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Blood, 15 November 2006, Vol. 108, No. 10, pp. 3237-3244. Prepublished online as a Blood First Edition Paper on July 20, 2006; DOI 10.1182/blood-2006-04-020271.
CHEMOKINES, CYTOKINES, AND INTERLEUKINS Stat1 and SUMO modificationFrom the Departments of Microbiology and Medicine, Columbia University, New York, and the Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY.
Many proteins are known to undergo small ubiquitin-related modifier (SUMO) modification by an E1-, E2-, and E3-dependent ligation process. Recognition that protein inhibitor of activated signal transducers and activators of transcription (STATs) (PIAS) proteins are SUMO E3 ligases raised the possibility that STATs may also be regulated by SUMO modification. Consistent with this possibility, a SUMO-ylation consensus site (  KxE; indicates hydrophobic residue, and x indicates any residue) was identified in Stat1 (ie, 702IKTE705), but not in other STATs. Biochemical analysis confirmed that Stat1 K703 could be SUMO modified in vitro. Mutation of this critical lysine (ie, Stat1K703R) yielded a protein that, when expressed in Stat1–/– mouse embryonic fibroblasts (MEFs), exhibited enhanced DNA binding and nuclear retention. This was associated with modest changes in transcriptional and antiviral activity. However, mutation of the second critical residue in the SUMO consensus site, E705 (ie, Stat1E705A), yielded a protein with wild-type DNA binding, nuclear retention, and transcriptional and antiviral activity. Similar observations were made when these mutants were expressed in primary Stat1–/– macrophages. These observations suggest that although Stat1 can uniquely be SUMO-ylated in vitro, this modification is unlikely to play an important role in regulating Stat1 activity in vivo.
Characterization of the ability of type I interferons (IFNs) (eg, IFN-  ) to rapidly activate genes led to the identification of ISGF-3, a transcription factor consisting of Stat1, Stat2, and an IFN regulatory factor-9 (IRF-9) DNA binding protein.1 Subsequently, IFN- was shown to induce genes through Stat1 homodimers.2 To date, 7 signal transducers and activators of transcription (STATs) have been identified in vertebrates, all of which are activated by phosphorylation on a single tyrosine (Y701 in Stat1; reviewed in Levy and Darnell3 and Kisseleva et al4). Activation drives STAT dimerization by directing a stable and specific association between the phosphotyrosine of one STAT and the src homology 2 (SH2) domain of a partner STAT.5 Residues located at positions +1, +3, +5, +6, and +7 carboxy terminal to this phosphotyrosine (ie, amino acids 702, 704, 706, and 707 for Stat1) determine the specificity of this interaction.6 Dimerized STATs translocate to the nucleus, where they bind to members of the gamma-activated site (GAS) family of enhancers, culminating in the induction genes.3,4
The regulation of STAT signal decay has also been an area of active investigation. Four major classes of counterregulatory molecules have been identified, including phosphatases,3,4,7 nuclear "transportases,"8-10 covalent modifiers,4,11,12 and specific STAT counterregulatory proteins (eg, suppressor of cytokine signaling [SOCS] and protein inhibitor of activated STATs [PIAS] proteins13,14). Studies on SOCS-1 have provided significant evidence for a critical role in down-regulating IFN
SUMOs are approximately 100–amino acid peptides which, like ubiquitin, become covalently attached to cellular target proteins (reviewed in Kim et al,17 Melchior et al,18 and Müller et al19). However, in contrast to ubiquitin, SUMO modifications do not target proteins for degradation, but rather promote protein–protein interactions and direct subcellular localization, and/or serve to antagonize ubiquitin-dependent degradation. SUMO conjugation entails the formation of a reversible isopeptide bond between the C-terminus of the SUMO peptide (SUMO-1, SUMO-2, or SUMO-3) and the
Sequence analysis revealed 2 potential SUMO modification sites, 109LKEE112 and 702IKTE705, in Stat1, but not in other STATs (Table 1). In vitro SUMO conjugation studies determined that Stat1 is SUMO modified at lysine 703, but not lysine 110. A subsequent functional analysis of 2 SUMO-ylation–resistant Stat1 mutants, Stat1K703R and Stat1E705A, revealed 2 distinct phenotypes. Stat1K703R exhibited enhanced DNA binding, prolonged nuclear retention, and modest changes in the biologic response to IFN- , as recently reported.23 In contrast, Stat1E705A exhibited wild-type DNA binding, nuclear retention, and biologic response to IFN-. These observations suggest that lysine 703, located at the critical interface between Stat1 homodimers,6 plays a fortuitous and structurally important role in Stat1 DNA binding activity, and that Stat1 activity is not likely to be significantly regulated by SUMO conjugation in vivo.
Cell culture HEK-293T, Vero, and L929 cells were from ATCC (Manassas, VA); and Stat1–/– mouse embryonic fibroblasts (MEFs) and macrophages were harvested from Stat1–/– mice (generously provided by D. Levy, New York University, NY). Cells were cultured in Dulbecco modified Eagle medium, supplemented with 10% fetal bovine serum from GIBCO Laboratories (Grand Island, NY). Bone marrow (from mouse femurs)–derived macrophages were cultured in 20% L929 cell–conditioned media for 5 to 10 days.24 After 16 hours in culture, adherent bone marrow cells were infected (3 times) with retrovirus freshly prepared from HEK-293T transfectants (below). Stat1–/– MEFs were infected twice with pMIG retroviruses encoding Stat1, Stat1K703R, and Stat1E705A in the presence of polybrene (8 µg/mL; Sigma, St Louis, MO), as previously reported.25 High-titer viral supernatants were prepared through transient transfection in HEK-293T cells by calcium phosphate precipitation.9 Retroviral infection efficiency, as determined by fluorescence-activated cell-sorter (FACS) (green fluorescent protein–positive [GFP+] cells; FACS-Calibur; BD Biosciences, San Jose, CA), varied between 90% to 95% in MEFs and 25% to 35% in bone marrow macrophages (BMMs).
For viral response assays, MEFs were infected with vesicular stomatitis virus (VSV, Indiana strain; gift from R. Pine, Public Health Research Institute, Newark, NJ) prepared in Vero cells. Viral yield was measured 24 hours after infection by titering on Vero cells overlayed with 1.5% methyl cellulose for 36 hours.26 Expression of the reporter GFP gene and major histocompatibility complex (MHC) II (after staining with anti-mouse I-Ab; BD Pharmingen, San Diego, CA) was evaluated by flow cytometry (FACS-Calibur).9,26 Nitric oxide (NO) production was evaluated at day 10 of culture and 72 hours after IFN- Biochemical studies Recombinant SUMO-1, E1, and E2 were expressed, purified, and assayed as previously described.20 The IR1-M-IR2 domains of Nup358 (aa 2596-2836) and full-length PIAS1 (aa 1-651) were cloned and expressed as Smt3 fusion proteins, cleaved by Ulp1, and purified by anion exchange and gel filtration chromatography.28 To assay for Stat1 SUMO-ylation, purified recombinant Stat1 (0.5 µM) was incubated with SUMO-1 (2 µM), E1 (Uba2/Aos1; 0.3 µM), E2 (Ubc9; 0.3 µM), and E3 (PIAS1 or Nup358 at 0.3 µM) in a buffer with 5 mM MgCl2, 20 mM HEPES (pH 7.5), 1 mM DTT, 2 mM ATP, and 0.5 U pyrophosphatase (Sigma) for 1 hour at 37°C. To assay for peptide SUMO-ylation, p53 (residues 323-393), p53K386M (residues 320-393) Stat1/phospho-Stat1 (residues 697-711), or Stat3 (residues 701-714) peptides (at 500 µM) were incubated with E1 and either wild-type or mutant Ubc9 for 5 or 24 hours at 37°C, as previously described.29 Samples were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue or Sypro (4-12–hour staining, > 1 hour destaining; Bio-Rad, Hercules, CA). Sypro-stained gels were imaged by UV illumination and the band intensity was quantified (Bio-Rad UV system; Quantity One Software; Bio-Rad).
Overexpression assays entailed transfecting protamine complementary DNA (pcDNA; Invitrogen, Carlsbad, CA) expression plasmids encoding hemagglutinin (HA)–SUMO-1, His–SUMO-1, Ubc9, and/or PIAS-y (gifts from S. Goff, Columbia University, New York, NY) into HEK-293T cells by calcium phosphate precipitation or MEFs with LipofectAmine Plus regent (Invitrogen). For oligonucleotide "pull-down" assays 20 µg GAS oligonucleotide (Table S1, available on the Blood website; see the "Supplemental Table" link at the top of the online article) coupled to 75 µL biotinylated agarose beads (Sigma) was incubated with 400 µL whole-cell extracts (WCEs) for 2 hours at 4°C, after blocking the beads with 1% bovine serum albumin (BSA) for 1 hour at 4°C, as previously reported.30 For immunoprecipitation, 400 µL WCEs was incubated with primary antibody for 2 to 16 hours followed by protein A agarose (Sigma), and then collected, as previously reported.9,26 WCE or precipitates were fractionated by SDS-PAGE and then evaluated by immunoblotting with the appropriate antibody.9 For nickel pulldown, 800 µL WCEs was incubated with 100 µL ProBond nickel beads, washed, and eluted as per the manufacturer's instructions (Invitrogen). For electrophoretic mobility shift assay (EMSA), extracts were incubated with a GAS probe and fractionated by native PAGE as previously described.9,26 Antibodies were directed against Stat1,1 phosphotyrosine Stat1 (Cell Signaling Technology, Beverly, MA), MHC II (BD Pharmingen), HA (Covance, Berkeley, CA), and Plasmid constructs pClEco and pMIG were provided by J. Luban (Columbia University).25 Murine Stat1, Stat1K703R, and Stat1E705A were cloned into the XhoI site in pMIG. Stat1K703R and Stat1E705A were prepared by site directed mutagenesis (Quickchange kit; Stratagene, La Jolla, CA) and confirmed by sequencing (Table S1 for primer sequences). Immunofluorescence Cells were cultured on sterile cover slips until 20% to 25% confluent, fixed in formaldehyde, and stained as previously reported9 with Stat1-specific antibodies (1:250 fold dilution) and a Cy3-conjugated secondary antibody (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). Slides were examined under a Nikon Eclipse TE300 microscope (Nikon, Melville, NY) after excitation at 550 nm (Cy3) and excitation at 495 nm (GFP). RNA expression analysis
Total RNA was prepared from MEFs with Trizol (Invitrogen) extraction. RNA (4 µg) was treated with RQ1 DNAse (Promega, Madison, WI) and then reverse-transcribed with SuperScript II (Invitrogen). The cDNA was quantitatively amplified in an ABI Prism 7700 polymerase chain reaction (PCR) system with a SYBR green master mix (Applied Biosystems, Foster City, CA) as previously described.24 Gene expression was normalized to a Luciferase reporter assay
MEFs were seeded in 24-well plates (7.5 x 105/well) and transfected with 400 ng of an IRF-driven luciferase reporter (ie, B2WT331) and 40 ng pRL-tk renilla in LipofectAmine Plus (Invitrogen). Later (24 hours), transfectants were stimulated with IFN-
Stat1 can be SUMO modified Recent studies identifying PIAS proteins as SUMO E3 ligases suggested that STATs may also be SUMO modified.17-19 Analysis of all mammalian STAT sequences identified 2 potential SUMO modification sites: Stat1 residues 109LKEE112 and 702IKTE705 (Table 1). Efforts to exploit SUMO-1–specific antibodies to detect endogenous SUMO-modified Stat1 were unsuccessful. To improve the chances of detecting this product, Ubc9 and HA–SUMO-1 were overexpressed in HEK-293T cells. Under these conditions SUMO-Stat1 conjugates were readily recovered in Stat1 immunoprecipitates (Figure 1A) as previously reported.32,33 Coexpression of PIAS-y, however, impeded recovery of SUMO-Stat1 conjugates, most likely because of reduced levels of SUMO-1 and Ubc9 expressed in triple transfectants.
One potential SUMO modification site, lysine 703 (K703), lies adjacent to the tyrosine that is phosphorylated during Stat1 activation, (ie, Y701). It was therefore important to determine whether SUMO conjugation affected Stat1 phosphorylation and subsequent DNA binding activity. As anticipated, phosphorylated Stat1 was readily recovered, by either immunoprecipitation or oligonucleotide precipitation, from prepared extracts of IFN-
Stat1 is SUMO modified at K703 To develop more direct evidence for Stat1 SUMO modification and to map the SUMO-ylation site, we turned to an effective in vitro conjugation assay that overcomes limitations imposed by SUMO deconjugating enzymes.20,22,34 Purified recombinant Stat1 was incubated with purified active preparations of human recombinant SUMO-1, E1 (Uba2/Aos1), E2 (Ubc9), and E3 (Nup358 or PIAS1).20,21,35 After 1 hour, the products were fractionated by SDS-PAGE and immunoblotted with a Stat1-specific antibody. As shown in Figure 2, the anticipated approximately 105-kDa SUMO-Stat1 conjugate was readily formed in an ATP-dependent, but not E3-dependent manner (Figure 2A). Analogous results were obtained with a purified p53 control (data not shown). The SUMO-Stat1 conjugates (approximately 3%-4% of the total Stat1) and the remaining nonreacted (ie, native) Stat1 were then excised, digested with trypsin, and evaluated by mass spectrometry. The specific loss of peptides spanning K703, but not K110 indicated that Stat1 had been SUMO modified at K703 (data not shown). To confirm that K703 was the SUMO-ylation site, a Stat1 peptide spanning residues 697-711 (ie, KGTGYIKTELISVS) was evaluated in an in vitro SUMO conjugation system where a defective E2 (ie, Ubc9C93S) served as a negative control.29 Several additional peptides were evaluated. This included the analogous peptide from Stat3 (ie, AAPYLKTKFICVT) and a Stat1 peptide in which K703 was mutated to arginine (ie, KGTGYIRTELISVS). To explore whether Stat1 Y701 phosphorylation precludes SUMO modification, as suggested by HEK-293T overexpression studies (Figure 1), a Stat1 phosphopeptide (ie, phospho-Y701) was also evaluated. Again, p53 peptides spanning a well-characterized SUMO-ylation site served as important controls.20 After 5 and 24 hours of SUMO conjugation, only the wild-type Stat1 peptide was SUMO-ylated as effectively as the control wild-type p53 peptide (Figure 2B). More detailed kinetic studies provided further evidence that wild-type Stat1 was an effective SUMO substrate, whereas Stat3, phospho-Stat1, and the mutant peptides were poor substrates (Figure 2C). These data confirmed K703 as the SUMO modification site, but only when the adjacent Y701 was not phosphorylated. The failure to SUMO-ylate the Stat3 peptide was consistent with our inability to modify purified preparations of recombinant Stat39 (data not shown), reflecting a critical divergence in the corresponding potential Stat3 SUMO conjugation site (Table 1). Stat1K703R exhibits enhanced DNA binding activity
To explore the potential role of Stat1 SUMO modification in vivo, a SUMO-ylation–resistant Stat1K703R mutant was generated. Retroviral vectors directed expression of Stat1K703R and wild-type Stat1 at physiologic levels in Stat1–/– MEFs36 in more than 95% of infected cells (data not shown; Figure 3A-B). Upon brief (ie, 0.5 hours) stimulation with IFN-
Analysis of additional Stat1 SUMO-ylation–defective mutant To develop additional evidence that Stat1 was SUMO modified at K703 in vivo, a second critical amino acid in the Stat1 SUMO consensus modification site (ie, E705), which is also not involved in dimerization, was mutated to alanine.6,17,18 Again, retroviral vectors directed a physiologic expression of Stat1K703R, Stat1E705A, and wild-type Stat1 in more than 95% of Stat1–/– MEFs (Figure 3A, top panel). To confirm that both mutants were defective in SUMO modification, HA–SUMO-1 and Ubc9 were overexpressed in these MEFs. Despite modest transfection efficiency in these MEFs (Figure 3A, bottom panel), the studies clearly demonstrate the formation of the approximately 105-kDa SUMO-Stat1 conjugate in cells expressing wild-type Stat1, but not in those cells expressing the SUMO-ylation–defective mutants Stat1K703R and Stat1E705A (Figure 3A, top panel).
Next, the IFN-
However, these modest changes in IFN-
The rapid and transient translocation of Stat1 to the nucleus is another characteristic feature of Stat1 signaling activity. Since several studies have implicated SUMO modification in the regulation of nuclear trafficking,34,37,38 a set of Stat1 immunolocalization studies were carried out. Analogous to the pattern observed with wild-type Stat1, both Stat1K703R and Stat1E705A exhibited a predominately cytoplasmic distribution in unstimulated cells and robust nuclear accumulation with IFN-
Evaluation of the biologic response mediated by Stat1E705A and Stat1K703R in MEFs
Next, several studies were undertaken to explore the potential role of SUMO-ylation in regulating biologic responses directed by Stat1. In the first set of studies, the ability of Stat1–/– MEFs expressing wild-type Stat1, Stat1K703R, or Stat1E705A to direct the IFN-
IFNs stimulate a potent and physiologically important antiviral response, which represents an integrated response of numerous target genes.4,26 To determine whether SUMO-ylation–defective Stat1 mutants direct an abnormal antiviral response to IFN-
Evaluation of the biologic response mediated by Stat1E705A and Stat1K703R in macrophages
Macrophages are an important physiologic target of IFN-
PIAS proteins were initially identified for their capacity to regulate STATs under conditions of overexpression.14 However, recent studies identifying PIAS proteins as SUMO E3 ligase(s) raised the possibility that STATs may be regulated through SUMO modification.19,34,37,38,41,42 When STAT sequences were scanned for the SUMO consensus modification site, only 2 potential sties were identified in Stat1, but not in other STATs (Table 1). Biochemical studies identified lysine 703, adjacent to the activation tyrosine (ie, Y701), as the SUMO conjugation site. However, the inability to recover tyrosine-phosphorylated SUMO-Stat1 conjugates in the HEK-293T cells raised concern over whether a single Stat1 molecule could be simultaneously modified at both Y701 and K703 (Figure 1). Consistent with this, a tyrosine-phosphorylated Stat1 peptide was a poor substrate for in vitro SUMO modification (Figure 2).
To evaluate the potential physiologic significance of SUMO Stat1 conjugation, SUMO-ylation–defective Stat1 mutants were generated. Fortuitously, the 2 most critical residues in the SUMO consensus modification site, K703 and E705, are exposed, suggesting they could be mutated without perturbing Stat1 activity.6,32 Consistent with this, expression of the first mutant, Stat1K703R, in Stat1–/– MEFs revealed a wild-type pattern of rapid IFN-
Upon completing our initial characterization of the Stat1 SUMO-ylation site, 2 other groups reported K703 as a Stat1 SUMO modification site.32,33 Both found that Stat1K703R exhibited relatively modest changes in Stat1K703R-dependent transcriptional activity in primate cells. Subsequently, 1 of these groups confirmed our observation of enhanced IFN-
Although the unusual properties of Stat1K703R could easily be exploited to argue that Stat1 activity is regulated by SUMO conjugation of K703, we believe that our data provide compelling evidence to the contrary. Notably, even under idealized reaction conditions, with purified Stat1, only a modest fraction of Stat1 was SUMO modified (Figure 2). This contrasted the fully penetrant-enhanced DNA binding activity of Stat1K703R, suggesting that this mutation causes a structural perturbation to Stat1 dimer that stabilizes DNA binding. Consistent with this, K703 residue lies in a critical location of the Stat1-Stat1 dimerization interface. More significantly, a second SUMO-ylation–defective Stat1 mutant fails to exhibit this phenotype, providing compelling evidence that the enhanced DNA binding is unrelated to a potential loss in the ability to be SUMO modified. Surprisingly however, the enhanced DNA binding activity of Stat1K703R correlated with relatively meek changes in biologic response, including a modestly enhanced antiviral activity at the lowest doses of IFN-
We would like to thank Dr Jutta Braunstein for purified preparations of recombinant Stat1 and Stat3, and Dr Steve Goff for reagents and advice.
Submitted January 6, 2006; accepted June 27, 2006.
Prepublished online as Blood First Edition Paper, July 20, 2006; DOI 10.1182/blood-2006-04-020271.
Supported by National Institutes of Health (NIH) grants AI05821 and GM65872, the Burroughs Wellcome Fund (APP no. 2010), and the Rita Allen Foundation.
The authors declare no competing financial interests.
L.S. and S.B. contributed equally to this study.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
Reprints: Christian Schindler, Columbia University, HHSC 1208, 701 West 168th St, New York, NY 10032; e-mail: cws4{at}columbia.edu.
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Abstract
Introduction
amino group of the lysine found in the consensus sequence 
KxE (
-actin (Sigma). 
