Evidence of STAT1 phosphorylation modulated by MAPKs, MEK1 and MSK1
Yiguo Zhang,
Yong-Yeon Cho,
Brandon L. Petersen,
Feng Zhu and
Zigang Dong1
Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA
1 To whom correspondence should be addressed Email: zgdong{at}hi.umn.edu
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Abstract
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Phosphorylation at Ser727 in signal transducer and activator of transcription 1 (STAT1) is essential for its activation and signal transduction. However, the upstream kinases responsible for phosphorylating Ser727 are still elusive. Here, we provide evidence showing that UVA-induced mitogen-activated protein kinase (MAPK) signaling pathways lead to STAT1 Ser727 phosphorylation. Our experimental results show that UVA-induced Ser727 phosphorylation of STAT1 was, to different degrees, diminished by PD98059 and U0126, two specific inhibitors of MEKs, and SB202190 and PD169316, inhibitors of p38 kinase and c-Jun N-terminal kinases (JNKs), respectively. STAT1 phosphorylation was also blocked by a dominant negative mutant of p38ß kinase or JNK1, JNK1- or JNK2-deficiency, or an N-terminal or C-terminal kinase-dead mutant of mitogen- and stress-activated protein kinase 1 (MSK1), a downstream kinase closer to p38 kinase and extracellular signal-regulated kinases (ERKs). In vitro kinase assays using the combined STAT1 proteins as substrates from immunoprecipitation and glutathione S-transferase pull down show that active ERK1, JNK1, p38 kinase, MEK1 and MSK1 stimulated phosphorylation of STAT1 (Ser727) indirectly through an unidentified factor or a downstream kinase. Overall, our data indicate that phosphorylation of STAT1 at Ser727 occurs through diverse MAPK cascades including MEK1, ERKs, p38 kinase, JNKs and MSK1 in the cellular response to UVA.
Abbreviations: CMVS, pCMV5-FLAG vector; DMEM, Dulbecco's modified Eagle's medium; DNM-JNK1, dominant negative mutant of JNK1; DNM-p38ß, dominant negative mutant of p38ß kinase; EGF, epidermal growth factor; ERKs, extracellular signal-regulated kinases; FBS, fetal bovine serum; GST, glutathione S-transferase; JAK, Janus kinase; JNKs, c-Jun N-terminal kinases; MAPK, mitogen-activated protein kinases; MSK1, mitogen- and stress-activated protein kinase 1; STAT1, signal transducer and activator of transcription 1
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Introduction
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Signal transducer and activator of transcription 1 (STAT1) is highly homologous to STAT3. It was identified as a transcription factor that mediates interferon action, but is now known to be involved in many other signaling pathways that play a role in regulating diverse cellular processes including growth, proliferation, differentiation and transformation, apoptosis, and is even involved in oncogenesis (15). When cells are stimulated with cytokines (e.g. interferon) or growth factors (e.g. epidermal growth factor, EGF), STAT1 activation is generally accepted to be initiated by tyrosine phosphorylation at a single site (Tyr701), which is carboxyl to the SH2 domain (1,3). Tyr701 phosphorylation by the Janus kinase (JAK) family members (e.g. JAK1, JAK2 and PYK2) (57) or receptor tyrosine kinases (e.g. EGF receptor, EGFR) (8,9) appeared to be all that was required for dimer formation, nuclear translocation and activation of STAT1. However, recent studies demonstrated substantial tyrosine phosphorylation-independent nuclear translocation of STAT1 in some signaling responses (1012). On the other hand, several interesting observations indicated that STAT1 activation might also require a secondary phosphorylation modification at the serine/threonine residues, possibly by extracellular signal-regulated kinases (ERKs) (13,14) or a H7-sensitive kinase (15,16). Furthermore, phosphorylation of STAT1 at serine 727, in addition to Tyr701 phosphorylation, was shown to be required for its maximal activation induction by cytokines (17,18). Importantly, stimulation of cells by diverse stresses (e.g. short wave ultraviolet irradiation) induced Ser727 phosphorylation of STAT1 resulting in its activation independently of Tyr701 phosphorylation (1924). These findings therefore indicate that Ser727 phosphorylation of STAT1 allows the integration of signals from multiple pathways, resulting in activation of STAT1-mediated target genes.
Ser727 is located within a potential mitogen-activated protein kinase (MAPK) consensus motif of the C-terminal transcriptional domain in STAT1 (17,18,24,25) and thereby, is postulated to be phosphorylated through activation of MAPKs, including ERKs, c-Jun N-terminal kinases (JNKs) and p38 kinase (26). However, the kinases responsible for catalyzing Ser727 phosphorylation of STAT1 are still elusive, although involvement of ERKs and p38 kinase in STAT1 (Ser727) phosphorylation induction was proposed previously based on preliminary experimental evidence (13,14,2224, 27,28). Furthermore, contribution of only STAT1 Ser727 phosphorylation to its constitutive activation was detected in some tumor cells (4,5,15). Ser727 phosphorylation and activation of STAT1 was also activated by carcinogens and tumor promoters, including UVC (200290 nm) and 12-O-tetradecanoylphorbol-13-acetate (15,23,24,28). Therefore, identifying the upstream kinases for serine phosphorylation of STAT1 will provide a clearer understanding of the mechanism of STAT1 signaling activation involved in oncogenesis, possibly leading to the development of novel preventive and therapeutic approaches to intervene in the process. Solar UV irradiation is believed to be one of the most important skin carcinogens. UVA (320400 nm) constitutes >90% of solar UV, of which all of the UVC and most of the UVB (290320 nm) are absorbed by the ozone layer of the earth's atmosphere. Thus, UVA is a major contributor to carcinogenesis. Different spectra of UV (UVA, UVB and UVC) induce different signal transduction pathways (29). However, the UVA-induced signaling pathways leading to Ser727 phosphorylation of STAT1 are unknown. Here, we show that a strong phosphorylation of STAT1 at Ser727 is induced in the intracellular response to UVA irradiation and this phosphorylation process occurs through diverse MAPK signaling pathways. Furthermore, we demonstrate that STAT1 is a potential substrate for Ser727 phosphorylation by ERK1, JNK1, p38 kinase or possibly by MEK1 or mitogen- and stress-activated protein kinase 1 (MSK1) in the presence of an unidentified cofactor and/or downstream kinase. In addition, the process is also negatively regulated by a pathway involving EGFR and/or ERKs.
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Materials and methods
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Amplification of the wild-type STAT1 cDNA
The cDNA fragment of STAT1, including open reading frame (2253 bp), was reverse-transcribed with an oligo-dT primer and SuperScript II RNase H Reverse Transcriptase (GibcoBRL, Grand Island, NY), and then amplified by the polymerase chain reaction with primers 5'-GCA GGA TCC TGT CTC AGT GGT ACG AAC T-3' (BamHI site underlined) and 5'-TCG ACG CGT TGC TCT ATA CTG TGT TCA TC-3' (MluI site underlined) and cloned into the pACT vector (Promega, Madison, WI) via BamHI and MluI restriction sites. The sequence of STAT1 was verified by comparison of restriction fragment length and DNA sequence analysis. The primer synthesis and DNA sequencing were performed by Sigma, St Louis, MO.
Construction and mutagenesis of glutathione S-transferase (GST)-fusion expression vectors
The STAT1 coding fragment digested with BamHI/EcoRV from the pACT was cloned into BamHI/SmaI sites of the pUC19 vector. To introduce the point mutation in the position of the 701(Y701F) or the 727(S727A) residue, a pair of sense and antisense primers for STAT1Y701F: 5'-CTA AAG GAA CTG GAT TTA TCA AGA CTG AGT TGA T-3' and 5'-A TCA ACT CAG TCT TGA TAA ATC CAG TTC CTT TAG-3', and another pair of the primers for STAT1S727A: 5'-C CTG CTC CCC ATG GCT CCT GAG GAG-3' and 5'-CTC CTC AGG AGC CAT GGG GAG CAG G-3' were synthesized (Sigma). Then, the nucleotide substitutions were accomplished using the QuickChange Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and then confirmed by DNA sequence analysis (Sigma). Further, the STAT1 Y701F and STAT1 S727A were ligated into the BamHI/MluI sites of the pGEX-5X-C modified multicloning sites from the pGEX-5X-1 vector. In addition, wild-type STAT1 was directly introduced into the BamHI/MluI sites of the pGEX-5X-C vector. The above-mentioned restriction endonucleases, ligases and related buffers were purchased from New English BioLabs (Beverly, MA).
GST-fusion STAT1 protein expression and pull down
The pGEX-5X-C plasmids encoding the wild-type full-length STAT1 (STAT1wt) and the point mutant STAT1 (STAT1Y701A or STAT1S727A) as GST-fusion protein sources were used to transform a DH5
competent Escherichia coli strain (Invitrogen Life Technologies, Carlsbad, CA). After induction of protein expression with 0.1 mM isopropyl-1-thio-ß-D-galactopyranoside (from Sigma) for 4 h, the bacteria were resuspended in a lysis buffer containing 50 mM TrisHCl pH 8.0, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% (v/v) Triton X-100, 10 µg/ml of aprotinin and leupeptin and 100 µM Na3VO4, and then were further disrupted by addition of 0.1 vol of 10 mg/ml lysozyme (Sigma) and subsequent sonication. Following centrifugation at 10 000 g for 20 min, the supernatant fractions containing the induced proteins were incubated with 50% slurry of pre-treated glutathioneSepharose 4B (Amersham, Piscataway, NJ, http://www1.amershambiosciences.com). After washing twice with the above-mentioned lysis buffer and an additional two times with kinase buffer (described below), the beads were subjected to SDSPAGE followed by western blotting with a GST or STAT1 antibody to determine expression of the GST-fusion STAT1 proteins. In subsequent in vitro kinase reactions, eluants of the beads with 20 mM reduced glutathione (GSH from Roche Molecular Biochemicals, Indianapolis, IN) were used as enzymatic substrates. Control experiments were performed with GSTSepharose beads generated by expression of GST alone, using the empty pGEX-5X-C vector.
Cell lines and cell culture
Mouse epidermal tumor promotion sensitive JB6 Cl 41 cells and related stable transfectants were cultured in Eagle's minimum essential medium (EMEM from BioWhittaker, Walkersville, MD) supplemented with 5% heat-deactivated fetal bovine serum (FBS from Gemini Bio-Products, Calabasas, CA), 2 mM L-glutamine, 100 U/ml of penicillin and 100 µg/ml of streptomycin at 37°C in humidified air with 5% CO2. Mouse wild-type (Egfr+/+ or Jnk+/+) and knockout (Egfr/, Jnk1/ or Jnk2/) embryonic fibroblast lines were generated and identified as described previously (30,31). The cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) (GibcoBRL Technologies) containing 10% FBS, 2 mM L-glutamine, 100 U/ml of penicillin and 100 µg/ml of streptomycin.
Stable transfectants
JB6 Cl 41 cell lines stably transfected with an empty CMV-neo vector (CMV-neo), or a construct containing a dominant negative mutant of ERK2 (DNM-ERK2), JNK1 (DNM-JNK1) or p38ß kinase (DNM-p38ß) was established and identified as reported previously (3236). Other transfected JB6 Cl 41 cell lines stably expressing pCMV5-FLAG vector (CMVS), pCMV5-FLAG-wild-type MSK1 (MSK1wt), pCMV5-FLAG-MSK1-A195/N-terminal kinase-dead (MSK1-Nd) or pCMV5-FLAG-MSK1-A565/C-terminal kinase-dead (MSK1-Cd) (from Dr D.R.Alessi) were generated and characterized according to previously described methods (29,37). The transfectants were selected in media containing 400 µg/ml of G418 (Gemini Bio-Products). Prior to the experiments preformed, we again confirmed that the above-mentioned cells expressed the desired activity by performing related assays for kinase activity or specific phosphorylation.
Treatment of cells
To diminish a basal level of protein phosphorylation or activity, JB6 Cl 41 cells and related transfectants were starved for 2448 h in 0.1% FBS EMEM, whereas other above-mentioned cells were starved in serum-free DMEM. Then, the cells were or were not incubated for 11.5 h with specific protein kinase inhibitors, including PD98059, SB202190 (Sigma), PD169316 (Alexis, San Diego, CA) or AG1478 (Calbiochem, San Diego, CA) and subsequently irradiated with UVA, UVB or UVC. A detailed description of UVA, UVB or UVC sources is available in our previous reports (32,33). Non-irradiated cell samples were used as negative controls. In an additional experiment, the cells were treated with EGF (Collaborative Research, Madison, WI).
Western blot analysis
Equal numbers of experimental cells (1 x 106 to 1.5 x 106) were cultured for 1224 h in 100-mm dishes. After 7080% confluence was reached, the cells were starved for 48 h in serum-free DMEM. At the indicated times after irradiation, the cells were harvested and washed once with ice-cold phosphate-buffered saline (PBS). Then the cell samples were disrupted in 200 µl of RIPA buffer [1x PBS, 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 of PMSF, 10 µg/ml of aprotinin and 100 µM Na3VO4]. The cell lysates were clarified by microcentrifuge and the supernatant fractions were saved. The samples containing equal amounts of proteins (Modified Lowry's method, Sigma) in an equal volume of RIPA buffer were diluted with 3x SDS sample buffer [187.5 mM TrisHCl pH 6.8, 6% (w/v) SDS, 30% (v/v) glycerol, 150 mM dithiothreitol (DTT) and 0.3% (w/v) bromophenol blue]. Then samples were subjected to separation by 8% SDSPAGE followed by western blot analysis according to the reported methods (32,33). A specific phospho-STAT1 (Ser727) antibody was purchased from Upstate Biotechnology (Lake Placid, NY), and the phospho-specific STAT1 (Tyr701) antibody was from Cell Signaling (Beverly, MA). Other antibodies against phospho-ERKs, p38 kinase, JNKs or p90RSK (Ser381), and against total STAT1, ERKs or ß-actin were from Cell Signaling. Total STAT1 or ß-actin was used as an internal control to verify basal level expression and equal protein loading. Net serine phosphorylation was calculated by dividing with total protein and then normalized to non-irradiated control cells and is presented as a fold change (1 of control value). In addition, the intensity of some western blots was quantified as reported previously (29).
Immunoprecipitation (IP) assay
After culturing for 1224 h, the experimental cells were starved for 24 h in 1% FBS-DMEM. The cells were harvested at the indicated times following irradiation and lysed in 250 µl of IP buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerol phosphate, 1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 1 mM PMSF]. The clarified supernatant fractions containing equal amounts of protein were subjected to IP followed by western blot analysis or kinase activity assays according to the described methods (29,32,33). The immune complex beads were washed with PBS. An antibody against total STAT1 was used for IP of STAT1 proteins.
Assay for in vitro phosphorylation by protein kinases
After starvation, JB6 Cl 41 cell lysates were prepared as described above and clarified by microcentrifuge. Equal amounts of protein in the supernatant fractions were subjected to IP for STAT1. Samples containing the immune-purified STAT1 proteins, GST-pull down STAT1 proteins, or both combined together were incubated at 30°C for 60 min with active MSK1, MEK1, ERK1, ERK2, JNK1, JNK2, p38
or p38ß kinase (Upstate Biotechnology) in kinase buffer (50 mM TrisHCl pH 7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT and 0.01 % Brij 35) (Cell Signaling) containing 5 mM ATP or 1 µCi of [
-32P]ATP. The reactions were stopped by adding 5x SDS sample buffer. Then phosphorylation of STAT1 was analyzed by western blotting with a specific antibody against phospho-STAT1 (Ser727) or autoradiography (38). Total STAT1 was utilized as an internal control to verify equal protein loading.
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Results
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A strong phosphorylation of STAT1 at Ser727 is induced by UVA
A recent report indicated that irradiation of human keratinocyte lines with UVA stimulated STAT1 activation through Tyr701 phosphorylation (39). On the other hand, previous experiments with UVC irradiation showed that Ser727 phosphorylation was required in the activation of STAT1 signaling, but the activation was independent of Tyr701 phosphorylation (20,23,24,28). However, whether UVA induces STAT1 Ser727 phosphorylation leading to its mediated signaling activation is not known. This question was investigated herein by using western blot analysis with specific antibodies to detect STAT1 phosphorylation at Ser727 or Tyr701. Our data show that compared with Tyr701 phosphorylation, a stronger phosphorylation of STAT1 at Ser727 was induced in a dose-dependent (Figure 1A) and time-dependent (Figure 1B) manner, following UVA exposure of mouse epidermal tumor promotion sensitive JB6 Cl 41 cells. The Ser727 phosphorylation occurred 5 min after irradiation with UVA (160 kJ/m2), increased to a maximal induction at 30 min, and then gradually decreased back to basal level by 240 min following irradiation (Figure 1A and B). In contrast, hardly any induction of phosphorylation of Tyr701 was detected 15 or 30 min after stimulation with UVA (Figure 1A and B). In the same experiments, a similar induction pattern of STAT1 phosphorylation at Ser727 by UVB or UVC irradiation was observed, but Tyr701 phosphorylation was undetectable (Figure 1A), consistent with earlier reported observations (20,23,24,28). In addition, treatment of cells with EGF was shown previously to stimulate both Tyr701 and Ser727 phosphorylation of STAT1 (1,4,26), but here we determined that stimulation of JB6 Cl 41 cells with EGF induced only Ser727 phosphorylation of STAT1 (Figure 1C). This is consistent with the previous observation that only serine phosphorylation stimulated with EGF (27) or a synergy of interleukin-2 and -12 (22) regulates induction of STAT1 signaling activation. Overall, these findings demonstrate that serine phosphorylation plays a role in stimulus-dependent activation of STAT1 signaling and thus indirectly reflects regulation of STAT1 signaling.

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Fig. 1. A strong dose-dependent and time-dependent phosphorylation of STAT1 (Ser727) induced in UVA-irradiated cells. After starvation for 36 h, JB6 Cl 41 cells were or were not irradiated with UVA, UVB or UVC at the indicated doses (A), or with UVA at 160 kJ/m2 (B), or were stimulated with EGF (100 ng/ml) (C). The cells were then harvested at 30 min (A) or the indicated times (B and C) after stimulation. In the cell lysates, phosphorylated STAT1 at Ser727 or Tyr701, as well as total STAT1, were separated by 8% SDSPAGE followed by western blot analysis with a specific antibody against phosphorylation of STAT1 at Ser727 or Try701, or against total STAT1. UVB/UVC- or EGF-stimulated samples were used as positive controls, whereas the non-irradiated samples served as negative controls. These data are representative of at least three independent experiments.
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UVA-induced STAT1 serine phosphorylation is independent of EGFR
Tyrosine phosphorylation of STAT1 was shown to require EGFR tyrosine kinase activity in response to EGF or other mitogens (8,9,40), but whether EGFR plays a role in modulating serine phosphorylation of STAT1 in the cellular responses to UVA, UVB, UVC, EGF or other mitogens (1924) is as yet unknown. Initiation of several potential EGFR signaling pathways, including Ras/MAPKs and phosphatidylinositol-3 kinase (PI-3 kinase), occurs after irradiation with UVA, UVB or UVC (41). Recently, we also observed that UVA activation of MAPK cascades occurred by both EGFR-dependent and -independent signaling mechanisms (42). Thus, whether EGFR signaling acts as a functional participant in the regulation of STAT1 Ser727 phosphorylation induced by UVA was further explored in the present study. We performed experiments using an identified EGFR-deficient (Egfr/) cell line (31) and a specific EGFR tyrosine kinase inhibitor, AG1478 (31). The results of the experiments showed that UVA-stimulated phosphorylation of STAT1 Ser727 was greater in Egfrsol; cells (Figure 2A) compared with wild-type (Egfr+/+) cells. Moreover, pre-treatment of JB6 cells with AG1478 also enhanced the UVA stimulation of STAT1 phosphorylation (Figure 2B). Total levels of STAT1 were unaffected in these experimental cells (Figure 2A and B). The data indicate that UVA-stimulated phosphorylation of STAT1 (Ser727) may be triggered through both EGFR-dependent and -independent signaling pathways. Taken together with previous data (42), our study suggests that the EGFR-independent JNKs or p38 kinase signaling pathways may play a positive regulatory role in UVA induction of STAT1 serine phosphorylation. However, another possibility cannot be ruled out that this STAT1 phosphorylation response is also regulated by an identified negative EGFR signaling pathway. Such negative regulation may make a significant contribution to deactivation versus phosphorylation effects.

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Fig. 2. UVA-stimulated STAT1 (Ser727) phosphorylation enhanced by inhibition or deficiency of EGFR. (A) The lysates were obtained at the indicated times following irradiation of Egfr+/+ and Egfr/ cells with UVA (80 kJ/m2) and then were assayed for phosphorylated STAT1 (Ser727) and total STAT1 levels. (B) After pre-treatment of JB6 cells with AG1478 at the indicated doses, the lysates were harvested 30 min following UVA irradiation (80 kJ/m2), and then were subjected to western blot analysis to determine phosphorylation of STAT1 (Ser727) and total levels of STAT1. These data are representative of at least three independent experiments.
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MEK1ERK cascades have a role in the induction of STAT1 Ser727 phosphorylation
ERKs are hypothesized to catalyze phosphorylation of Ser727 in STAT1 (17,18). Indeed, serine phosphorylation of STAT1 has been associated with activation of ERK2 (13,14), but no direct evidence was provided for a direct kinase role of ERKs. Here, we further examine a role for the ERKs pathway in the intracellular phosphorylation response. Experimental results showed that pre-treatment of JB6 cells with PD98059 or U0126, two specific inhibitors of MEK1 (MAPK kinase 1) and/or MEK2, significantly blocked UVA-induced phosphorylation of STAT1 (Ser727) (Figure 3A and B). The basal levels of STAT1 were unaffected by these treatments (Figure 3C and D). Recently, Ser727 phosphorylation of STAT3, which is highly homologous to STAT1, was determined to require stress-activated protein kinase/ERK kinase 1 (SEK1), MAPK kinase 4 (MKK4) (43,44) and MEK kinase 1 (MEKK1) (45). These findings, therefore, suggest that the MKKMEKERK cascade may be involved in modulating the STAT1 serine phosphorylation response to UVA irradiation.

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Fig. 3. Blockage of UVA-stimulated phosphorylation of STAT1 (Ser727) by MEK1 inhibition. After pre-treatment with PD98059 (A) or U0126 (B) at the indicated doses, JB6 cells were or were not irradiated with UVA (160 kJ/m2) and then harvested 30 min following irradiation. Phosphorylation of STAT1 (Ser727) and total STAT1 levels in the cell lysates was determined with specific antibodies. These data are representative of at least three independent experiments.
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JNKs are required for STAT1 serine phosphorylation
Both in vivo and in vitro serine phosphorylation of STAT3 were shown to require JNK, a stress-responsive MAPK (46,47). These findings were challenged by the study of Schuringa et al. (43). However, to date, whether JNKs phosphorylate STAT1 is unknown. Here, to further explore whether JNKs have a role in the intracellular STAT1 phosphorylation response to UVA, we performed experiments using JB6 Cl 41 cell lines stably expressing DNM-JNK1 and embryonic fibroblast lines from Jnk1 or Jnk2 knockout mice. These cell lines were engineered and identified as reported previously (30,32,33). The results showed that UVA-stimulated phosphorylation of STAT1 at Ser727 was significantly prevented by expression of DNM-JNK1 (Figure 4A and B), and was also markedly abolished by deficiency of JNK1 or JNK2 (Figure 4C) compared with their corresponding control cells. On the other hand, expression of total STAT1 was unaffected by DNM-JNK1 (Figure 4A and B) or knockout of Jnk1 or Jnk2 (Figure 4C). In addition, UVA-stimulated STAT1 phosphorylation at Ser727 was also markedly reduced by pre-treatment of cells with PD169316 (Figure 4D), an inhibitor of JNKs and p38 kinase (33). Overall, the data indicate that JNKs may be required for mediating UVA-induced serine phosphorylation of STAT1.

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Fig. 4. Inhibition of UVA-induced phosphorylation of STAT1 in DNM-JNK1 or JNKs-deficient cells. (A and B) JB6 cell lines stably expressing an empty vector (CMV-neo), or DNM-JNK1 were harvested at 30 min (A) or the indicated times (B) after irradiation with UVA at the indicated doses (A), or at 160 kJ/m2 (B). (C) After starvation in serum-free DMEM, the cells from wild-type (Jnk+/+) or knockout (Jnk1/ or Jnk2/) mice were or were not irradiated with UVA (80 kJ/m2) and then were harvested 15 or 30 min following irradiation. (D) JB6 cells were or were not pre-incubated for 1.5 h with PD169316 at the indicated doses and then were or were not irradiated with UVA (160 kJ/m2) and then harvested 30 min following irradiation. Subsequently, the cell lysates were subjected to assays for phosphorylation of STAT1 (Ser727) and total STAT1. These data are representative of at least three independent experiments.
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p38 kinase is also required for mediating STAT1 serine phosphorylation
Theoretically, p38 kinase, another stress-responsive MAPK, may phosphorylate STAT1 at Ser727, which lies within a proline-flanked consensus sequence on the C-terminal domain of STAT1 (17,18). In fact, a weaker phosphorylation of a STAT1 C-terminal peptide (aa 711750) was induced in vitro by activated p38 kinase (23). However, whether p38 kinase is involved in the Ser727 phosphorylation in vivo is still controversial (26). For example, some studies indicated that p38 kinase is involved in the serine phosphorylation response leading to intracellular STAT1 signaling activation (2224,28), but others showed that p38 kinase was not required for serine phosphorylation (48,49). Here, to assess the role of p38 kinase in mediating serine phosphorylation induction by UVA, we prepared and identified JB6 Cl 41 cell lines stably expressing DNM-p38ß as reported previously (32,33). The results showed that UVA-stimulated phosphorylation of STAT1 (Ser727) was inhibited by expression of DMN-p38ß (Figure 5A and B) compared with the corresponding control JB6 cells that only expressed the empty CMV-neo vector (CMV-neo). Furthermore, a similar inhibitory effect was also observed after pre-incubation of JB6 cells with SB202190, a specific p38 kinase inhibitor (Figure 5C), or PD169316 (Figure 4D). These observations, therefore, demonstrate that in addition to JNKs, p38 kinase is required for STAT1 serine phosphorylation in the JB6 cellular response to UVA irradiation.

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Fig. 5. Suppression of UVA-stimulated STAT1 (Ser727) phosphorylation by p38 kinase/MSK1 inhibition. (A and B) JB6 cells were stably transfected with an empty vector (CMV-neo) or DNM-p38. The two cell lines were harvested at 30 min (A) or the indicated times (B) following irradiation with UVA at the indicated doses (A) or at 160 kJ/m2 (B). (C) JB6 cells were or were not pre-incubated for 1.5 h with SB202190 (C) at the indicated doses and then were or were not irradiated with UVA (160 kJ/m2) and then harvested 30 min following irradiation. (D) JB6 cells were stably transfected with an empty vector (CMVS), MSK1wt or MSK1-Nd or -Cd. Then the cells were or were not irradiated with UVA (160 kJ/m2) and were harvested at 30 min following irradiation. Subsequently, the cell lysates were subjected to assays for phosphorylation of STAT1 (Ser727) and total STAT1 levels. These data are representative of at least three independent experiments.
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MSK1 is involved in UVA-stimulated phosphorylation of STAT1
To further explore the role of ERK/p38 kinase-dependent serine/threonine kinases in serine 727 phosphorylation of STAT1, we established and identified JB6 Cl 41 cell lines stably expressing an N-terminal or C-terminal kinase-dead mutant (Nd or Cd) of MSK1, as well as wild-type MSK1 (MSK1wt) (37). These cells were exposed to UVA and the results showed that the UVA-induced serine phosphorylation of STAT1 (Ser727) was enhanced by expression MSK1wt (Figure 5D) compared with the induction in control cell lines expressing an empty vector pCMV5-neo (CMVS). However, expression of MSK1-Cd or MSK1-Nd markedly prevented the UVA-stimulated serine phosphorylation of STAT1 (Figure 5D), in contrast to the response induced in MSK1wt or CMVS cells. No change in total STAT1 expression was observed in the same experiments. Together, these findings suggest that ERK1/p38-mediated MSK1 may also be involved in mediating UVA-induced STAT1 Ser727 phosphorylation.
In vitro identification of potential protein kinases responsible for phosphorylating STAT1 proteins
To date, the upstream kinases responsible for phosphorylating Ser727 in STAT1 are still elusive. For example, some studies showed that STAT1 was a relatively poor substrate for activated ERKs obtained from IP (27). Additionally, a weaker phosphorylation of a STAT1 C-terminal peptide (aa 711750) was induced by activated p38 kinase in vitro (23). Here, we further examined whether protein kinases in the MAPK cascades are responsible for phosphorylating STAT1 proteins using the immune-purified or GST-pull down STAT1 proteins as kinase substrates. Our results of western blot analysis for the in vitro kinase reactions with STAT1 immunoprecipitates showed that active JNK1, MEK1 and MSK1 stimulated phosphorylation of the STAT1 proteins (Ser727) (Figure 6A and B), but the phosphorylation was undetectable in the ERKs or p38 kinase reactions (data not shown). Furthermore, GST-pull down STAT1 proteins, including GST-fusion wild-type full-length STAT1 (GSTSTAT1wt) and point-mutant STAT1 Y701F (GSTSTAT1Y701F) and S727A (GSTSTAT1S727A) proteins, were identified as shown in Figure 7A. Subsequently, using a phospho-STAT1 (Ser727) antibody or autoradiography, the in vitro kinase reactions showed that none of the experimental kinases phosphorylated the GST-pull down STAT1 proteins (data not shown). Interestingly, additional kinase experiments utilizing combined substrates of the STAT1 proteins from GST-pull down and IP followed by autoradiographic analysis revealed that the immunoprecipitated STAT1 proteins from cell lysates, were to different degrees, phosphorylated by active ERK1, JNK1, p38
kinase, p38ß kinase, MEK1 or MSK1, but not ERK2 or JNK2 (Figure 7B). At the same time, GST-fusion wild-type full-length STAT1 proteins in the presence of IP-STAT1 proteins were also, to different extents, phosphorylated by JNK1, JNK2, p38
kinase or p38ß kinase, but not ERK1, ERK2, MEK1 or MSK1 (Figure 7B). The data suggest that ERKs, JNKs and p38 kinases, as well as MEK1 or MSK1, may participate in the phosphorylation of STAT1 (Ser727) in the presence of an unidentified cofactor or downstream kinase.

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Fig. 6. Western blot analysis for phosphorylation in immunoprecipitated STAT1 proteins induced by protein kinases in vitro. After starvation for 36 h, the cells were harvested. Equal amounts of JB6 cell lysates were subjected to IP with a STAT1 antibody. Then, these STAT1 immunoprecipitates were incubated with the indicated amounts of active purified JNK1 (A), MEK1 (B) or MSK1 (A and B). The phosphorylated STAT1 (Ser727) and total levels of STAT1 in these reactions were determined by western blotting with their corresponding specific antibodies. mU, milliunits. These data are representative of at least three independent experiments.
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Fig. 7. Autoradiographic analysis of STAT1 phosphorylation induced by protein kinases in vitro. (A) A GST-fusion wild-type full-length STAT1 (GSTSTAT1wt), and point mutant STAT1 (GSTSTAT1Y701F or GSTSTAT1S727A) proteins from pull down assays were confirmed by cDNA sequencing (data not shown) and western blotting with specific antibodies against STAT1 (A, upper panel) or GST (A, lower panel). WB, western blotting. (B) A combination of wild-type STAT1 proteins from GST-pull down (GSTSTAT1) and IP (IP-STAT1) was used as a substrate of activated protein kinases, including ERK1, ERK2, JNK1, JNK2, p38 kinase and p38ß kinase (50 mU each), MEK1 (100 mU) and MSK1 (80 mU). The kinase reactions were carried out in a kinase buffer containing [ -32P]ATP and then the reactive products were subjected to 8% PAGE resolution followed by autoradiography. (C) A mixture of each of the indicated GST-fusion STAT1 proteins with IP-STAT1 proteins was used as a kinase substrate and then reacted with 50 mU of JNK1 (C, upper panel) or p38ß kinase (C, lower panel). Subsequently, the kinase reactive products were determined by autoradiography as described for B. These data are representative of at least two independent experiments.
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Moreover, further studies showed that a strong phosphorylation of GSTSTAT1wt or GSTSTAT1Y701F combined with IP-STAT1 proteins was stimulated by either JNK1 (Figure 7C, upper panel) or p38ß kinase (Figure 7C, lower panel), whereas a significantly lower phosphorylation level in the GSTSTAT1S727A proteins was also observed (Figure 7C). These results suggest that JNK1 and p38ß kinase may mediate phosphorylation of STAT1 mainly at the serine 727 residue, as well as possible non-Ser727 residues. In addition, phosphorylation of IP-STAT1 by JNK1 or p38ß kinase was blocked markedly by addition of GSTSTAT1S727A (Figure 7C), suggesting that STAT1S727A may be used as a dominant negative mutant form of the STAT1 protein. Taken together, our study suggests that the EGFR-independent JNKs or p38 kinase signaling pathways play a positive regulatory role in UVA induction of STAT1 serine phosphorylation, and the phosphorylation induction is differentially regulated by the EGFR-mediated MEK1/ERKs signaling pathways (Figure 8). Further, phosphorylation of STAT1 (Ser727) catalyzed by ERKs, JNKs and p38 kinases, as well as MEK1 or MSK1 may occur in the presence of an unidentified cofactor or downstream kinase.

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Fig. 8. A hypothetical model for regulation of phosphorylation of STAT1 (Ser727) by diverse MAPK cascades in the UVA response. The solid arrows indicate activation. The broken arrows represent an uncertain activation. The question marks indicate an unknown. The p and pS/T indicate phosphorylation at the serine 727 and a non-ser727 residue, respectively.
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Discussion
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Most information regarding STAT1 signaling regulation is focused heavily on phosphorylation of Tyr701 catalyzed by JAKs or related protein tyrosine kinases. This resulted in the assumption that Tyr701 phosphorylation was indispensable for the STAT1 activation process (1,3). However, this idea is challenged by recent findings revealing the existence of Tyr701 phosphorylation-independent STAT1 activation mechanisms (11,12). Here, we demonstrate a stronger Ser727 phosphorylation and a relatively lower or no Tyr701 phosphorylation induced by stimulation of JB6 cells with UVA, UVB or UVC, as well as EGF. These observations, together with previous findings showing that Ser727 phosphorylation results in a significant increase in STAT1 activity (4,1724,26), indicate that Ser727 phosphorylation plays a pivotal role in activation of STAT1 signaling. But so far, the kinase directly responsible for the Ser727 phosphorylation involved in this activation process has been not fully identified. In the present report, using pharmacological and genetic approaches, we provide a possible mechanism for signal transduction towards Ser727 phosphorylation. A proposed model of the signaling pathways is presented in Figure 8.
Ser727 is located in a proline-directed consensus motif of the C-terminal domain of STAT1 and the motif makes it a potential target for phosphorylation by MAPKs (17,18). Indeed, its phosphorylation has been shown to be correlated with activation of ERKs (27), p38 kinase (2224) or JNKs (22). However, except for one report indicating that a C-terminal peptide was weakly phosphorylated by p38 kinase (23), no substantial direct evidence has been presented showing that any of the MAPKs act as a direct kinase for STAT1. Here, we performed a series of in vitro kinase reactions using the immunoprecipitated or GST-pull down STAT1 proteins, or combined together as substrates followed by autoradiography or western blotting with a phospho-specific STAT1. The results showed that Ser727 phosphorylation in intact immunoprecipitated STAT1 proteins by active purified JNK1 was detected, but the phosphorylation by active ERK1, ERK2, JNK2 or p38
kinase was not detected by western blotting. However, the more sensitive autoradiography demonstrated that the STAT1 immunoprecipitates are, to different degrees, phosphorylated by active ERKs, JNKs or p38 kinases in vitro. But a GST-fusion wild-type full-length STAT1 (GSTSTAT1wt) was not phosphorylated by any of the experimental kinases (data not shown), consistent with the results of Chung et al. (27). Chung et al. performed in vitro kinase reactions using ERKs, JNKs or p38 kinase immunoprecipitated from Swiss 3T3 cells stimulated with fetal calf serum or sorbitol. The discrepancies in the phosphorylation of STAT1 proteins from IP and GST-pull down assays may be related to docking interactions that contribute to regulation of the specificity and efficiency of the enzymatic reactions with substrates (50). For example, a recombinant STAT3 protein was shown to be phosphorylated by purified JNK in the report of Lim and Cao (45), but the phosphorylation was not detected in the experiments of Chung et al. (27). In fact, experimental evidence is provided that the GSTSTATwt proteins combined with immunoprecipitated STAT1 proteins are, to different extents, phosphorylated by JNK1, JNK2, p38
or p38ß kinases, suggesting that a conformational change in GSTSTAT1wt may be triggered by a yet-to-be unidentified cofactor in STAT1 immunoprecipitates. Furthermore, the phosphorylation of GSTSTAT1 by JNK1 or p38ß kinase was unaffected by a point mutation at the tyrosine 701 residue (GSTSTAT1Y701F), but was significantly decreased by the point mutation at the serine 727 residue (GSTSTAT1S727A). Taken together, these observations indicate that ERKs, JNKs and p38 kinases may catalyze phosphorylation of STAT1 at Ser727 in the presence of an unidentified factor. However, a weaker phosphorylation in GSTSTAT1S727A proteins suggests that the experimental kinases may be also involved in catalysis of STAT1 phosphorylation at non-Ser727 residues.
Further evidence is presented that UVA-stimulation of Ser727 phosphorylation of STAT1 in JB6 cells was blocked by a dominant negative mutant of JNK1 (DMN-JNK1) or p38ß kinase (DNM-p38ß), or by biochemical inhibition of JNKs and/or p38 kinase, and also abolished by deficiency of Jnk1 or Jnk2. These data, together with in vitro kinase reactions, indicate that JNKs and p38 kinases are required for mediating serine phosphorylation of STAT1 stimulated by UVA.
Interestingly, our results revealed that the presence of MEK1/2 inhibitors, PD98059 and U0126, significantly reduced STAT1 phosphorylation induced by UVA, suggesting an involvement of MEK1/ERK1 in the process. In addition, SEK1/MEK4, MKK6 and/or MEKK1 have been shown to participate directly or indirectly in regulating Ser727 phosphorylation of STAT3 (4345). Our in vitro experiments provide evidence showing that active MEK1 may induce phosphorylation of immunoprecipitated STAT1 proteins, but not STAT1 proteins from GST-pull down. These findings, together with the fact that the MAPK activation motif, in which Ser727 is located, is significantly different from the MEK activation motif (YXS/T), indicate that MEK could play a role in mediating STAT1 phosphorylation indirectly via ERK1 or an unidentified downstream kinase. Overall, these results suggest that the MAPK cascades, including MKKMEKERKs, may have different effects in the regulation of phosphorylation of STAT1 (Ser727). Additionally, the UVA-stimulated serine phosphorylation of STAT1 was also prevented by an N-terminal or C-terminal kinase-dead mutant of MSK1, a downstream serine/threonine of both p38 kinase and ERKs, but the phosphorylation was enhanced when wild-type MSK1 was over-expressed. These findings appear to suggest that MSK1 may also be required for UVA-stimulated STAT1 phosphorylation (Ser727). Further, in vitro kinase experiments showed that phosphorylation in immunoprecipitated STAT1, but not GSTSTAT1 proteins, was induced by active MSK1. These findings, together with the fact that the MSK1 activation motif (37) is starkly different from the MAPK activation motifin which Ser727 is located, suggest that mediation of STAT1 phosphorylation by ERKs/p38 kinase-mediated MSK1 may occur indirectly through a downstream kinase. In addition, our experiments showed that both inhibition and deficiency of EGFR kinase also promoted the enhancement in the UVA-stimulated phosphorylation of STAT1 (Ser727), suggesting that both EGFR-dependent and -independent MAPK signaling pathways lead towards STAT1 Ser727 phosphorylation in the UVA response.
In summary (Figure 8), STAT1 (Ser727) phosphorylation appears to be a point of convergence and is eventually stimulated following the integration of signals from multiple pathways. These signals from EGFR-independent MAPK-mediated pathways to STAT1 are triggered and transduced in the cellular response to UVA. Further, the phosphorylation process by MAPKs, including ERKs, JNKs and p38 kinases, as potential direct kinases for STAT1 (Ser727) phosphorylation, may occur in the presence of an unidentified factor. Additionally, unlike MAPKs, MEK1, an upstream kinase closer to ERKs, and MSK1, a downstream kinase of ERKs/p38 kinase, may also play a role in modulating STAT1 phosphorylation indirectly via an unidentified downstream kinase or other signaling mechanisms. Overall, STAT1 phosphorylation at Ser727 and/or possibly at non-ser727 sites is differentially mediated by diverse MAPK cascades in the UVA response.
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Acknowledgments
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This work was supported in part by the Hormel Foundation and National Institutes of Health grants CA77646 and CA81064. We are very grateful to Dr Dario R.Alessi for providing the pCMV5-FLAG vector (CMVS), pCMV5-FLAG-wild-type MSK1 (MSK1wt), pCMV5-FLAG-MSK1-A195/N-terminal kinase-dead (MSK1-Nd), or pCMV5-FLAG-MSK1-A565/C-terminal kinase-dead (MSK1-Cd) (37). We thank Dr Nanyue Chen for preparation and identification of Jnk+/+, Jnk1/ or Jnk2/, as well as Egfr+/+ and Egfr/ fibroblasts (30,32). We thank Dr Ann M.Bode for editorial assistance and Ms Andria Hansen for her secretarial assistance.
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Received November 25, 2003;
revised January 26, 2004;
accepted February 4, 2004.