bands showing phosphorylated
levels of STAT3 or STAT1 was quantified. These data are representative
of at least three similar independent experiments.
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Fig. 2.
Effects of ATM kinase activity on regulation
of phosphorylation and activation of MAPKs in the UVA cellular
response. Following irradiation with UVA (80 kJ/m2)
(A, C, D, and E) or UVC (60 J/m2) (B), ATM+ and
ATM cells were harvested at the indicated times
and then the cell lysates were analyzed by Western blotting using a
specific antibody to detect phosphorylation of ERKs, JNKs, or p38 kinase (A and B), as well as
total levels of ERKs, JNKs, or p38 kinase (A and
B). These data represent at least three similar independent
experiments. Furthermore, the lysates from UVA-irradiated cells were
subjected to immunoprecipitation and subsequent kinase activity assays
for JNKs (C), ERKs (D), or p38 kinase
(E). The UVA-stimulated MAPK activity is expressed as a fold
change after subtraction of background and normalization to the
non-irradiated control cells (value of 1). Each column and bar
represents the mean and standard deviation from three independent
experiments performed in duplicate (C, D, and E).
UVA-induced JNKs (C), ERKs (D) or p38 kinase
(E) activity was significantly (*, p < 0.05) reduced and delayed in ATM-deficient cells compared with
corresponding ATM-expressing cells.
) compared with ATM-expressing control
cells (ATM+). Thus, these data suggest an
involvement of ATM kinase in modulating activation and phosphorylation
of RSK1 and RSK2 in the intracellular response to UVA but not UVC.
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Fig. 3.
Prevention of UVA-stimulated activity and
phosphorylation of RSKs in ATM-deficient cells. After starvation
in serum-free Dulbecco's modified Eagle's medium and subsequent
irradiation with UVA (80 kJ/m2) (A,
C, and E) or UVC (60 J/m2)
(B, D, and E), ATM-expressing
(ATM+) and ATM-deficient
(ATM ) cells were harvested at the indicated
times (A, B, C, and D) or
at 30 min (E). Then, phosphorylated RSKs (Ser380)
(A and B), as well as total levels of RSK1 or
RSK2 (A and B), in the cell lysates was detected
by Western blotting with corresponding specific antibodies. In addition, the
intensity of bands showing phosphorylated levels of RSKs was
quantified. These data are representative of at least four similar
independent experiments. Furthermore, the cell lysates were subjected
to immunoprecipitation followed by kinase activity assays for RSK1
(C and D) or RSK2 (E). After
subtraction of background, the UV-stimulated activity was normalized to
the non-irradiated control cells (value of 1) and is expressed as a
fold change. Each column and bar represents the mean and standard
deviation from three independent experiments performed in duplicate
(C, D, and E). UVA-induced RSK1
(C) or RSK2 (E) activity was significantly (*,
p < 0.05) inhibited in ATM-
cells compared with corresponding ATM+
cells.
), leading to Coffin-Lowry syndrome (35).
Western blot analysis of intracellular protein phosphorylation showed
that RSK2
cells are defective for Ser727
phosphorylation of STAT3 stimulated by UVA or UVC (Fig.
4, A and
B). On the other hand, STAT1
(Ser727) phosphorylation by UVA or UVC was unaffected or,
conversely, increased to a modest level in
RSK2
cells (Fig. 4, A and
B). Additionally, no change in total levels of STAT3 or
STAT1 expression (Fig. 4, A and B) was observed
in the two cell lines.
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Fig. 4.
Requirement of RSK2 in mediating
UV-stimulated phosphorylation of STAT3 but not STAT1. After
starvation for 36-48 h in 0.5% FBS in RPMI 1640 medium, human
wild-type and RSK2 lymphoblast lines were or were
not irradiated with UVA (80 kJ/m2) (A, C, D, and
E) or UVC (60 J/m2) (B, D,
and E). Then, the cells were harvested at the indicated
times (A, B, D, and E), or
at 30 min (C) and the cell lysates were resolved by 8%
SDS-PAGE. Then, the total and phosphorylated (Ser727)
levels of STAT3, or STAT1 (A and B), as well as
phosphorylated levels of RSKs (Ser380), and total levels of
RSK1, RSK2, RSK3 or
-actin (C) were assayed by Western
blotting with corresponding specific antibodies. In addition, the
intensity of bands showing phosphorylated levels of STAT3 or STAT1 was
quantified. These data are representative of at least five independent
experiments. In addition, RSK2
cells were further
characterized by using functional kinase activity assays. The cell
lysates harvested at the indicated times (D and
E) were subjected to immunoprecipitation with a RSK1
(D) or RSK2 (E) antibody followed by RSK1 or RSK2
activity assays. After subtraction of background, the UV-stimulated
activity was normalized to the non-irradiated wild-type control (value
of 1) and is expressed as a fold change. Each column and bar represents
the mean and S.D. of three independent experiments performed in
duplicate (D and E). UV-induced RSK2 activity was
significantly (*, p < 0.05) blocked in
RSK2
cells compared with corresponding wild-type
cells (E).
cell line from a
Coffin-Lowry syndrome patient was also confirmed here. As expected,
these cells had a significantly lower expression level of RSK2 compared
with the wild-type cell line from a normal human subject (Fig.
4C). However, expression of RSK1, RSK3, or
-actin was not
different between the two cell lines (Fig. 4C). Moreover,
UVA-stimulated phosphorylation of RSK (Ser380) was
apparently prevented in RSK2
cells (Fig.
4C). These results, together with our previously reported
data (39), may indirectly reflect a marked decrease in total levels of
RSK activity in the response of the cell to UVA. Indeed, further
studies revealed that RSK2 activity stimulated by UVA or UVC was
defective in RSK2
cells (Fig. 4E),
but a modest compensatory increase in RSK1 activity was observed when
the RSK2
cells were exposed to UVA or UVC
irradiation (Fig. 4D). Overall, our results suggest that
RSK2 may be required for UVA- and UVC-stimulated phosphorylation of
STAT3 (Ser727), whereas the phosphorylation of STAT1
appears to occur through both RSK2-independent and
-dependent pathways.
Cells--
Here, we further assess a requirement for RSK2 in
phosphorylation of STAT3. As expected, a significant ectopic expression of the wild-type RSK2 protein (Fig.
5A) was determined in the RSK2
cells transfected with a construct
containing a human wild-type full-length RSK2 (RSK2-wt) (41), compared
with control RSK2
cells transfected with an
empty vector. Further, a marked restoration in the UVA-stimulated
phosphorylation of STAT3 (Ser727) was determined to
correspond to the ectopic expression of RSK2-wt (Fig. 5B).
On the other hand, no induction of the STAT3 phosphorylation was
observed in the original RSK2
cells or the
RSK2
cells transfected with an empty vector
only (Fig. 5B). Additionally, total STAT3 levels were
unaffected by the transfection (Fig. 5, A and B).
These data further indicate an involvement of RSK2 in UVA-stimulated
phosphorylation of STAT3 (Ser727).
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Fig. 5.
Ectopic expression of full-length RSK2
resulting in restoration of phosphorylation of STAT3
(Ser727) in the RSK-deficient cells.
RSK2 cells were transfected with the indicated
amounts of an empty vector and/or a construct containing a wild-type
full-length RSK2 gene as described under "Experimental Procedures."
Wild-type, non-transfected RSK2
, and transfected
RSK2
cells (A and B) were
starved and then irradiated with UVA as described for Fig. 4.
Subsequently, the cell lysates were subjected to Western blot analysis
with a specific antibody against Ser727 phosphorylation or
total STAT3 (B), as well as total RSK2 (A). In
addition, a nonspecific band was presented as an internal control
(A). These data are representative of at least two
independent experiments.
-32P]ATP labeling (Fig. 6, B,
D, and E). The results showed that phosphorylation (Ser727) of intact STAT3 immunoprecipitates
(IP-STAT3) was significantly triggered by active RSK2, similar to the
positive control JNK1 (Fig. 6A). On the other hand, a lower
level of phosphorylation was initiated by active RSK1 (Fig.
6A), and total levels of STAT3 were unaffected (Fig.
6A). Moreover, autoradiograph analysis revealed a high
phosphorylation level of IP-STAT3 proteins by activated JNK1, RSK1, or
RSK2 but not ERK2 as a negative control (Fig. 6B). The
discrepancies in the degrees of detectable phosphorylation in the above
mentioned two different assays appear to be related to phosphorylation
at an additional residue, besides Ser727, of STAT3.
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Fig. 6.
Induction of phosphorylation of STAT3 or
STAT1 (Ser727) indirectly by RSKs in
vitro. The STAT3 proteins from immunoprecitation
(IP-STAT3) (A, B, D, and E)
or a GST pull-down assay (GST-STAT3) (C, D, and
E) were prepared as described under "Experimental Procedures." (A) After incubation of
IP-STAT3 with active RSK1, RSK2, or JNK1 at the indicated doses, the
reactive products were analyzed by Western blotting with a specific
antibody to detect total and Ser727 phosphorylation levels
of STAT3. Additionally, the intensity of the blots representing
phosphorylated STAT3 was calculated. (B) The kinase
reactions of JNK1, RSK1, RSK2, or ERK2 with IP-STAT3 as a substrate
were carried out in a kinase buffer containing -32pATP.
Then, the reactive products were subjected to 8% PAGE resolution
followed by autoradiography. (C) The indicated GST-fusion
STAT3 proteins were assayed by Western blotting with a specific STAT3
or GST antibody. (D and E) IP-STAT3 proteins were
mixed together with each of the indicated GST-fusion STAT3 proteins and
the mixtures were used as substrates and then were reacted with RSK1,
RSK2, or JNK1 at the indicated doses. Subsequently, the kinase reactive
products were determined by radioisotope
-32pATP
(D) or a phospho-specific STAT3 (Ser727)
antibody (E). In addition, a nonspecific IgG blot was used
as an internal control (E). These data are representative of
at least three independent experiments.
(GST-STAT3
wt) or a dominant-negative STAT3
variant (STAT3
wt) (34) as well as STAT3
point-mutated at the
Ser727 or Tyr705 residue (named STAT3
S727A
or STAT3
Y705F, respectively). These constructs were confirmed by
sequencing (data not shown). The expressed STAT3 proteins from the GST
pull-down were primarily characterized by Western blotting with a STAT3
or GST antibody, and the results were as expected (Fig. 6C).
Subsequently, our protein kinase assays using the pull-down precipitate
as an enzymatic substrate showed that GST-STAT3
wt, GST-STAT3
wt,
GST-STAT3
S727A, GST-STAT3
Y705F, or GST alone was not
phosphorylated directly by active JNK1, RSK1, or RSK2 in
vitro (data not shown). However, phosphorylation of
GST-STAT3
wt, but not GST-STAT3
wt, in a mixture with IP-STAT3 was
stimulated by JNK1, but not RSK1 or RSK2 (Fig. 6D).
Interestingly, a significant increase in the JNK1-stimulated phosphorylation of GST-STAT3
S727A or GST-STAT
Y705F in the
presence of IP-STAT3 was observed (Fig. 6D). Conversely, the
GST-STAT3
S727A or GST-STAT3
Y705F phosphorylation was undetectable
in the RSK1 or RSK2 kinase reactions (Fig. 6D). Furthermore,
phosphorylation of IP-STAT3 by RSK1, RSK2, or JNK1 was shown as
described above (Fig. 6, A and B) but was
diminished by the addition of GST-STAT3
wt (Fig. 6D) and
was also to different degrees affected by the addition of
GST-STAT3
S727A or GST-STAT3
Y705F (Fig. 6D). As
expected, Western blotting with a phosphospecific antibody showed that
Ser727 phosphorylation in JNK1-induced GST-STAT3
wt or
GST-STAT3
Y705F (Fig. 6E) in the presence of IP-STAT3 was
detectable, but Ser727 phosphorylation in GST-STAT3
or
GST-STAT3
S727A was not observed (Fig. 6E). These
observations, together with previously reported data (29), demonstrate
that the antibody specifically recognized Ser727
phosphorylation of STAT3. The addition of GST fusion STAT3 protein substrates also interfered with Ser727 phosphorylation of
IP-STAT3 by JNK1 (Fig. 6E). Overall, the results of these
in vitro kinase assays suggest that JNK1 only in the presence of a cofactor may catalyze phosphorylation at the C-terminal Ser727 and/or non-Ser727 residues of STAT3, and
the Ser727/non-Ser727 phosphorylation may be
also stimulated indirectly by RSK2 as well as RSK1 in cooperation with
a RSK downstream kinase or/and a cofactor binding to IP-STAT3 precipitates.
Cy1luc reporter contains a partial motif of STAT
binding. Our studies showed that phosphorylation of STAT3 and STAT1,
but not other STATs (data not shown), was stimulated by UVA and that
the intracellular phosphorylation of STAT3, but not STAT1, was markedly
dependent on RSK2 activation by UVA. These findings thus suggest that
changes in Cy1luc luciferase activity in RSK2 cells may reflect a
specific STAT3-dependent transcriptional activation.
Experimental results revealed that basal and UVA-stimulated luciferase
activities of STAT3 were significantly induced in wild-type RSK2 cells
transfected with the Cy1luc reporter (Fig.
7A). Additionally, a modest
level of luciferase activity in wild-type RSK2 cells transfected with the
Cy1luc reporter containing only an incomplete STAT binding motif
was determined compared with the original Cy1luc reporter (Fig.
7A). On the other hand, both basal and stimulated luciferase activities were defective (Fig. 7A) in corresponding
RSK2-deficient cells transfected with Cy1luc or
Cy1luc. These
observations further indicate that RSK2-mediated Ser727
phosphorylation of STAT3 is required in the functional regulation of
STAT3 transcriptional activation.
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Fig. 7.
Requirement of RSK2 in regulating STAT3
transcripitional activation induced by serine phosphorylation.
Wild-type (A and B) and RSK2
(A) cells (1 × 106) were or were not
transfected or co-transfected with indicated Cylluc,
Cylluc
(A, 2 µg; B, 1.4 µg), and STAT3
, or
STAT3
(B, 0.7 µg) constructs. The experimental cells
were cultured for 24 h in normal complete media and then starved
for 24 h in serum-free media. The cells were or were not
irradiated with UVA (80 kJ/m2) and harvested 4 h after
irradiation and then placed for 1 h in 100 µl of lysis buffer.
The lysates were subsequently subjected to assays for STAT3-mediated
luciferase acitivity as described under "Experimental Procedures."
The results of the luciferase activity are expressed in relative
luminescence units (RLU). Each column and bar represents the
mean and standard deviation of two independent experiments performed in
triplicate. * indicates a significant difference (p < 0.05) from corresponding controls.
Cy1luc or Cy1luc reporters and STAT3
or STAT3
vectors to
further assess STAT3-dependent transcriptional activation. Results showed that overexpression of STAT3
in wild-type RSK2 cells
partially enhanced UVA-stimulated luciferase activity of Cy1luc or
Cy1luc reporters (Fig. 7B) compared with cells with only
the reporters transfected. These stimulated luciferase activities were,
however, partially suppressed by expression of STAT3
, a dominant
negative form containing the Tyr705 but not the
Ser727 site (Fig. 7B). However, basal levels of
luciferase activities were not significantly affected by STAT3
expression. Overall, these data indicate that Ser727
phosphorylation of STAT3 is required in the functional regulation of
STAT3-mediated transcriptional activation in the UVA response and that
the intracellular phosphorylation process may occur through a pathway
involving ATM, MAPKs, and/or RSK2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), which leads to Coffin-Lowry syndrome.
The RSK2
cells are confirmed to be defective
for basal RSK2 expression and UVA/UVC-induced RSK2 activity as well as
UVA-stimulated phosphorylation (Ser380) of total RSKs.
Further, we present evidence showing a significant decrease in
UVA/UVC-stimulated Ser727 phosphorylation of STAT3 in
RSK2-deficient cells compared with the wild-type control cells.
Importantly, UVA-stimulated phosphorylation of STAT3
(Ser727) was restored after the transfected RSK-deficient
cells ectopically expressed wild-type full-length RSK2. Moreover,
in vitro phosphorylation of immunoprecipitated STAT3
complexes was induced by active RSK2. On the other hand, RSK2
deficiency did not cause a decrease in the intracellular STAT1
phosphorylation (Ser727) stimulated by UVA/UVC. In
addition, basal levels of STAT3 or STAT1 expression were unaffected by
deficiency of RSK2. Taken together, these observations indicate that
activation of RSK2 may be required for phosphorylation of STAT3
(Ser727) in the intracellular response to UVA/UVC
irradiation, whereas STAT1 phosphorylation (Ser727) appears
to occur principally through a RSK2-independent pathway (e.g. RSK1 or MSK1).
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Fig. 8.
A hypothetical model for RSK-mediated
phosphorylation of STAT3 (Ser727) induced by UVA or UVC
irradiation. ATM or other kinases such as ATR (ATM- and
Rad3-related) that lie at the beginning of multiple signal transduction
pathways are activated by UVA or UVC irradiation. Subsequently, MAPKs
including JNKs and ERKs are stimulated and contribute to RSK-mediated
STAT3 signaling activation. RSKs or JNKs only in the presence of a
downstream kinase and/or a cofactor may act as upstream kinases
responsible for phosphorylation of STAT3 at Ser727, as well
as at an unidentified non-Ser727 residue. STAT3 , a
dominant negative variant, has not yet reported to be phosphorylated by
so far known kinases and but inhibits induction of phosphorylation of
STAT3. The arrows or bars indicate activation or
inhibition, respectively. The broken arrow or question
markers represent unidentified or unknown. The p,
pS727, or pS/T indicates phosphorylation,
STAT3 phosphorylation at Ser727 or at a
non-Ser727 serine-threonine site, respectively.
Ser727 is shown to be located within a potential MAPK
consensus (PMS727P) motif of the C-terminal transcriptional domain in
STAT3 (9, 11, 12), predicting that MAPKs are direct kinases for catalyzing Ser727 phosphorylation. Here, to further
determine a role for RSK2 or JNK1 in the phosphorylation catalysis, we
prepared GST-fusion wild-type or mutant STAT3 proteins as enzymatic
substrates for in vitro kinase assays. Phosphorylation of
the kinase reactive products was determined by both Western blotting
with a phosphor-Ser727 antibody and radioisotope labeling.
We present evidence showing that active JNK1 may stimulate
phosphorylation of the STAT3 proteins from immunoprecipitation, but not
of GST-fusion STAT3wt from the pull-down assay. But, GST-fusion
STAT3
wt combined with immunoprecipitated STAT proteins was
phosphorylated by JNK1. Moreover, Cao's laboratory (30) reported that
JNKs can phosphorylate Ser727 in the STAT3 protein, which
is a recombinant C-terminal peptide-deleted bigger STAT3 fragment, but
not a wild-type full-length STAT3 (personal correspondence). These
observations indicate that a cofactor in the STAT3 immunoprecipitates
may be required for JNK1-catalyzed phosphorylation of STAT3 and the
action occurs probably by modulation of the molecular conformation of
the kinase-substrate docking interaction.
In a mixture of STAT3 proteins from both immunoprecipitation and GST
pull-down, GST-STAT3wt, a dominant negative STAT3 variant (34), was
not phosphorylated by JNK1, suggesting that the C-terminal 55 amino
acid sequence is essential for both total and Ser727
phosphorylation of STAT3. Importantly, radioisotope assays showed that
JNK1 can induce total phosphorylation of a STAT3 point mutated at the
Y705 (GST-STAT3
Y705F) or Ser727 site (GST-STAT3
S727A)
mixed with IP-STAT3 precipitates, whereas Western blotting revealed
that JNK1 only stimulates Ser727 phosphorylation of
GST-STAT3
Y705F, but not GST-STAT3
S727A. Moreover, our recent
report (29) showed that Ser727 phosphorylation of IP-STAT3
is suppressed by preincubation of the phospho-STAT3
(Ser727) antibody with a Ser727
phospho-specific blocking peptide (sc-7993P), but not a
Tyr705 phospho-specific blocking peptide (sc-8001P), a C
terminus with no Ser727 or Tyr705 residues
(sc-482P), or an internal domain (sc-483P) of STAT3
. These data
indicate that the phospho-STAT3 (Ser727) antibody
specifically recognizes Ser727 phosphorylated STAT3
proteins. The discrepancies in the STAT3 phosphorylation determined in
the above-mentioned two kinase assays suggest an occurrence of
phosphorylation at a non-Ser727 residue, in addition to
Ser727, in the C-terminal domain of STAT3. In addition, the
autoradiograph observations showing a markedly stronger enhancement in
JNK1-catalyzed phosphorylation of GST-STAT3
Y705F or
GST-STAT3
S727A only when mixed with IP-STAT3 further demonstrate
that other non-Ser727 residues may be phosphorylated by
JNK1 in the presence of a cofactor.
Furthermore, RSK2 is postulated to not be a kinase directly responsible
for catalyzing Ser727 phosphorylation, based on the fact
that the PMS727P motif in STAT3 is very different from the basic
consensus sequence R/KXRXXS/T or RRXS/T motif recognized by RSKs (49),
which is present in the thus far identified RSK substrates that include
c-Fos, CREB and Bad. Here, we present experimental evidence showing
that RSK2, as well as RSK1, cannot catalyze phosphorylation of each of
the above-described four recombinant GST-fusion STAT3 proteins in the
presence or absence of IP-STAT3 immunoprecipitates. However, the
results of intracellular phosphorylation and kinase activity assays
reveal a requirement for RSK2 in UV-stimulated phosphorylation of STAT3
(Ser727). Phosphorylation of STAT3 immunoprecipitates from
cell lysates was triggered by RSK2. These findings suggest that RSK2 is
not a direct kinase for Ser727 phosphorylation of STAT3.
However, an involvement of RSK2 in mediation of intracellular STAT3
phosphorylation (Ser727) may occur indirectly
via a downstream kinase binding to a STAT3 signal complex.
Additionally, phosphorylation of STAT3 immunoprecipitates by RSK2 or
JNK1 vary with the addition of GST-fusion STAT3 proteins, likely being
associated with changes of functional configurations of substrate
complexes. For example, addition of GST-fusion STAT3wt may result in
a competitive inhibition of substrates in the phosphorylation reactions, because GST-fusion STAT3
wt as a dominant negative form
with no Ser727 site suppresses phosphorylation of intact
STAT3 protein. Overall, our results indicate that UVA-stimulated
intracellular phosphorylation of STAT3 (Ser727) occurs
through ATM-dependent or -independent MAPKs/RSK2 pathways, and that JNK1 or RSK2 only in the presence of a cofactor and/or a
downstream kinase could induce phosphorylation of STAT3 at
Ser727, as well as at an unidentified
non-Ser727 residue in vitro. Furthermore,
expression and activation of RSK2 is required for both basal and
UVA-stimulated STAT3-dependent transcriptional activities
and the stimulated activity is partially reduced by STAT3
expression, indicating that phosphorylation of STAT3
(Ser727) induced through the RSK2 pathway is involved in
functionally regulating STAT3-mediated transcriptional activation.
![]() |
ACKNOWLEDGEMENTS |
---|
We are very grateful to Dr. Yosef Shiloh for
the generous gift of Atm+ (Ft-pEBS7-YZ5) and
Atm (Ft-pEBS7) cell lines (36, 37). We gratefully
acknowledge Drs. Morten Frodin and Aviva Symes for generously providing
the full-length RSK2 in the vector pMT2 (41) and the Cy1luc reporter (12), respectively. We thank Dr Wei-Ya Ma for some experimental technical assistance. We also thank Ms. Andria Hansen for secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Hormel Foundation and National Institutes of Health Grants CA77646 and CA81064.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) XM_032859.
To whom correspondence should be addressed: The Hormel Institute,
University of Minnesota, 801 16th Ave. NE, Austin, MN 55912. Tel.:
507-437-9600; Fax: 507-437-9606; E-mail: zgdong@hi.umn.edu.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M210368200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: STAT3 or STAT1, signal transducer and activator transcription 3 or 1, respectively; RSKs, p90 ribosomal protein S6 kinases; MAPKs, mitogen-activated protein kinases; ERKs, extracellular signal-regulated kinases; JNKs, c-Jun N-terminal kinases; UVA or UVC, ultraviolet A or C; FBS, fetal bovine serum; GST, glutathione S-transferase.
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REFERENCES |
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1. | Ihle, J. N. (1996) Cell 84, 331-334[Medline] [Order article via Infotrieve] |
2. | Horvath, C. M. (2000) Trends Biochem. Sci. 25, 496-501[CrossRef][Medline] [Order article via Infotrieve] |
3. | Ihle, J. N. (2001) Curr. Opin. Cell Biol. 13, 211-217[CrossRef][Medline] [Order article via Infotrieve] |
4. | Williams, J. G. (2000) Curr. Opin. Genet. Dev. 10, 503-507[CrossRef][Medline] [Order article via Infotrieve] |
5. | Bromberg, J., and Darnell, J. E., Jr. (2000) Oncogene 19, 2468-2473[CrossRef][Medline] [Order article via Infotrieve] |
6. | Bromberg, J. (2001) Bioessays 23, 161-169[CrossRef][Medline] [Order article via Infotrieve] |
7. | Frank, D. A. (1999) Mol. Med. 5, 432-456[Medline] [Order article via Infotrieve] |
8. | Bowman, T., Garcia, R., Turkson, J., and Jove, R. (2000) Oncogene 19, 2474-2488[CrossRef][Medline] [Order article via Infotrieve] |
9. | Wen, Z., Zhong, Z., and Darnell, J. E., Jr. (1995) Cell 82, 241-250[Medline] [Order article via Infotrieve] |
10. | Zhang, X., Blenis, J., Li, H. C., Schindler, C., and Chen-Kiang, S. (1995) Science 267, 1990-1994[Medline] [Order article via Infotrieve] |
11. |
Wen, Z.,
and Darnell, J. E., Jr.
(1997)
Nucleic Acids Res.
25,
2062-2067 |
12. | Yokogami, K., Wakisaka, S., Avruch, J., and Reeves, S. A. (2000) Curr. Biol. 10, 47-50[CrossRef][Medline] [Order article via Infotrieve] |
13. | Decker, T., and Kovarik, P. (2000) Oncogene 19, 2628-2637[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Lim, C. P.,
and Cao, X.
(1999)
J. Biol. Chem.
274,
31055-31061 |
15. |
Gatsios, P.,
Terstegen, L.,
Schliess, F.,
Haussinger, D.,
Kerr, I. M.,
Heinrich, P. C.,
and Graeve, L.
(1998)
J. Biol. Chem.
273,
22962-22968 |
16. |
Bode, J. G.,
Gatsios, P.,
Ludwig, S.,
Rapp, U. R.,
Haussinger, D.,
Heinrich, P. C.,
and Graeve, L.
(1999)
J. Biol. Chem.
274,
30222-30227 |
17. | Zhu, X., Wen, Z., Xu, L. Z., and Darnell, J. E., Jr. (1997) Mol. Cell. Biol. 17, 6618-6623[Abstract] |
18. |
Ng, J.,
and Cantrell, D.
(1997)
J. Biol. Chem.
272,
24542-24549 |
19. |
Ceresa, B. P.,
and Pessin, J. E.
(1996)
J. Biol. Chem.
271,
12121-12124 |
20. | Fukuzawa, M., Araki, T., Adrian, I., and Williams, J. G. (2001) Mol. Cell 7, 779-788[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Lillemeier, B. F.,
Koster, M.,
and Kerr, I. M.
(2001)
EMBO J.
20,
2508-2517 |
22. | David, M., Petricoin, E., III, Benjamin, C., Pine, R., Weber, M. J., and Larner, A. C. (1995) Science 269, 1721-1723[Medline] [Order article via Infotrieve] |
23. |
Ceresa, B. P.,
Horvath, C. M.,
and Pessin, J. E.
(1997)
Endocrinology
138,
4131-4137 |
24. | Chung, J., Uchida, E., Grammer, T. C., and Blenis, J. (1997) Mol. Cell. Biol. 17, 6508-6516[Abstract] |
25. |
Turkson, J.,
Bowman, T.,
Adnane, J.,
Zhang, Y.,
Djeu, J. Y.,
Sekharam, M.,
Frank, D. A.,
Holzman, L. B.,
Wu, J.,
Sebti, S.,
and Jove, R.
(1999)
Mol. Cell. Biol.
19,
7519-7528 |
26. | Kuroki, M., and O'Flaherty, J. T. (1999) Biochem. J. 341, 691-696[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Uddin, S.,
Lekmine, F.,
Sharma, N.,
Majchrzak, B.,
Mayer, I.,
Young, P. R.,
Bokoch, G. M.,
Fish, E. N.,
and Platanias, L. C.
(2000)
J. Biol. Chem.
275,
27634-27634 |
28. | Schuringa, J. J., Jonk, L. J., Dokter, W. H., Vellenga, E., and Kruijer, W. (2000) Biochem. J. 347, 89-96[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Zhang, Y.,
Liu, G.,
and Dong, Z.
(2001)
J. Biol. Chem.
276,
42534-42542 |
30. |
Jain, N.,
Zhang, T.,
Kee, W. H.,
Li, W.,
and Cao, X.
(1999)
J. Biol. Chem.
274,
24392-24400 |
31. |
Lim, C. P.,
and Cao, X.
(2001)
J. Biol. Chem.
276,
21004-21011 |
32. |
Schuringa, J. J.,
Dekker, L. V.,
Vellenga, E.,
and Kruijer, W.
(2001)
J. Biol. Chem.
276,
27709-27715 |
33. |
Boeuf, H.,
Merienne, K.,
Jacquot, S.,
Duval, D.,
Zeniou, M.,
Hauss, C.,
Reinhardt, B.,
Huss-Garcia, Y.,
Dierich, A.,
Frank, D. A.,
Hanauer, A.,
and Kedinger, C.
(2001)
J. Biol. Chem.
276,
46204-46211 |
34. | Yoo, J.-Y., Huso, D. L., Nathans, D., and Desiderio, S. (2002) Cell 108, 331-344[Medline] [Order article via Infotrieve] |
35. |
Sassone-Corsi, P.,
Mizzen, C. A.,
Cheung, P.,
Crosio, C.,
Monaco, L.,
Jacquot, S.,
Hanauer, A.,
and Allis, C. D.
(1999)
Science
285,
886-891 |
36. | Ziv, Y., Bar-Shira, A., Pecker, I., Russell, P., Jorgensen, T. J., Tsarfati, I., and Shiloh, Y. (1997) Oncogene 15, 159-167[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Banin, S.,
Moyal, L.,
Shieh, S.,
Taya, Y.,
Anderson, C. W.,
Chessa, L.,
Smorodinsky, N. I.,
Prives, C.,
Reiss, Y.,
Shiloh, Y.,
and Ziv, Y.
(1998)
Science
281,
1674-1677 |
38. |
Zhang, Y.,
Dong, Z.M.,
Nomura, M.,
Zhong, S.,
Chen, N.,
Bode, A. M.,
and Dong, Z.
(2001)
J. Biol. Chem.
276,
20913-20923 |
39. |
Zhang, Y.,
Zhong, S.,
Dong, Z.M.,
Chen, N.,
Bode, A. M.,
Ma, W.,
and Dong, Z.
(2001)
J. Biol. Chem.
276,
14572-14580 |
40. |
Zhang, Y.,
Mattjus, P.,
Schmid, P. C.,
Dong, Z.M.,
Zhong, S.,
Ma, W.Y.,
Brown, R. E.,
Bode, A. M.,
Schmid, H. H.,
and Dong, Z.
(2001)
J. Biol. Chem.
276,
11775-11782 |
41. |
Frodin, M.,
Jensen, C. J.,
Merienne, K.,
and Gammeltoft, S.
(2000)
EMBO J.
19,
2924-2934 |
42. |
Zhang, Y.,
Ma, W.-Y.,
Kaji, A.,
Bode, A. M.,
and Dong, Z.
(2002)
J. Biol. Chem.
277,
3124-3131 |
43. |
Abraham, R. T.
(2001)
Genes Dev.
15,
2177-2196 |
44. | Shiloh, Y. (2001) Curr. Opin. Genet. Dev. 11, 71-77[CrossRef][Medline] [Order article via Infotrieve] |
45. | Kastan, M. B., and Lim, D. S. (2000) Nat. Rev. Mol. Cell. Biol. 1, 179-186[CrossRef][Medline] [Order article via Infotrieve] |
46. | Kovarik, P., Stoiber, D., Eyers, P. A., Menghini, R., Neininger, A., Gaestel, M., Cohen, P., and Decker, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1956-13961 |
47. |
Kovarik, P.,
Mangold, M.,
Ramsauer, K.,
Heidari, H.,
Steinborn, R.,
Zotter, A.,
Levy, D. E.,
Muller, M.,
and Decker, T.
(2001)
EMBO J.
20,
91-100 |
48. |
Dufresne, S. D.,
Bjorbaek, C.,
El-Haschimi, K.,
Zhao, Y.,
Aschenbach, W. G.,
Moller, D. E.,
and Goodyear, L. J.
(2001)
Mol. Cell. Biol.
21,
81-87 |
49. | Frodin, M., and Gammeltoft, S. (1999) Mol. Cell. Endocrinol. 15, 65-77 |
50. |
Akira, S.
(1999)
Stem Cells
17,
138-146 |
51. | Durbin, J. E., Hackenmiller, R., Simon, M. C., and Levy, D. E. (1996) Cell 84, 443-450[Medline] [Order article via Infotrieve] |
52. |
Pages, G.,
Guerin, S.,
Grall, D.,
Bonino, F.,
Smith, A.,
Anjuere, F.,
Auberger, P.,
and Pouyssegur, J.
(1999)
Science
286,
1374-1377 |
53. | Bromberg, J. F., Wrzeszczynska, M. H., Devgan, G., Zhao, Y., Pestell, R. G., Albanese, C., and Darnell, J. E., Jr. (1999) Cell 98, 295-203[Medline] [Order article via Infotrieve] |
54. | Blume-Jensen, P., and Hunter, T. (2001) Nature 411, 355-365[CrossRef][Medline] [Order article via Infotrieve] |