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J. Biol. Chem., Vol. 280, Issue 23, 21700-21705, June 10, 2005
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From the
Institut für Biochemie, Universitätsklinikum der Rheinisch-Westfälischen Technischen Hochschule Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany, the Institute for Molecular Biology and Tumor Research (IMT), Philipps-University Marburg, Emil-Mannkopff-Strasse 2, 35033 Marburg, Germany, and the ¶Laboratoire de Biologie et Physiologie Intégrée, Faculté des Sciences, de la Technologie et de la Communication, Université du Luxembourg, 162A, avenue de la Faïencerie, 1511 Luxembourg, Luxemburg
Received for publication, December 21, 2004 , and in revised form, March 25, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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90 kDa), which are specifically activated to regulate gene transcription when cells encounter cytokines and growth factors. Subsequent to receptor binding, the STAT proteins are phosphorylated on a single tyrosine residue (Tyr701 in STAT1 and Tyr705 in STAT3). The phosphorylated STATs dissociate from the receptors, form active dimers through interactions of their SH2 domains, and translocate to the nucleus to induce gene transcription. In addition to tyrosine phosphorylation, STATs 1, 3, and 5 also require phosphorylation on a serine residue in the C-terminal region to achieve maximal transactivation potential (1–3).
Previously, a model has been proposed suggesting that STAT1 methylation on arginine at position 31 is functionally necessary since it suppresses the interaction with the negative regulatory protein PIAS (4). Later, it was published by the same group that methylation of Arg31 of STAT1 is necessary for its dephosphorylation by the phosphatase TcPTP (5). Similarly, for STAT6, arginine methylation has been described to be important for STAT6 function (6). Mostly, these studies have made use of antibodies directed against methylarginine, STAT mutants, and inhibitors of methylation, methylthioadenosine (MTA) (4) or N-methyl-2-deoxyadenosine (MDA), in the presence of adenosine and DL-homocysteine (5–7). In addition, STAT3 has been shown to be recognized by an anti-methylarginine antibody (8).
The model proposed by Mowen et al. (4) could provide an explanation for the observed interferon resistance of cancer cells that have lost expression of methylthioadenosine phosphorylase (MTAP), the enzyme that inactivates the metabolite MTA implicated in inhibition of methylation. We recently showed that MTAP expression is reduced in melanoma cells (9). Since STAT3 is involved in growth inhibition of melanoma cells (10), we wondered whether a potential arginine methylation of STAT3 might be inhibited in an analogous way in MTAP-negative cells, which could provide an explanation for cytokine resistance of late stage melanoma cells. Therefore, we started to investigate the potential arginine methylation of STATs.
Our results did not confirm the occurrence of arginine methylation on STAT1 or STAT3 in cell culture or in vitro. Therefore, the data presented in this communication question the validity of the model published by Mowen et al. (4). In agreement with a very recent report by Meissner et al. (11) we present further compelling evidence which suggests the lack of arginine methylation on STATs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ cells (murine embryonic fibroblasts kindly provided by Dr. V. Poli, Torino, Italy) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen). All media were supplemented with 100 mg/liter streptomycin and 60 mg/liter penicillin. Cells were grown at 37 °C in a water-saturated atmosphere at 5% CO2. Transient transfections of MEF / and U3A cells were performed using FuGENE (Roche Applied Science) and Superfect (Qiagen) according to the manufacturer's instructions.
Cytokines and Reagents—IFN
and OSM were obtained from IntronA and from Peprotech, respectively. Recombinant IL-6 and soluble IL-6 receptor was prepared as described previously (12, 13). Adenosine, DL-homocysteine, MDA, and 5'-methylthioadenosine were purchased from Sigma and dissolved in culture medium or Me2SO.
Plasmids—The constructs of human STAT1 R31K and murine STAT3 R31K were generated by site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA). pBS-STAT1 and pBS-STAT3 were kindly provided by Dr. T. Hirano (Osaka, Japan) and were used as a template in PCR. cDNA fragments corresponding to human STAT1wt, STAT1R31K, murine STAT3wt, or STAT3R31K were then inserted into KpnI and NotI sites of pcDNA3 (Invitrogen). The plasmid encoding a GST fusion protein of full-length STAT1 was generated by excision of an EcoRV/NotI fragment from pBS-STAT1 and subsequent ligation into the SmaI/NotI sites of pGEX-5X-2 (Amersham Biosciences). PGEX-HRMT1L2 (v2) (= pGEX-PRMT1, human) has been described previously (14). pGEX-GAR was a gift from Dr. S. Clarke (15). pcDNA3.1 hPRMT1 full-length wild type was cloned from the pGEX-HRMT1L2 (v2) by PCR introducing a BamHI site at the 5'-end and an EcoRI site at the 3'-end of the HRMT1L2 cDNA (deleting the stop at the same time). Subsequently the HRMT1L2 cDNA was subcloned with BamHI and EcoRI into pcDNA3.1-B (Invitrogen) generating a C-terminal myc/His-tag. The point mutations S69A, G70A, and T71A were introduced into pcDNA3.1 hPRMT1 via site-directed mutagenesis using the QuikChange kit to give rise to pcDNA3.1 PRMT1 full-length catalytic mutant. All constructs were verified by DNA sequencing.
Cell Lysis, Immunoprecipitation, and Western Blot Analysis—Cells were lysed on the dish with 500 µl of lysis buffer containing 20 mM HEPES, pH 7.4, 1% Triton X-100, 100 mM NaCl, 50 mM NaF, 10 mM 
-glycerophosphate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 µg/ml aprotinin, 3 µg/ml pepstatin, and 5 µg/ml leupeptin. Lysates were clarified at 13,000 rpm for 10 min at 4 °C, and protein concentration in the supernatant was determined by the Lowry method (Bio-Rad protein assay). Equal amounts of total cellular proteins were separated by 7.5% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Pall) and probed with the appropriate antibodies. Rabbit anti-phospho-STAT1 (Tyr701) (9171), rabbit anti-phospho-STAT3 (Tyr705) (9131), rabbit anti-phospho-MAP-KAPK-2 (Thr222) (3044), and mouse anti-phospho-Erk1/2 (9106) were from Cell Signaling Technology Inc. Mouse anti-STAT1 (610185) and mouse anti-STAT3 (610189) were purchased from Transduction Laboratories. Rabbit anti-STAT1 (E-23), rabbit anti-STAT3 (C-20), rabbit anti-p38 (C-20), goat anti-Erk1/2 (C-16 and C-14), goat anti-SOCS3 (M-20), anti-GST (B-14, mouse IgG1), and anti-Lamin A/C (346, mouse IgM) were from Santa Cruz Biotechnology, Inc. Rabbit anti-phospho-p38 (V1211) was obtained from Promega (Madison, WI). ab412 (mono- and dimethylarginine antibody, mouse IgG1) and ab413 (dimethylarginine antibody, mouse IgM) were supplied from Abcam. The horseradish peroxidase-conjugated secondary antibodies were purchased from Dako. Signals were detected using the ECL system (Amersham Biosciences). Before reprobing, blots were stripped in 2% SDS, 100 mM -mercaptoethanol in 62.5 mM Tris-HCl, pH 6.7, for 20 min at 75 °C. For immunoprecipitations, cell lysates (1 ml containing 1.5 mg of protein) were incubated with 5 µl of monoclonal mono-/dimethylarginine antibody (Abcam) or an isotype control antibody overnight at 4 °C. The resulting immune complexes were precipitated with 5 mg protein A-Sepharose (CL-4B, Amersham Biosciences) for 1 h, washed three times with lysis buffer, and boiled in Lämmli buffer for 5 min at 95 °C. Proteins were separated by SDS-PAGE, followed by Western blot analysis.
Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared as described previously (16), and EMSAs were performed as described previously (17) using the m67SIE probe binding STAT1 and STAT3.
GST Fusion Protein Expression—GST fusion proteins were expressed in Escherichia coli strain BL21 using the pGEX (Amersham Biosciences) vector system. Purification of GST fusions from crude bacterial lysates was performed by immobilization on glutathione-Sepharose (Amersham Biosciences). Subsequently, fusion proteins were eluted from the beads in the presence of 25 mM glutathione, 50 mM Tris-HCl, pH 8 solution. Eluates were dialyzed against phosphate-buffered saline, 10% glycerol.
In Vitro Methyltansferase Assays—Eluted and dialyzed GST-PRMT1 was incubated with eluted/dialysed GST-GAR and GST-STAT1, respectively, in the presences of 1 µl of S-adenosyl-L-[methyl-14C]methionine (Amersham Biosciences, 60 mCi/mmol) and phosphate-buffered saline at 37 °C for 2 h. Reaction products were resolved by SDS-PAGE, blotted onto nitrocellulose, and visualized by autoradiography.
Reporter Gene Assay—U3A cells were transfected with 3 µg of IRF-1 luciferase reporter construct, 1 µg of -galactosidase control plasmid, and 100 ng of STAT1wt or 1 µg of STAT1R31K using Superfect (Qiagen). MEF / cells were transfected with 3 µg of c-fos luciferase reporter construct, 1 µg of -galactosidase control plasmid, and 100 ng of STAT3 wt or 1 µg of STAT3R31K using FuGENE 6 transfection reagent (Roche Applied Science). Twenty-four hours after transfection, cells were stimulated with 1000 units/ml IFN or 20 ng/ml recombinant human IL-6 for 16 h. Cell lysis and luciferase assays were performed using Promega's dual luciferase kit according to the manufacturer's instructions. Luciferase activity values were normalized to transfection efficiency monitored by the co-transfected -galactosidase expression vector pCH110 (Amersham Biosciences).
Molecular Modeling of the N-terminal Domains of STAT1 and STAT3—For molecular modeling and graphic representation of the protein structures, the programs WHAT IF (18), Ribbons (19), and Rasmol (20) were used. The structure of the N-terminal domain of STAT4, Brookhaven data bank entry code 1BGF (21), was used as a template for the model structures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- or OSM-mediated STAT1 and STAT3 phosphorylation and DNA binding activity (Fig. 1A, first through fifth panels). Interestingly, when reprobing the blots with antibodies against phosphorylated p38 and Erks, we found that MDA had a serious impact on these kinases, i.e. it resulted in a dramatic decrease of p38 and Erk1/2 phosphorylation (Fig. 1A, seventh and tenth panels). Moreover, phosphorylation of MAPKAP2 was reduced, and it abrogated inducible SOCS3 expression in WM9 cells (Fig. 1A, sixth and ninth panels). The effect was detectable at a concentration of 0.2 mM MDA (Fig. 1B), which is below the concentrations that have previously been used to inhibit methylation (0.3 mM up to 2 mM (5, 6, 22)). The inhibition of signal transduction by preincubation with MDA occurred rapidly, i.e. in less than 1 h (Fig. 1B, lower panels). In fibrosarcoma cells, MDA pretreatment similarly attenuated IFN- and OSM-mediated STAT phosphorylation as well as p38 and SHP-2 phosphorylation. In this cellular system, however, MDA treatment stimulated Erk1/2 phosphorylation (Fig. 1C). The Jak/STAT signaling cascade is well known to be subject to cross talk with signaling pathways involving Erks, p38, and SHP-2 (1, 23–26). Therefore, effects of MDA treatment on STAT function do not necessarily mean that STAT arginine methylation is affected.
Since the solvent, culture medium or Me2SO, was reported to influence the effect of the methylation inhibitor MTA on STAT phosphorylation (4, 7, 11), we compared the effects of MDA using either culture medium or Me2SO as carrier. As shown in supplemental Fig. 1A, phosphorylation of STATs, Erk1/2, and p38 was diminished upon MDA treatment, irrespective of the solvent used. We also tested the effects of MTA, dissolved in culture medium or in Me2SO (supplemental Fig. 1B). Phosphorylation of p38 and Erk1/2 was unchanged by MTA treatment. We observed in 729 melanoma cells an inhibitory effect of MTA on STAT phosphorylation, in particular STAT3, but this effect was independent of the solvent, which does not correspond to the findings by Mowen and David (7).
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/) and recognized in STAT3-specific Western blots, indicating again a specificity problem. Using another STAT3-specific antibody for detection we observed no band corresponding to STAT3 neither in wild-type MEFs nor in STAT3 knock-out MEFs (Fig. 2B, lower panel). Furthermore, the dimethylarginine-specific antibody ab413 did not lead to a specific precipitation of STAT1 or STAT3 (data not shown). Taken together, these data do not provide any evidence for arginine methylation of STATs but indicate a specificity problem of the mouse anti-STAT antibodies and underline the importance of negative control cells (i.e. STAT1/3-deficient cells).
Exchange of Arg31 Renders STATs Unstable—Replacement of STAT1 Arg31 by alanine or by glutamic acid or of STAT6 Arg27 by alanine rendered the proteins unstable (4, 6). Moreover, the solved structure of the STAT4 N-terminal region (21) suggests that Arg31 plays a structural role as it has multiple contacts with other amino acids in the N-terminal domain, which are also conserved in STAT1 and STAT3 (supplemental Fig. 2, A and B). To generate a mutation that would minimize structural effects we decided to exchange Arg31 by Lys. Structural models of the N-terminal regions of STAT1 and STAT3 indicated that this residue could mimic at least one of the two salt bridges built up by Arg31 (supplemental Fig. 2, A and B). However, upon heterologous expression the mutant STAT1 and STAT3 proteins still turned out to be expressed to a much lower extent (Fig. 2, C and D, upper panels), an effect that could at least partly be attributed to the reduced half-life of the R31K mutants, which was investigated by cycloheximide treatment and Western blot analysis (lower panels).
In the case of STAT1, the RK mutation slightly reduced STAT1-mediated gene expression but had no effect on IFN-mediated STAT1 phosphorylation or induction of STAT1 DNA binding activity (supplemental Fig. 3A). The RK mutation in STAT3 led to a decrease in STAT3-mediated gene expression and possibly in IL-6/sIL-6R-mediated STAT3 phosphorylation (supplemental Fig. 3B). However, the relevance of these effects is questionable considering the 10-fold excess of DNA encoding the R31K mutants of STAT1 or STAT3, which was needed to obtain approximately equivalent expression levels of the wild-type and mutant proteins.
No Evidence for in Vitro Methylation of STAT by PRMT1 and for Effects of PRMT1 on STAT1-mediated Gene Expression—To assess arginine methylation directly we performed in vitro methylation assays using purified PRMT1 as well as GST-STAT1 and GST-STAT3 fusion proteins. Although PRMT1-mediated 14C-labeled methyl group incorporation was readily detectable for histone H4 and GST-GAR (glycine-arginine-rich domain of fibrillarin), which were used as positive controls, we failed to detect methylation of the STAT fusion proteins (Fig. 3A and data not shown). We further tested whether STAT3 could be a substrate for other PRMT family members, like PRMT2, -3, -4, and -6, but could not detect any in vitro methylation of STAT3 by these enzymes (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(i) We did not observe an in vitro methylation of a full-length GST-STAT1 fusion protein by PRMT1, although the catalytic activity of our enzyme preparation was readily demonstrated for the glycine-arginine rich protein. It should be noted that Mowen et al. (4) used a GST fusion protein of STAT1 truncated after the N-terminal 129 amino acids to demonstrate in vitro methylation, which might explain the divergent results. Moreover, we did not observe in vitro methylation of a GST-STAT3 fusion protein by active enzyme preparations of PRMT1, -2, -3, -4, or -6, although FLAG-tagged STAT3 had been used as a positive control in a study on in vivo arginine methylation of the hepatitis C virus NS3 protein (8). Furthermore, we did not observe a difference in STAT1-mediated reporter gene activity upon co-expression of PRMT1 or a catalytically inactive form of PRMT1. Interestingly, in previous reports a similar PRMT1 co-expression substantially enhanced reporter gene activity controlled by NIP45 co-activator and nuclear hormone receptors, respectively (28, 29). Thus, our data indicate that PRMT1 does not affect STAT1 function with respect to transcriptional regulation.
(ii) Certain STAT antibodies can apparently recognize proteins that are precipitated by anti-methylarginine antibodies but that are different from STAT1 or STAT3, since they were also observed in cells lacking STAT1 and STAT3, respectively. The reason for this unexpected cross-reactivity of potentially arginine-methylated proteins with monoclonal antibodies directed against STAT1 or STAT3 is currently unknown. A potential unspecific detection of proteins, which are co-migrating with STAT1 and STAT3, makes it difficult to interpret Western blot results (4, 5, 7, 27).
(iii) Our data certainly underline the importance of Arg31 for the structural integrity and stability of the STAT protein, a fact already implied by the solved crystal structure of the N-terminal domain of STAT4 (21). Analyzing the three-dimensional structure it is difficult to envisage how this arginine residue can post-translationally be modified. When the DNA amounts of the STAT wild-type and R31K mutant expression vectors were adapted to obtain equal overall expression levels, we observed a suppressive effect of the mutation on STAT-mediated gene expression. Our findings are contrasting those describing a gain-of-function upon mutation of Arg31 in GST-STAT1 (4) but would match data describing loss-of-function of STAT1-Arg31 and STAT6-Arg27 mutants, which both showed defective nuclear translocation (6, 11).
(iv) We have shown that MDA treatment not only affects STAT phosphorylation and DNA binding activity but also rapidly reduces phosphorylation of p38 and Erk MAP kinases. MTA primarily inhibited phosphorylation of STAT3 and weakly also of STAT1. The MDA effects can be observed at concentrations below those usually used for inhibition of arginine methylation (5, 6, 22). Moreover, MDA treatment suppressed luciferase reporter gene expression under the control of the IRF-1 promoter, but it also led to a reduction of -galactosidase expression regulated via the cytomegalovirus promoter (data not shown) further underlining the wide range of MDA effects. The Jak/STAT signal transduction cascade is known to be influenced by other pathways (1, 23–26). Therefore, effects of MDA treatment on STAT function could well be explained by the influence on these and possibly other signaling molecules and do not necessarily imply an involvement of STAT arginine methylation.
N-Methyl-2-deoxyadenine is a nucleoside that naturally only occurs in DNA from prokaryotes. Long term incubation with this compound has previously been shown to affect various cell types: it induces differentiation of P19 teratocarcinoma cells and of C6.9 glioma cells, neurite outgrowth of PC12 cells, and also stimulates C2C12 cells to undergo myogenic differentiation. Based on inhibitor studies, a possible involvement of adenosine A2a receptors and cAMP-, MAPK-, and rapamycin-sensitive pathways has been discussed (30–32). It clearly warrents further investigations to identify the molecular mechanisms underlying the observed effects of MDA and MTA on STAT function and the p38 and Erk pathways, as well as the role protein arginine methylation may play.
In conclusion, and together with the very recently published data (11), our results do not support the proposed model of STAT arginine 31 methylation.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.
|| To whom correspondence should be addressed. Tel.: 352-466644-361; Fax: 352-466644-435; E-mail: iris.behrmann{at}uni.lu.
1 The abbreviations used are: STAT, signal transducer and activator of transcription; EMSA, electrophoretic mobility shift assay; Erk, extracellular signal-regulated kinase; GAR, glycine-arginine-rich domain of fibrillarin; GST, glutathion S-transferase; IFN, interferon; IL, interleukin; Jak, Janus kinase; MTA, methylthioadenosine; MDA, N-methyl-2-deoxyadenosine; MEF, murine embryonic fibroblast; MTAP, methylthioadenosine phosphorylase; OSM, oncostatin M; PRMT, protein arginine methyltransferase; SAM, S-adenosylmethionine; SHP-2, Src homology domain containing phosphatase-2; SIE, sis-inducible element.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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