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(The FASEB Journal. 2008;22:4296-4305.)
© 2008 FASEB

Identification of a nuclear localization signal in suppressor of cytokine signaling 1

Andrea Baetz, Christian Koelsche, Julia Strebovsky, Klaus Heeg and Alexander H. Dalpke1

Department of Hygiene and Medical Microbiology, Hygiene Institute, University of Heidelberg, Heidelberg, Germany

1Correspondence: Department of Hygiene and Medical Microbiology, Hygiene-Institute, University Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany. E-mail: alexander.dalpke{at}med.uni-heidelberg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of janus kinase and signal transducer and activators of transcription signaling pathways. In addition, SOCS1 has been identified to regulate stability of nuclear NF-{kappa}B subunits. However, details about the regulation of the nuclear pool of SOCS1 are unknown. Using different experimental approaches, we observed that SOCS1 but no further SOCS family members localized to the nucleus when expressed in various cell lines. Nuclear transport was confirmed for endogenous SOCS1 in macrophages stimulated with IFN-{gamma}. Sequence analysis revealed a bipartite nuclear localization signal (NLS) located between the src-homology 2 (SH2) domain and the SOCS box of SOCS1. Deletion of this region, introduction of a series of R/A point mutations, or substitution of this sequence with the respective region of SOCS3 resulted in loss of nuclear localization. Fusion of the SOCS1-NLS to cytokine-inducible SH2 region containing protein (CIS) resulted in nuclear localization of this otherwise cytoplasmic protein. SOCS1 mutants with loss of nuclear localization were still effective in suppressing IFN-{alpha}-mediated STAT1 tyrosine phosphorylation. However, they showed decreased inhibition of IFN-{gamma}-mediated induction of CD54. The results identify a hitherto unknown transport of SOCS1 into the nucleus which extends the spectrum of SOCS1 inhibitory activity.—Baetz, A., Koelsche, C., Strebovsky, J., Heeg, K., Dalpke, A. H. Identification of a nuclear localization signal in suppressor of cytokine signaling 1.


Key Words: cell activation • cytokine receptors • inflammation • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SUPPRESSOR OF CYTOKINE SIGNALING (SOCS) proteins constitute a class of inducible negative regulators of cytokines activating janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathways. All 8 proteins (SOCS1 to SOCS7, CIS) belonging to the SOCS family are composed of a central src-homology 2 (SH2) region that mediates binding to tyrosine-phosphorylated residues within cytokine receptor chains or JAKs themselves (1 , 2) . Moreover, SOCS proteins possess a carboxy-terminal SOCS box, which is also found in further protein families (3) . SOCS1 and SOCS3 additionally contain a kinase inhibitory region (KIR) juxtaposed to the SH2 region. SOCS proteins serve as potent inhibitors of type I and II cytokine receptors. For SOCS1 and SOCS3, this is achieved by inhibiting JAK activity, whereas CIS and other SOCS family members seem to act via competition for STAT recruitment. SOCS1 has been suggested to bind directly to JAK2 and to act as a pseudosubstrate (4) . In contrast, SOCS3 has been reported to bind to cytokine receptor chains with high affinity, especially in gp130, and this serves as a platform to gain access to JAKs (5 , 6) . Moreover, it has been shown that the SOCS box binds to elongin BC, Cul, and Rbx proteins, thereby functioning as part of an ECS-type ubiquitin ligase complex (7 8 9 10) . This results in degradation of bound proteins and contributes to termination of signal transduction.

Recently, however, an additional function of SOCS1 has been identified, which required localization to the nucleus. Two independent reports showed that SOCS1 is recruited to NF-{kappa}B subunits thereby, regulating stability via ubiquitination (11 , 12) , and this interaction took place in the nucleus. Indeed, it had already been described before that ubiquitination and proteasomal degradation of promoter-bound p65/RelA is essential for transcriptional termination of NF-{kappa}B signaling, and SOCS1 had been suggested to act as mediator (13) . However, as SOCS1 so far was thought to be expressed in the cytoplasm exclusively, this idea was discarded again. Another recent observation also suggests that SOCS1 might play an additional role in the nucleus: It was reported that SOCS1 promotes degradation of human papilloma virus E7 protein, thereby limiting proliferation of HeLa cells (14) . This interaction was dependent on a functional SOCS box and was suggested to take place in the nucleus.

Support for the hypothesis that SOCS1 might have a broader range of activity than so far anticipated comes from the finding that although SOCS1–/– mice die due to multiorgan inflammation induced by unlimited IFN-{gamma}/STAT1 signaling (15 , 16) , alleviation from the lethal phenotype was also obtained by backcrossing to STAT4–/– or STAT6–/– mice (17 , 18) . In addition, backcrossing of SOCS1–/– mice to IFN-{gamma}–/– mice results in a rescue from acute lethality; however, these mice show a polycystic kidney disease of unknown cause, as well as chronic inflammation (19) . Partial deletion of the SOCS box of SOCS1 results in a much milder phenotype, which indicates that the SOCS-box domain has a significant function of its own (20) .

Thus, evidence is present suggesting that SOCS1 has activities different from its well-characterized function on inhibition of JAK activity and might even be operative in compartments different from plasma membrane. This was the starting point for our experiments. Using different methods, we here show that SOCS1 has a specific NLS that mediates transport of SOCS1 into the cell nucleus, thereby expanding the inhibitory spectrum of SOCS1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and transfection
HEK293, HeLa, and NIH/3T3 cells were maintained in Dulbecco modified Eagle medium (DMEM; Biochrom AG, Berlin, Germany). RAW 264.7 and BEAS-2B cells were cultured in RPMI 1640 (Biochrom AG). All media were further supplemented with 10% (v/v) fetal calf serum (Biowest, Nuaillé, France), 100 U/ml penicillin, and 100 µg/ml streptomycin (PAA Laboratories, Pasching, Austria). Transient transfection of cells was carried out using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany).

Cloning and mutations
Plasmids encoding full-length murine or human SOCS1, SOCS3 (supplied by D. J. Hilton, WEHI, Melbourne, Australia), or CIS (supplied by A. Yoshimura, Kurume, Japan) were used to amplify SOCS sequences by polymerase chain reaction (PCR) using primer pairs with 5'-XhoI and 3'-BamHI (SOCS1 and SOCS3) or 5'-BglII und 3'-SalI sites (CIS). The amplified DNA was digested with the respective enzymes and inserted into pEGFP-C1 and pEGFP-N1 (BD Clontech, Heidelberg, Germany). Point mutations, deletions, and insertions were generated using QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, Netherlands). To insert the NLS of human SOCS1 into pEGFP-C1-hCIS, a SpeI site was generated at position 204 (A/S). At this position, the NLS was inserted using 2 complementary oligonucleotides with SpeI sites. The protein coding regions of all constructs were verified by automated DNA sequencing (MWG, Martinsried, Germany).

Immunofluorescence microscopy
Cells were grown on poly-D-lysine (Sigma-Aldrich, Taufkirchen, Germany) -coated glass coverslips and transfected. After overnight incubation, the cells were fixed, and nuclei were stained with 4',6'-diamidino-2-phenylidole (DAPI)/ethanol (3 µM; Molecular Probes Inc., Eugene, OR, USA). When using flag- and myc-tagged plasmids, cells were fixed with 4% paraformaldehyde (PFA)/PBS for 20 min at room temperature (RT) and permeabilized with ice-cold 90% methanol for 20 min at –20°C. Cells were washed with Tris-buffered saline (TBS) and blocked with 10% (v/v) goat serum (Sigma-Aldrich) in TBS for 45 min at RT. After 4 rinses, cells were incubated with fluorescein isothiocyanate (FITC) -conjugated monoclonal anti-Flag M2 (1:100, Sigma-Aldrich) or polyclonal anti-c-myc (1:1000, Cell Signaling Technology, Frankfurt, Germany) antibodies overnight at 4°C. After one more rinse, the nuclei were stained with DAPI (3 µM in PBS). Glass coverslips were mounted in AF1 (Citifluor Ltd., London, UK) and analyzed using a Leica DMI 6000B microscope with HCX PL APO x63/1.4 objective (Leica Microsystems, Wetzlar, Germany).

Alternatively, live cells were examined and analyzed with a Leica TCS SP5 confocal microscope (Leica Microsystems) containing a 488-nm laser, spectrophotometer prism, tuneable detectors, and an x63 water-corrected objective. Live cell imaging was performed using a POCmini chamber system (PeCon GmbH, Erbach, Germany).

Western blot analysis
HEK293 cells (2x106) transfected with the respective plasmids were lysed with 200 µl of lysis buffer containing 50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM each of EDTA, NaF, Na3VO4, and PMSF; and 1 µg/ml each of aprotinin, pepstatin, and leupeptin. Cleared lysates were analyzed by SDS-PAGE and immunoblotting using anti-green fluorescent protein-horseradish peroxidase (GFP-HRP) (1:500; Santa Cruz Biotechnology, Heidelberg, Germany). Signals were detected using the enhanced chemiluminescence (ECL) system Immobilon Western Chemiluminescence HRP Substrat (Millipore, Schwalbach, Germany).

Cell fractionation
All fractionation and centrifugation steps were performed at 4°C using ice-cold buffers. HEK293 cells (3x106) transfected with the respective plasmids were harvested, washed with PBS, and resuspended in 750 µl of hypotonic lysis buffer [10 mM Tris/HCl, pH 7.5; 10 mM NaCl; 3 mM MgCl2; 1 mM Na3VO4; 5 µg/ml aprotinin and leupeptin; 3 µg/ml pepstatin; 1 mM EDTA; and 1 mM dithiothreitol (DTT)]. Cells were incubated on ice for 30 min and intensively vortexed in between. The nuclei were removed by two 15-min centrifugation steps at 800 g. The supernatant was centrifuged at 12,500 g for 10 min to yield the cytoplasmic fraction. The crude nuclear pellet was resuspended in 750 µl of nuclear isolation buffer (hypotonic lysis buffer from above supplemented with 0.5% Nonidet P-40) and incubated on ice for 5 min, after which nuclei were pelleted by centrifugation at 800 g for 15 min. This procedure was repeated once. The purified nuclei were lysed using 750 µl of 2% Triton-containing nuclear lysis buffer (2% Triton X-100; 20 mM Tris/HCl, pH 7.5; 280 mM NaCl; 10 mM NaF; 1 mM Na3VO4; 5 µg/ml aprotinin and leupeptin; 3 µg/ml pepstatin; 1 mM EDTA; and 1 mM DTT) and the lysate was cleared by centrifugation at 12,500 g for 10 min. The nuclear fraction was solubilized in 300 µl of 1x Laemmli buffer by heating and vigorous mixing for 10 min at 95°C. Cytoplasmic and nuclear fractions were analyzed by Western blotting. The purity was determined using anti-lamin A/C (1:1000, BD Biosciences, Heidelberg, Germany) and anti-GAPDH (1:200; Chemicon International, Ltd., Hampshire, UK) antibodies.

Detection of endogenous SOCS1
RAW264.7 cells (5x107) were stimulated with 100 ng/ml IFN-{gamma} (ImmunoTools, Friesoythe, Germany) for 6 h. Cell fractionation was performed as described above, with the following modifications. Cytoplasm and nuclear extracts were adjusted to 150 mM NaCl prior to the addition of the antibodies for immunoprecipitation. Each extract was incubated with 6 µl anti-SOCS1 monoclonal antibody (2E1/2D4; obtained from D. J. Hilton) for 30 min on ice before 60 µl protein A-agarose (Pierce Biotechnology, Rockford, IL, USA) was added. After 2 h of rotation at 4°C, complexes were pelleted and washed 3 times with the appropriate buffers. Bound proteins were solubilized in 60 µl of 2x Laemmli buffer and analyzed by Western blotting using anti-JAB/SOCS1 antibody (1:50; IBL, Hamburg, Germany).

Bioinformatics
Human and murine cDNA sequences for SOCS1, SOSC3, and CIS were analyzed using PredictNLS algorithm (21) . All available SOCS1 sequences were aligned using ClustalW.

Reporter gene assay
HEK293 cells were seeded into 24-well plates at 2 x 105 cells/well and transfected with the different SOCS1 expression plasmids (0.6 µg) together with an ISRE-luciferase reporter plasmid (0.3 µg; Stratagene) in duplicates. After overnight incubation, the cells were stimulated with 100 ng/ml human IFN-{alpha} for 6 h. The cells were lysed using Luminescence Reporter Gene Assay System (LucLite; PerkinElmer, Rodgau-Jügesheim, Germany) and analyzed. Alternatively, HEK293 cells were analyzed using dual luciferase kit (Promega, Mannheim, Germany). Cells were transfected as above using the various SOCS1 expression plasmids (0.4, 0.05, 0.025, or 0.005 µg), 0.1 µg of pISRE reporter plasmid, and 0.1 µg of pRL-TK (renilla luciferase). After overnight incubation, the cells were stimulated with IFN-{alpha} for 6 h, and both renilla and firefly luciferase activities were measured.

Flow cytometry of CD54 expression
BEAS-2B were seeded into 12-well plates at 2 x 105 cells/well in RPMI 1640 and maintained to achieve 60% confluency. Transfection was carried out by using 2 µl of FugeneHD (Roche, Mannheim, Germany) and 1 µg of different SOCS1 expression plasmids. One day later, BEAS-2B cells were exposed for 30 h to 15 U/ml IFN-{gamma}. Subsequently, cells were stained with allophycocyanin-anti-human CD54 (BD Biosciences) antibody and analyzed using a FACSCanto flow cytometer (BD Biosciences).

STAT1 tyrosine phosphorylation
HEK293 cells (4x105) transfected with the different SOCS1 plasmids were split into 2 equal parts, one of which was stimulated with 100 ng/ml human IFN-{alpha} for 40 min. Cells were fixed with 4% PFA/PBS for 20 min at RT and permeabilized overnight at –20°C with ice-cold 90% methanol. Cells were stained in 100 µl of PBS/2% FCS with 8 µl anti-STAT1 (pY701) Alexa Fluor 647 (BD Biosciences) antibody for 1 h at RT and analyzed. A gate was set on viable GFP+ cells; mean fluorescence intensity (MFI-APC) was measured, and {Delta}MFI = MFIIFN – MFIcontrol was calculated and normalized to the empty vector control (pEGFP-C1).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of SOCS1 results in nuclear localization
To study temporal-spatial aspects of SOCS signaling, we constructed GFP-fusion proteins for SOCS1, SOSC3, and CIS with the GFP-tag positioned either at the amino or the carboxy terminus. Constructs were functional when analyzing their effects on JAK/STAT inhibition (see below). Expression of the fusion proteins revealed that SOCS1 could be found in the nucleus (Fig. 1A ). Nuclear expression was observed for both N- and C-terminal-tagged SOCS1 (data not shown). For SOCS1, we observed a mostly homogenous nuclear staining pattern with spaces resembling the nucleoli. Infrequently, cells displayed a speckled, nuclear SOCS1 expression pattern. Bleaching experiments employing multiple iterations of spot bleaching showed that total homogenous nuclear staining was rapidly lost when parts of the nuclei were bleached, whereas the dot-like structures were stable (Fig. 1B ). This argues for a rapid exchange or movement of GFP-SOCS1 within the nucleus. The dot-like structures do not participate in rapid exchange. Performing fluorescence recovery after photobleaching experiments with a short pulse of bleaching, we observed half-life of recovery to be ~10 s, with nearly complete recovery and only a minor immobile fraction (Fig. 1C ). Increasing the exposure time during image acquisition revealed that SOCS1 was also expressed in low amounts within the cytoplasm (Fig. 1D ). SOCS3 showed a uniform staining within cytoplasm and nuclei. CIS showed homogenous, nearly exclusive cytoplasmic staining. All of the constructs were expressed at the expected size as analyzed by Western blotting (Fig. 1E ). No hints for cleavage or degradation could be observed. In addition, all constructs were expressed in nearly equal quantities, as judged by quantitative RT-PCR and flow cytometry (data not shown). We further examined different cell lines for expression of the fluorescent SOCS fusion proteins. Expression in NIH/3T fibroblasts, RAW264.7 macrophages, and HeLa cells confirmed that SOCS1 was predominantly localized in the nucleus (data not shown). Again, SOCS3 was found in cytoplasm and nucleus, whereas CIS was expressed exclusively in the cytoplasm. To exclude that the nuclear staining pattern was produced by the bulky GFP-tag, we resorted to flag- and myc-tagged proteins (Fig. 1F ). We were able to completely confirm the above-described expression pattern: SOCS1 was found in the nucleus, partly with dot-like structures. Infrequently, perinuclear dots as reported by others (22) were observed. SOCS3 showed uniform staining and CIS was expressed in the cytoplasm.


Figure 1
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  		Figure 1. SOCS1 localizes to the nucleus. A) HEK293 cells were transfected with the indicated human SOCS expression plasmids containing an N-terminal GFP-tag and were visualized by microscopy. B) Live HEK293 cells expressing GFP-tagged SOCS1 were visualized by confocal microscopy. Images were taken immediately before and after bleaching of the indicated region (circle) with multiple bleach iterations of high intensity. C) Fluorescence recovery after photobleaching within a circular region in the nucleus after 3 short bleach pulses (mean+SD of n=5 normalized experiments). D) HEK293 cells expressing GFP-SOCS1 were analyzed as in A; image exposure time was increased. E) HEK293 cells expressing either human GFP-SOCS fusion proteins or GFP alone were analyzed by Western blotting using anti-GFP antibody. F) Flag-tagged murine SOCS1 or SOCS3 and myc-tagged murine CIS were expressed in HEK293 cells. Cells were fixed and stained with the respective FITC-labeled antibodies as well as DAPI to visualize nuclei.

To confirm the localization pattern, we performed biochemical fractionation and Western blotting experiments. We were able to observe that GFP-SOCS1 was prominently expressed in the nucleus (Fig. 2A ), which contrasts the pattern of SOCS3 and CIS. Cytoplasmic and nuclear fractions had high purities as revealed by detection of GAPDH and lamin A/C, respectively.


Figure 2
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  		Figure 2. SOCS1 is detectable in the nuclear fraction. A) Cytoplasmic and nuclear fractions of HEK293 cells transfected with the indicated GFP-SOCS constructs were analyzed by Western blotting using anti-GFP, anti-GAPDH, or anti-lamin A/C antibodies. B) RAW264.7 cells were stimulated with murine IFN-{gamma}. Cell fractionation and immunoprecipitation for SOCS1 protein were performed. The accumulated murine SOCS1 protein was detected by Western blotting.

Endogenous SOCS1 is found in the nucleus
We next tried to analyze the localization of endogenous SOCS1, although it is known that SOCS1 is expressed at low levels and is relatively short-lived (23) . From previous experiments we knew that RAW264.7 macrophages stimulated with IFN-{gamma} increased SOCS1 transcription rapidly (24) . Cells stimulated in this way were used for biochemical fractionation and immunoprecipitation. Using different antibodies we were able to detect a faint, yet specific band of the expected size, indicating the presence of endogenous SOCS1 protein in the nucleus (Fig. 2B ). These results thus confirm the data obtained by microscopy. None of the available SOCS1 antibodies was of sufficient quality to detect endogenous SOCS1 by immunofluorescence.

Identification of a putative NLS in SOCS1
As only SOCS1 showed a prominent nuclear localization, we hypothesized that a so-far unknown specific NLS might be present in SOCS1. Using the PredictNLS algorithm (21) and employing human as well as murine sequences of SOCS1, SOSC3, and CIS, we identified a putative NLS in SOCS1 that fit to a known consensus sequence R[MNQ]X(4 ,8) R[MNQ]RR, which should mediate localization to the nucleus. This putative NLS was located at the SH2/SOCS-box border from aa 159 to aa 172 (Fig. 3A ). The sequence resembled a bipartite NLS composed of 2 basic stretches. Neither SOCS3 nor CIS contained this consensus motif, which paralleled our observation of specific nuclear expression of SOCS1. Alignment of published SOCS1 sequences revealed that this bipartite signal was evolutionary conserved. Moreover, there were additional basic amino acids slightly more carboxy-terminal (Fig. 3B ).


Figure 3
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  		Figure 3. Identification of a putative NLS in SOCS1. A) Schematic drawing of SOCS1 showing the KIR, SH2, and SOCS-box domain as well as the putative NLS. Bottom: alignment of human SOCS1, SOCS3, and CIS with the identified putative NLS, marked in red. The two basic amino acid stretches from SOCS1 are boxed. B) Alignment of SOCS1 sequences of the indicated species. The putative NLS is shown in red; basic amino acid residues within the NLS are blue. Arginine residues within a third basic region are light orange.

Mutation of the putative NLS diminishes nuclear expression of SOCS1
We next performed an extensive mutational analysis of the putative NLS and analyzed localization of the newly generated constructs by transfection in HEK293 cells (Fig. 4A ). In addition, we quantitatively classified the expression pattern according to whether nuclear or cytoplasmic expression was observed (Fig. 4B ). All constructs were verified by automated sequencing. Equal and correct expression was controlled by Western blotting (data not shown).


Figure 4
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  		Figure 4. NLS mutation abrogates nuclear expression of SOCS1. A)The indicated GFP-SOCS1 constructs were expressed in HEK293 cells and visualized by microscopy. Nuclei are counterstained with DAPI. A schematic drawing of each mutant is shown (dark gray, KIR region; light gray, SH2 domain; hatched, NLS; black, SOCS box). B) At least 100 different cells from A were judged for the pattern of expression. C) Human GFP-CIS as well as CIS with the inserted NLS from SOCS1 was expressed in HEK293 cells.

Mutating essential amino acids within the KIR domain (F58A) or the SH2 region (R104K) of human SOCS1, which have been reported to impede the function of SOCS1, did not alter the cellular expression pattern: As for the wild-type construct, both mutants showed a predominant nuclear localization. To analyze the role of the putative NLS we first introduced a set of carboxy-terminal deletions, which covered the NLS sequence to differing degrees. Deletion of the entire SOCS box (R172X), which did not alter the putative NLS still resulted in predominant nuclear localization of SOCS1. However, the cells showed a slightly increased staining of the cytoplasm. Increasing the deletion by introducing a stop codon in the center of the supposed NLS (P165X) resulted in a more uniform expression within the entire cell. Quantification confirmed that a significant number of cells showed cytoplasmic and nuclear localization. Deletion of the entire NLS (Y154X) showed complete loss of nuclear expression. However, this construct showed expression of a bright cytoplasmic spot and we were unsure whether the protein was expressed properly (data not shown). Therefore, another mutant (E152X) was generated, which also showed a strong decrease of nuclear localization.

We decided to perform a second approach by introducing point mutations within the NLS. We replaced increasing numbers of arginine residues by alanine (R/A). We observed that mutating 1 to 3 arginine residues (1R/A, 3R/A) did not alter the localization significantly. However, when we mutated 5 (5R/A) (data not shown) or 6 (6R/A) of the respective arginine residues, we observed a complete reversal of the expression pattern. These constructs showed predominant if not exclusive cytoplasmic localization. Expression in the cytoplasm was uniform with some vesicular structures cut out. Sometimes accumulation within a perinuclear spot was observed.

In a third approach, we specifically deleted the putative NLS (hS1/Del) or substituted it with the counterpart of SOCS3 (hS1/Del+hS3). In both cases, localization was predominantly cytoplasmic and resembled that of the 6R/A mutant.

SOCS1-NLS transfers nuclear expression to CIS
To prove that the sequence identified above functions as a nuclear localization signal, we introduced the NLS of human SOCS1 between the SH2 domain and the SOCS box in otherwise cytoplasmic CIS (hCIS/NLS) (Fig. 4B, C ). Introducing this mutation resulted in an impressive reversal of localization pattern of CIS. While CIS was mainly expressed in the cytoplasm, the CIS construct with the human SOCS1-NLS was expressed predominantly in the nucleus. The nuclear expression was only slightly weaker than that of wild-type SOCS1, the nuclear pattern was similar.

NLS-mutants of SOCS1 are not impeded in inhibiting IFN-mediated STAT1 tyrosine phosphorylation
We speculated that inhibition of nuclear localization of SOCS1 should not affect the ability of the various SOCS1 mutants to inhibit STAT1 tyrosine phosphorylation, as this is known to occur at the level of the receptor/janus kinase complex. Therefore, we first stimulated HEK293 cells that were transfected with the various mutant constructs with IFN-{alpha} and analyzed tyrosine phosphorylation of STAT1 (Fig. 5A ). Using a flow cytometry-based approach, we could differentially analyze STAT1 activity in transfected (GFP+) and nontransfected (GFP–) cells, the latter serving as internal control. Wild-type SOCS1 led to a complete inhibition of tyrosine phosphorylation. Mutation of the KIR region (F58A) led to loss of inhibitory activity. Mutating the SH2 domain (R104K mutant) resulted in 40–50% reduction of the STAT1 inhibitory capacity. Deleting the SOCS box (R172X mutant) did not have any effects on the inhibitory activity. Thus, in these readout systems, the function of SOCS1 as part of an E3 ubiquitin ligase that is mediated by the SOCS-box domain is not necessary for STAT1 inhibition. Among the constructs with altered nuclear expression, the mutants 6R/A and hS1/Del+hS3 still efficiently inhibited STAT1 tyrosine phosphorylation even when analyzing different time points (10, 30, 60, 120 min, data not shown). Interestingly, deletion of the entire NLS without substitution by the homologous SOCS3 region (hS1/Del) abolished the inhibitory capacity.


Figure 5
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  		Figure 5. NLS mutants are not impeded in inhibition of STAT1 tyrosine phosphorylation but show decreased inhibition of IFN-mediated gene induction. A) HEK293 cells were transfected with the indicated mutants of GFP-SOCS1. After overnight incubation, cells were stimulated with IFN-{alpha} for 45 min and analyzed by flow cytometry. Mean fluorescence intensity of pY-STAT1-Alexa 647 within GFP+ cells was determined (n≥3 experiments, mean+SD). B) HEK293 cells were transfected with differing amounts of the various GFP-SOCS1 constructs and analyzed by dual luciferase assay for reporter gene activity (n=3, mean+SD). C, D) CD54 expression on BEAS-2B cells, transfected with various GFP-SOCS1 mutants and stimulated with IFN-{gamma} for 30h, was measured by flow cytometry. C) Selected dot plots are displayed. Horizontal bars indicate mean fluorescence intensity of CD54 expression in GFP– and GFP+ cells; the box exemplifies the gating strategy for quantitative analysis of n = 3 experiments (mean+SD) in D.

Changing localization of SOCS1 alters inhibition of IFN-mediated reporter gene activity
Next, we hypothesized that mutants of SOCS1 with altered nuclear localization might contribute to the overall capacity of SOCS1 to inhibit IFN-induced gene induction by acting downstream in the signaling cascade. We analyzed the mutants with altered nuclear localization for inhibition of reporter gene activity using an ISRE-promoter (Fig. 5B ) and different amounts of plasmid to examine possible shifts in dose-response curves. It turned out that both mutants with loss of nuclear expression (6R/A, hS1/Del+hS3) were severely less inhibitory than wild-type SOCS1.

Changing localization of SOCS1 alters inhibition of IFN-{gamma}-mediated CD54 regulation
To further analyze the need of nuclear localization for complete inhibition of IFN activity and to substantiate our hitherto existing findings, we resorted to the analysis of IFN-{gamma}-mediated modulation of CD54 expression in human bronchial epithelial cells. BEAS-2B cells were transfected with the GFP-tagged mutants and analyzed by flow cytometry for CD54 expression. This approach allows for the parallel analysis of SOCS-expressing and control cells in one sample (Fig. 5C, D ). Prior to activation by IFN-{gamma}, expression of CD54 was similar in all experimental conditions (control vs. transfected wild-type or mutant constructs). On IFN-{gamma} activation cells increased CD54 expression. This increase was completely abolished in wild-type SOCS1-expressing cells. In contrast, 6R/A and hS1/Del+hS3 mutants failed to inhibit IFN-{gamma}-induced CD54 increase. Thus, the results indicate that nuclear localization does play a role for full inhibition of IFN-mediated JAK/STAT signaling and gene induction.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We here report for the first time the identification of a specific nuclear localization signal in SOCS1 located between the SH2 region and the SOCS box, which accounts for the nuclear transport of SOCS1. Furthermore, we confirm and extend earlier reports that suggested a localization of SOCS1 within the nucleus (11 , 12 , 25 , 26) . Incidentally, it has been observed by other groups that SOCS1 appears to be expressed in the nucleus but to date no mechanisms have been identified. It was reported that SOCS1 localizes to the microtubule organizing center as well as to the 20S proteasome (22) , but a closer look at the published data also reveals a prominent nuclear expression. In our hands, we rarely observed a similar localization pattern with a strong cytoplasmic dot; mostly, the expression was restricted to the nucleus. Yoshimura’s group (14) also observed nuclear localization of SOCS1 when they overexpressed SOCS1 in HeLa cells. Moreover, nuclear localization of SOCS1, but not SOCS2, SOCS3, or CIS was also found in other reports (25 , 27 , 28) ; however, no underlying mechanisms have been identified to date.

The following points regarding our findings are noteworthy: predominant nuclear localization did only occur for SOCS1, but not for SOCS3 or CIS, which are highly homologous and of nearly equal size (1 , 2) ; we observed the same expression pattern in a variety of different human and murine cell lines, and confirmed the findings using different tags (flag, myc), biochemical fractionation, and detection of endogenous nuclear SOCS1. We have now identified a specific nuclear localization signal using the ‘PredictNLS’ algorithm (21) , thereby providing the mechanistic basics for nuclear SOCS1 activity. All proteins expressing the consensus sequence R[MNQ]X(4 ,8) R[MNQ]RR were reported to be expressed in the nucleus, and no non-nuclear proteins with this sequence were found. The NLS was evolutionary conserved for SOCS1 (although modified in fish SOCS1 sequences) and was not found in SOCS3 or CIS. It resembled a bipartite NLS motif (29) characterized by two separated clusters of basic residues, the latter having a known role in binding to importins (30) . Mutation of the NLS by deletion, disruption of the basic residues, or substitution by the corresponding SOCS3 sequence resulted in loss of nuclear localization. Moreover, the SOCS1-NLS transferred nuclear expression to otherwise cytoplasmic CIS. Both facts clearly identify this motif as a functional NLS (31) . Therefore, nuclear localization of SOCS1 is specific property of this protein and does not occur only if increased cytoplasmic levels are available, as suggested recently (26) . In fact, we observed the predominant localization to the nucleus only for the NLS-harboring SOCS1, but not for SOCS3 and CIS, although all proteins were expressed in equal quantities. However, the ratio of nuclear vs. cytoplasmic localization was lower for endogenous wild-type SOCS1 as compared to overexpressed GFP-SOCS1.

The NLS identified here locates between the SH2 region and the SOCS box. No protein structure for SOCS1 has been reported, but for SOCS2/elongin BC and SOCS4 it has been shown that this region is unstructured and seems to be well-accessible from the outside (9 , 32) . Moreover, this region serves as spacer that allows for juxtaposition of the extended SH2 region and the SOCS box. Deletion of the NLS sequence (hS1/Del) resulted in a complete loss of function which confirms that this specific region indeed serves an important structural need.

How does nuclear expression of SOCS1 fit to the well-described function of inhibiting proximal JAK/STAT signaling? First, SOCS1 was not completely found in the nucleus, as microscopy also revealed a faint cytoplasmic staining pattern, and endogenous SOCS1 could be clearly detected in the cytoplasmic fraction. Together with the observation that endogenous SOCS1 protein occur at low levels (23) , it might well be that quite low amounts of proteins are sufficient to mediate effects at the level of cytokine receptors and JAKs. Indeed, we observed that inhibition of tyrosine phosphorylation of STAT1 was not different for wild-type and mutant SOCS1. This observation also shows that the generated mutants were still functional and mutating the respective amino acids did not interfere with overall protein activity.

However, full inhibition of IFN-{alpha} reporter gene activity and IFN-{gamma}-mediated CD54 expression required nuclear localization. One explanation for this requirement might be found in reports showing that IFNGR1 and IFNAR receptor chains can be cleaved (33) and translocated (34) to the nucleus. This novel kind of signal transduction contributes to IFN-{gamma} and type I IFN transcriptional activity (35) . It could be that in this setting nuclear SOCS1 plays a role and is responsible for inhibition of STAT1/2-dependent transcriptional activity. In this context, SOCS1 by means of its SOCS box could act as a E3 ubiquitin ligase that regulates stability of nuclear transcription factors. Indeed, it has been shown that ubiquitination of STAT1 contributes to signal termination (36) . It is also well known that IFN-{gamma} activates additional signaling pathways apart from JAK/STAT, including MAP kinases and PI3 kinase (37) as well as NF{kappa}B (38) , and these pathways might be sensitive to nuclear SOCS1 action. Indeed, SOCS1 has been reported to decrease stability of nuclear NF{kappa}B p65 (11) ; NF{kappa}B cooperates with STAT1 in the induction of CD54 (39) , which here was shown to be inhibited by nuclear SOCS1. Analyzing the whole IFN transcriptome in the presence of wild-type or mutant (6R/A) SOCS1 will reveal whether nuclear SOCS1 has effects in this setting.

Further functions of nuclear SOCS1 include degradation of human papilloma virus protein E7 within the cervix cancer cell line HeLa on IFN-{gamma} application, resulting in loss of proliferation. This effect was due to SOCS1 which functions as E3-ubiquitin ligase. It has been proposed that SOCS1 might be responsible for nuclear tagging and subsequent export and cytoplasmic degradation of E7 (14) . Indeed, this might fit to our observations of nuclear expression of SOCS1 as well as nuclear function distinct of JAK/STAT inhibition.

Furthermore, it was reported that NF{kappa}B p65 stability is regulated by proteasomal degradation and it was found by immunoprecipitation that SOCS1 interacts with p65 (12) . A more recent report then identified binding of SOCS1 to p65 by means of the protein COMMD1, and this interaction occurred in the nucleus (11) . We now provide the underlying structural features in SOCS1 that are responsible for nuclear transport. Identification of a specific NLS in SOCS1 that mediates nuclear import supports the anticipated role of SOCS1 for termination of NF-{kappa}B signaling (13) .

It seems probable that a potential nuclear function of SOCS1 is linked to the ubiquitin ligase function of SOCS1. Indeed, the SOCS box plays a role of its own as shown by the fact that knockout mice with a selective deletion of the SOCS1 box show a specific and hitherto not fully understood phenotype (20) .

Our results show that SOCS1 localizes to the nucleus and that this is due to the presence of a specific NLS. Altering the localization by mutating the NLS affected the capacity of SOCS1 to inhibit gene induction by IFN-{alpha} and IFN-{gamma}. The results suggest that the spectrum of inhibitory activity of SOCS1 is broader than initially anticipated with a specific function also within the cell nucleus.


   ACKNOWLEDGMENTS
 
This work was supported by grants of the German Research Foundation to A.D. (Da592/2, -3).

Received for publication June 30, 2008. Accepted for publication July 31, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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