MOLECULAR AND CELLULAR BIOLOGY, Oct. 2005, p. 9115–9126
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 20
SOCS2 Can Enhance Interleukin-2 (IL-2) and IL-3 Signaling by
Accelerating SOCS3 Degradation
Gillian M. Tannahill,1† Joanne Elliott,1† Anna C. Barry,1Linda Hibbert,2Nicolas A. Cacalano,3
and James A. Johnston1*
Infection and Immunity Group, Centre for Cancer Research and Cell Biology, Queens University, 97 Lisburn Rd., Belfast BT9 7BL,
United Kingdom1; DNAX Research Institute, 901 California Avenue, Palo Alto, California 943042; and Department of
Radiation Oncology, UCLA Center for Health Sciences, Los Angeles, California 900953
Received 1 March 2005/Returned for modification 13 April 2005/Accepted 18 July 2005
Cytokine responses can be regulated by a family of proteins termed suppressors of cytokine signaling
(SOCS) which can inhibit the JAK/STAT pathway in a classical negative-feedback manner. While the SOCS
are thought to target signaling intermediates for degradation, relatively little is known about how their
turnover is regulated. Unlike other SOCS family members, we find that SOCS2 can enhance interleukin-2
(IL-2)- and IL-3-induced STAT phosphorylation following and potentiate proliferation in response to cytokine
stimulation. As a clear mechanism for these effects, we demonstrate that expression of SOCS2 results in
marked proteasome-dependent reduction of SOCS3 and SOCS1 protein expression. Furthermore, we provide
evidence that this degradation is dependent on the presence of an intact SOCS box and that the loss of SOCS3
is enhanced by coexpression of elongin B/C. This suggests that SOCS2 can bind to SOCS3 and elongin B/C to
form an E3 ligase complex resulting in the degradation of SOCS3. Therefore, SOCS2 can enhance cytokine
responses by accelerating proteasome-dependent turnover of SOCS3, suggesting a mechanism for the gigan-
tism observed in SOCS2 transgenic mice.
Cytokines such as interleukin-2 (IL-2) regulate the immune
response via interaction with cell surface receptors on target
cells. These receptors interact with cytoplasmic tyrosine ki-
nases, specifically, members of the Janus kinase (JAK) family,
which subsequently phosphorylate signal transducer and acti-
vator of transcription (STAT) proteins. Phosphorylation of
STATs results in their dimerization and translocation to the
nucleus and subsequent transcriptional activation of genes im-
portant for proliferation and differentiation (11). Inhibition of
these signaling pathways is crucial for the control of the in-
flammatory response (16). The suppressors of cytokine signal-
ing (SOCS/SSI/CIS) are thought to play a key role in this
process and are upregulated by and inhibit the JAK/STAT
pathway in a classic negative-feedback manner (7, 32, 39).
Eight SOCS family proteins have been described, CIS (cyto-
kine-inducible SH2 domain-containing protein) and SOCS1 to
SOCS7 (10, 22, 29). These proteins are characterized by two
common structural motifs, an SH2 domain and a C-terminal
SOCS box. The SOCS box is thought to interact with elongin
B/C, part of an E3 ubiquitin ligase complex that targets asso-
ciated proteins for degradation through the ubiquitin pathway
(27). As well as SOCS, a number of other protein subfamilies
including the von Hippel-Lindau (VHL) tumor suppressor
protein contain this SOCS box motif, indicating that it may
have an important and conserved role (17). The SOCS box of
VHL associates with an E3 ligase complex and induces the
proteasomal degradation of hypoxia-inducible factor 1? (40).
More recently, Asb, an adipocyte-specific ankyrin and SOCS
box-containing protein, has been shown to interact with the
adaptor protein APS (adapter protein with PH and SH2 do-
main) to enable recruitment of elongin B/C to the insulin
SOCS1 and SOCS3 are induced rapidly by a range of cyto-
kines including prolactin (PRL), growth hormone (GH), leu-
kemia inhibitory factor, IL-2, and IL-6 and act to negatively
regulate the strength and duration of cytokine responsiveness
(6, 45). SOCS1 and SOCS3 achieve this by binding to phos-
phorylated JAKs within the activation loop, thereby blocking
kinase activity (44). SOCS1 is also thought to target JAK2 for
proteasomal degradation by recruiting an elongin-containing
Skp-cullin-F-box-like (SCF) E3 ligase complex to the phos-
phorylated JAK (41), a degradative mechanism similar to that
reported for the TEL-JAK2 oncogene (translocation erythro-
blast transform leukemia-JAK2 fusion protein) (9, 23).
SOCS2 can inhibit GH and PRL signaling when expressed at
very low levels. However, higher concentrations of SOCS2
restored responsiveness to these growth factors, perhaps by
antagonizing the inhibitory effect of SOCS1 (5, 8, 34). This
dual role of SOCS2 is reinforced by the observation that both
knockout (31) and transgenic (12) SOCS2 mice displayed in-
creased organ mass and excess weight gain and are therefore
substantially (30%) larger than their wild-type littermates.
These observations suggest that SOCS2 may have a dual sup-
pressive and stimulating effect depending on its concentration
in the cell (8, 12). Although the biochemical mechanism by
which SOCS2 suppresses GH signaling remains unknown, the
fact that SOCS2?/?mice are phenotypically similar to GH and
IGF-1 transgenic mice supports the idea that SOCS2 may act
to negatively regulate growth-promoting cytokines (18, 28, 34,
* Corresponding author. Mailing address: Centre for Cancer Re-
search and Cell Biology, 2nd floor, Whitla Medical Building, 97 Lis-
burn Rd., Belfast BT9 7BL, Northern Ireland. Phone: 02890272260.
Fax: 02890325839. E-mail: firstname.lastname@example.org.
† G.M.T. and J.E. contributed equally to this work.
Here, we investigate the ability of SOCS2 to enhance cyto-
kine signaling and focus specifically on the effect of SOCS2 on
the expression on other SOCS proteins. We show that SOCS2
can enhance IL-2- and IL-3-induced STAT phosphorylation
and that while both SOCS2 and SOCS3 are induced following
IL-2 treatment, SOCS3 was induced earlier and disappeared
when SOCS2 was present. Expression of SOCS2 markedly
reduced SOCS3 protein levels in response to cytokines, while
SOCS3 mRNA remained unaffected. Therefore, SOCS2 can
regulate SOCS3 protein expression in a proteasome-depen-
dent manner and thus potentiate signaling by inducing degra-
dation of other SOCS proteins.
MATERIALS AND METHODS
Antibodies. Polyclonal rabbit anti-SOCS2 and -SOCS3 antibodies were sup-
plied by Fusion Antibodies (Belfast, United Kingdom). FLAG M2 monoclonal
antibody was purchased from Sigma Aldrich (Poole, United Kingdom). Anti-
phosphotyrosine clone 4G10 was purchased from Upstate Biotechnology Inc.
(Lake Placid, NY). Polyclonal rabbit anti-STAT5b was a generous gift of J. J.
O’Shea (NIH, Bethesda, MD). Polyclonal anti-phospho-STAT3 was purchased
from New England Biolabs (Hertfordshire, United Kingdom), and polyclonal
anti-STAT3 was purchase from Santa Cruz Biotechnologies (Santa Cruz, CA).
Monoclonal anti-phospho-extracellular signal-regulated kinase (ERK) and anti-
ERK were purchased from Cell Signaling (Beverley, MA), and polyclonal anti-
IL-2R? was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mono-
clonal anti-Myc and anti-His were purchased from Sigma Aldrich (Dorset,
Quantitative real-time PCRs. Quantitative PCR analysis was performed using
the TaqMan Universal PCR Master Mix kit (Applied Biosystems, Foster City,
CA). The probes were labeled with a reporter fluorescent dye, VIC, at the 5? end
and with a quencher fluorescent dye, 6-carboxytetramethylrhodamine, at the 3?
end. The resulting relative increase in reporter fluorescent dye emission was
monitored in real time during PCR amplification using a sequence detection
system (ABI Prism 7900 HT Sequence Detection; Applied Biosystems, Foster
City, CA). Actin was used as an internal standard. The relative expression of
target mRNA was computed from the target cycle threshold values and the actin
cycle threshold value using the standard curve method (User Bulletin 2; Applied
Biosystems, Foster City, CA). The following TaqMan forward (F) and reverse
(R) primers and probes (P) were used (the nucleotide position for each open
reading frame is given in parentheses: for SOCS2, (99)GGCGCGTCTGGCGA
(F), (166)TAACAGTCATACTTCCCCAGTACCAT (R), and (118)CCCTGC
GGGAGCTCGGTCAGA (P); for CIS, ATCTGCTGTGCATAGCCAAGAC
(F), CGTAATGGAACCCCAATACCA (R), and AGCTCCCTTGTGTCCGT
TTCCTGCC (P); for SOCS1, (448)GCGACTACCTGAGCTCCTTCC (F),
(682)AACACGGCATCCCAGTTAATG (R), and (625)TCCAGATTTGACCG
GCAGCGC (P); for SOCS3, (75)CAGCTCCAAGAGCGAGTACCA (F), (139)
AGAAGCCGCTCTCCTGCAG (R), and (118)TGCGCACTGCGTTCACC
RT-PCR analysis. RNA samples were extracted from 10 ? 106Ba/F3-SOCS2
cells using Stat60 reagent (Tel-Test Inc., Friendswood, Texas). Reverse tran-
scription-PCR (RT-PCR) was carried out using the One Step RT-PCR system
(QIAGEN, Crawley, United Kingdom). The forward and reverse primers used to
amplify mouse cDNA are as follows: for CIS, (200)CTGGACTCTTAACTGCT
TGTC (F) and (576)TAGGCAGCACCGAGTCAC (R); for SOCS1, (328)AA
CTGCTTTTTCGCCCTTAGC (F) and (390)AAAGTGCACGCGGATGCT
(R); for SOCS2, (265)TCAGCTGGACCGACTAATCT (F) and (402)CAGGT
GAACAGTCCCATTCC (R); for SOCS3, (77)GCTCCAAAAGCGAGTA
CCAG (F) and (286)GGATGCGTAGGTTCTTGGTC (R); for GAPDH (glyc-
eraldehyde-3-phosphate dehydrogenase), (58)CCACATCGCTCAGACACCAT
(F) and (115)TGACCAGGCGCCCAAT (R); for Pim-1 (6536)CCCGAGCTA
TTGAAGTCTGA (F) and (6900)CTGTGCAGATGGATCTCAGA (R); for
BCL-xL, (26)TGGTCGACTTTCTCTCCTAC (F) and (580)GAGATCCACAA
AAGTGTCCC (R); for OSM, (99)GCTGCTCCAACTCTTCCTC (F) and (725)
GACCCAGATTCTGCGGGTTC (R); for ?-actin, (663)CATCACTATTGGC
AACGAGC (F) and (1067)ACGCAGCTCAGTAACAGTCC (R).
Constructs. SOCS2 and SOCS3 cDNAs were tagged with the FLAG epitope
at their C termini by standard PCR-based methods. Each cDNA was amplified
by PCR using a 5? oligonucleotide containing an EcoRI site, an ATG codon, and
a 3? oligonucleotide containing a ClaI restriction site. The EcoRI/ClaI PCR
fragment was subcloned between the EcoRI and ClaI sites of a modified
pME18S vector in frame with the FLAG epitope. The pUHD plasmids express-
ing SOCS2 and SOCS3 were constructed by subcloning the human SOCS2 or
SOCS3 FLAG cDNA. SOCS2 dSB, in which the consensus elongin B/C binding
region was disrupted, contained the following mutations: L163A, C167Q, and
I171Q. This construct was made using the QuikChange Multi site-directed mu-
tagenesis kit (Stratagene, CA). Plasmids expressing elongin B and C were a kind
gift from D. Hilton (WEHI, Melbourne, Australia).
Cells and transfections. Peripheral blood mononuclear cells (PBMCs) were
purified by Ficoll density gradient centrifugation from consenting healthy do-
nors. Isolated PBMCs were plated in 100 ml of RPMI growth medium containing
10% fetal calf serum (FCS) in 175-cm2flasks for 90 min to allow contaminating
monocyte/macrophage to adhere. T lymphocytes were removed by pipetting
RPMI growth medium over the base of the flask five times. The cells were
activated for 72 h in 100 ml of 10% FCS RPMI growth medium (Gibco-BRL)
supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin,
and 1.25 ?g/ml phytohemagglutinin (PHA) (mitogenic stimulus that triggers
synthesis of T lymphocytes) at 37°C in 5% CO2and 100% humidity. Prior to
stimulation with cytokine, the cells were washed and incubated for 12 h in 2%
FCS RPMI medium at 37°C in 5% CO2and 100% humidity.
The IL-3-dependent pro-B-cell line Ba/F3 cells expressing the TA transacti-
vation repressor (Ba/F3TA) were maintained in RPMI medium supplemented
with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 200
?g/ml puromycin, and 10 U/ml IL-3. SOCS2 and SOCS3 stable transfectants
were generated by electroporation of Ba/F3TA (5 ? 106cells/ml) with
pUHD10-3 containing the relevant SOCS using a Bio-Rad GenePulser (260 V,
960 mF) and selected using 1.25 ?g/ml hygromycin. Cells were maintained in 4
?g/ml tetracycline and were removed from tetracycline 48 h prior to stimulation
to allow gene expression. Transfections were performed in 293T cells grown to
60% confluence on p90 tissue culture plates in 5 ml of Dulbecco’s modified
Eagle’s medium (Gibco-BRL) with 10% FCS using Fugene (Lewes, United
Kingdom) transfection reagents according to the manufacturer’s instructions.
The 293T cells were transfected with 1 to 5 ?g of either wild-type (WT) or SOCS
box deletion plasmids in the presence of 1 ?g of SOCS3 plasmid. After 24 h, the
transfection medium was replaced with fresh medium. Cells were harvested 24 h
later as outlined above.
Immunoprecipitation and immunoblotting. Ba/F3 cells were maintained in
RPMI medium supplemented with 5% FCS and 10 U/ml IL-3. Thereafter, the
cells were collected, washed twice, and resuspended in RPMI medium containing
2% FCS. After 12 h of incubation, the cells were stimulated with 100 U/ml of
IL-3 for different periods. Following treatment, cells were washed in 1? phos-
phate-buffered saline (PBS). If phosphorylation of the protein was being as-
sessed, the 1? PBS was supplemented with Na3VO4(0.2 mM) and lysed by
adding a buffer composed of 0.875% (vol/vol) Brij 97, 50 mM Tris, pH 7.4, 150
mM NaCl, 5 mM EDTA, 1 mM Na3V04, 10 ?g/ml leupeptin, 10 ?g/ml aprotinin,
and 1 mM phenylmethylsulfonyl fluoride. After 15 min, cell lysates were clarified
by centrifugation (13,400 ? g), and the supernatants were subjected to immu-
noprecipitation. This was performed by exposure to either SOCS3 polyclonal
antibody (1 ?g) or STAT5b polyclonal antibody (1 ?g) that was precoupled to
protein A-Sepharose beads. After 1.5 h of incubation, the beads were subse-
quently collected by centrifugation and washed three times in the lysis buffer.
The beads were then resuspended in Laemmli sample buffer containing 5%
?-mercaptoethanol and boiled under reducing conditions for 5 min. For immu-
noblot analysis, the proteins were subjected to electrophoresis by 7.5% or 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to polyscreen polyvinylidene difluoride transfer membranes. The
membranes were blocked in PBS supplemented with 0.2% Tween 20 and either
3% bovine serum albumin or 5% Marvel milk and then incubated for 1 h (or
overnight at 4°C) with a primary antibody (1:500 dilution of the anti-SOCS2
antibody, 1:1,000 dilution of anti-SOCS3, 1:1,000 dilution of anti-STAT3, 1:1,000
dilution of anti-ERK1/2, 1:1,000 dilution of anti-Myc, 1:1,000 dilution of anti-
STAT5b, 1:5,000 dilution of the antiphosphotyrosine antibody, 1:2,000 dilution
of anti-FLAG and phospho-STAT3 antibodies, and 1:3,000 dilution of anti-His).
Thereafter, the membranes were washed three times for 15 min in PBS supple-
mented with 0.2% Tween and were subsequently incubated for 1 h with perox-
idase-conjugated anti-mouse or anti-rabbit immunoglobulin G (1:10,000) in PBS
supplemented with 0.2% Tween 20 and 3% bovine serum albumin or 5% milk.
The blots were extensively washed, and antibody binding was visualized by
enhanced chemiluminescence (ECL; Amersham Biosciences, Chalfont, St. Giles,
Interaction between SOCS2 and SOCS3. For fusion protein pull-down exper-
iments, 293T cells were transfected as described above. Cells were lysed in buffer
containing 50 mM Tris-HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl,
9116TANNAHILL ET AL.MOL. CELL. BIOL.
1 mM EDTA, 1 mM Na3V04, 10 ?g/ml leupeptin, 10 ?g/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride. Lysates were centrifuged at 13,400 ? g for 10
minutes at 4°C, and supernatants were incubated with 3 ?g of His-tagged SOCS2
fusion protein (Fusion Antibodies, Belfast, United Kingdom) which had been
precoupled to 50 ?l of 20% nickel-nitrilotriacetic acid beads (Promega, Madison,
WI). Reaction mixtures were incubated for 2 to 4 h at 4°C in protein interaction
buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole), and precipitates
were washed with protein interaction buffer. Beads were resuspended in Laem-
mli sample buffer containing 5% ?-mercaptoethanol and analyzed by SDS-
PAGE as described above.
The endogenous association between SOCS2 and SOCS3 was analyzed in
Ba/F3-SOCS2 cells grown in the presence and absence of tetracycline for 48 h.
Cells were incubated overnight in 2% FCS and then stimulated with 100 U/ml
IL-3. Proteasome inhibitors MG132 (0.5 ?M) and LLnL (0.5 ?M) were added
1 h before lysing. Cell lysates were immunoblotted with anti-FLAG or immuno-
precipitated and immunoblotted with a SOCS3 antibody or anti-FLAG as de-
Trypan blue exclusion assay. Ba/F3-SOCS2 cells were seeded at a density of
1 ? 105cells per ml and grown in RPMI medium containing 5% FCS and 10
U/ml IL-3 in the presence or absence of tetracycline (4 ?g/ml). Every 12 h, 100-?l
aliquots of cells were incubated at a 50:50 ratio with 0.1% trypan blue for 5 min,
after which viable cells (able to exclude trypan blue) were counted using an
hematocytometer (Invitrogen, Paisley, United Kingdom). Cell counts were car-
ried out in triplicate.
MTT assay. Ba/F3-SOCS2 cells were seeded at a density of 1 ? 105cells per
ml and grown in a 96-well plate in RPMI medium supplemented with 5% FCS
and 10 ng/ml IL-3 in the presence or absence of tetracycline (4 ?g/ml). After 24 h
and 48 h, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT;
0.5 mg/ml) was added to a 100-?l cell aliquot and incubated at 37°C. After 2 h,
the cell suspension was placed in a 1.5-ml Eppendorf tube, and the cells were
pelleted by low-speed centrifugation (300 ? g for 3 min). The supernatant was
discarded using a Hamilton syringe, and 200 ?l dimethyl sulfoxide was added to
the cell pellet. Cells were mixed, placed in a 96-well plate, and incubated for a
further 10 min at 37°C. The optical density of each sample was read at 570 nm
using a microplate reader.
SOCS2 is highly expressed in T cells. Although SOCS1 and
SOCS3 are known to be expressed in many leukocyte lineages,
the abundance of SOCS2 expression in the immune system has
not been explored. To address this, a panel of cDNA libraries
from various immune and nonimmune tissues was analyzed for
expression of the SOCS mRNA. The copy number of individ-
ual genes within the cDNA libraries should not be altered, as
they had not been amplified. SOCS3 was expressed in a num-
ber of libraries tested, appearing predominantly in cells of the
monocyte lineage and PBMCs (Fig. 1A). SOCS3 expression
was increased in some T-cell populations by activation with
IL-2 ? anti-CD3 or PHA (data not shown). Expression of
SOCS3 in activated PBMCs was confirmed by Northern blot
analysis (data not shown). In contrast, expression of SOCS2
was more restricted to T cells and again particularly evident in
activated T cells of both T-helper 1 (Th1) and T-helper 0 (Th0)
lineages (Fig. 1A). Moreover, the relative levels of SOCS2
expression were over 3 logs higher in T-cell lineages than in
other cells examined, suggesting that it may have an important
function in regulating T-cell activation.
SOCS2 and SOCS3 expression is reciprocally regulated in T
cells. In light of the real-time PCR data, SOCS protein expres-
sion in primary human PBMCs was analyzed. Treatment of
PBMCs with PHA induced SOCS expression, but incubation in
2% FCS for 12 h reduced this to background levels. SOCS3
expression was observed 1 h following IL-2 stimulation and was
greatly reduced at 4 h (Fig. 1B). In contrast, SOCS2 was in-
duced at 4 h with a substantial increase in protein expression
between 4 h and 8 h. A similar pattern of expression was
observed in all samples examined, suggesting that it was not
donor specific. These observations indicated that SOCS2 and
SOCS3 were induced in T cells by IL-2 but with different
kinetics, with SOCS3 being detectable much earlier and tran-
siently and SOCS2 appearing later and for a more sustained
period. Interestingly, SOCS3 expression was rapidly reduced to
background levels following the appearance of SOCS2, sug-
gesting that these proteins may be reciprocally regulated.
These findings imply that SOCS3 plays an early role in regu-
lating T-cell responses to IL-2 prior to SOCS2 induction and
that SOCS2 may have a role later in the response.
FIG. 1. (A) Analysis of SOCS expression. A panel of cDNA librar-
ies from different cell types were analyzed for SOCS expression by
real-time PCR using specific primers for SOCS2 and SOCS3 as de-
scribed in Materials and Methods. The abbreviations used in the figure
are as follows: Th0, naive CD4?T cells; B21 and T2023, Th1 clones;
Mot72, CD4?MEL14 naive T cells; LPS, lipopolysaccharide. (B) IL-
2-induced expression of SOCS2 and SOCS3 proteins in PBMCs.
PBMCs were PHA blasted for 72 h, incubated for 12 h in 2% FCS, and
stimulated with 100 U/ml IL-2 for the indicated periods. Lysates were
immunoblotted (IB) with anti-SOCS2 (top) or immunoprecipitated
(IP) and immunoblotted with anti-SOCS3 (bottom).
VOL. 25, 2005 SOCS2 ACCELERATES SOCS3 DEGRADATION9117
9118 TANNAHILL ET AL.MOL. CELL. BIOL.
SOCS2 potentiates cytokine-induced STAT5 phosphoryla-
tion. We next investigated whether SOCS2 could regulate
IL-2- and IL-3-induced STAT5 activation. Ba/F3 cells express-
ing either SOCS2 or SOCS3 under the control of a tetracy-
cline-inducible promoter were incubated overnight in 2% FCS
followed by stimulation with IL-3 for the time course shown.
Figure 2A (top) demonstrates the expression of SOCS3 when
tetracycline was removed from the cells. To analyze whether
STAT5b was phosphorylated upon stimulation with IL-3, ly-
sates were immunoprecipitated with a STAT5b antibody and
blotted with anti-phosphotyrosine. When cells were stimulated
with IL-3, elevated levels of STAT5b tyrosine phosphorylation
were observed between 0.5 h and 2 h (Fig. 2A, middle). Ex-
pression of SOCS3 decreased the level of STAT5 phosphory-
lation at 0.5 h and 1 h, which was almost undetectable 2 h
following IL-3 treatment. In contrast, expression of SOCS2
increased and prolonged the level of STAT5 tyrosine phos-
phorylation following IL-3 treatment, with STAT5 phosphor-
ylation being more pronounced 2 h following cytokine treat-
ment (Fig. 2B, middle). The data suggest that SOCS2 could
enhance STAT5 phosphorylation in response to IL-3. We
therefore investigated whether other signaling responses, such
as STAT3 phosphorylation, are potentiated in the presence of
SOCS2 (Fig. 2C). To verify this, Ba/F3-SOCS2 and Ba/F3-
SOCS3 cells were incubated as before and stimulated with IL-3
for the period shown. Again, expression of SOCS3 clearly
inhibited phosphorylation of STAT3 at 0.5 h and 1 h (Fig. 2C,
top), whereas expression of SOCS2 enabled the levels of phos-
phorylation to be maintained (Fig. 2C, bottom right).
Similarly, we tested the effect of SOCS2 expression on cy-
tokine-induced ERK phosphorylation. Lysates from Ba/F3-
SOCS2 cells were directly immunoblotted with anti-phospho-
ERK. SOCS2 had little effect on the phosphorylation of ERK
in response to IL-3 (Fig. 2D), suggesting that the target of
SOCS2 may be limited to the JAK/STAT pathway.
Signaling in response to IL-2 was also investigated in Ba/F3
cells that stably express the IL-2 receptor (Fig. 2E). In the
absence of SOCS3, levels of phosphorylation increased at 0.5 h
and were still observed 1 h following IL-2 treatment. Again,
SOCS3 expression strongly inhibited STAT5 tyrosine phos-
phorylation following IL-2 stimulation (Fig. 2E, top), whereas
expression of SOCS2 prevented this inhibition (Fig. 2E, bot-
tom). We also observed an increase in IL-2 receptor ?-chain
phosphorylation in the presence of SOCS2 (data not shown).
Overall, these data suggest that SOCS2 can potentiate IL-2-
and IL-3-mediated responses (5, 8).
SOCS2 augments cytokine-induced proliferation. These
findings raised the possibility that SOCS2 expression may also
affect proliferation in response to these cytokines. To examine
this, Ba/F3-SOCS2 cells were seeded at 1 ? 105cells per ml in
the presence and absence of tetracycline and cultured in RPMI
1640 medium supplemented with 5% FCS and 10 U/ml of IL-3.
Samples were taken every 12 h over 48 h, and numbers of
viable cells were determined using the trypan blue exclusion
assay. At 48 h, cells expressing SOCS2 proliferated at double
the rate of controls (Fig. 3A). Results were taken in triplicate
and are representative of three separate experiments. This
result demonstrates that SOCS2 can enhance cytokine-induced
proliferation following IL-3 stimulation. To confirm this ob-
servation, proliferation was also examined in Ba/F3-SOCS2
cells using the MTT assay. Again, cells expressing SOCS2 pro-
liferated at almost double the rate of the control cells when
they were cytokine stimulated (Fig. 3B). These results confirm
that SOCS2 can enhance IL-3-induced proliferation, in con-
trast to other members of the SOCS family, and support the
observation that SOCS2 can promote lymphokine signaling
and proliferative responses as has been reported for GH and
PRL (8, 33).
SOCS2 drives SOCS3 degradation by a proteasome-depen-
dent mechanism. Given that SOCS2 and SOCS3 are induced
under similar conditions in T cells, but with different kinetics,
we can assume that SOCS3 functions prior to SOCS2 in the
cytokine response. Since these proteins are reciprocally regu-
lated in T cells, we investigated whether SOCS2 may enhance
signaling by regulating SOCS3 expression.
To investigate the effects of SOCS2 on endogenous SOCS3,
FIG. 2. SOCS2 and SOCS3 have reciprocal effects on cytokine-
induced STAT5 phosphorylation. Ba/F3 cells lacking SOCS3 (A) or
SOCS2 (B) (with tetracycline [?tet]) or in which expression of SOCS3
(A) or SOCS2 (B) was induced by removal from tetracycline (?tet)
were incubated for 12 h in 2% FCS before stimulation with IL-3 for the
periods shown. Lysates (WCLs) were immunoblotted (IB) with anti-
SOCS3 (A, top) or anti-SOCS2 (B, top). Alternatively, lysates were
immunoprecipitated (IP) with anti-STAT5b and immunoblotted for
anti-phosphotyrosine (A and B, middle). STAT5b levels were checked
using a STAT5b antibody (A and B, bottom). (C) Ba/F3 cells treated
as described above were stimulated with IL-3 as indicated, and lysates
were immunoblotted with a phospho-STAT3 antibody (top). STAT3
levels were verified by immunoblotting with an anti-STAT3 antibody
(bottom). (D) Lysates from Ba/F3-SOCS2 cells were treated as de-
scribed above and immunoblotted with phospho-ERK monoclonal an-
tibody, and protein levels were confirmed by immunoblotting with
anti-ERK. (E) Ba/F3 cells which stably express the IL-2 receptor with
SOCS2 (bottom) or SOCS3 (top) were stimulated with 100 U/ml IL-2
as indicated, and lysates were immunoprecipitated with anti-STAT5b
and immunoblotted with antiphosphotyrosine. STAT5 levels were ver-
ified using an anti-STAT5b.
VOL. 25, 2005 SOCS2 ACCELERATES SOCS3 DEGRADATION9119
Ba/F3-SOCS2 cells were used. In these cells, endogenous
SOCS3 is rapidly induced upon IL-3 stimulation. Ba/F3-
SOCS2 cells were cultured for 48 h in the presence or absence
of tetracycline before being incubated for 12 h in RPMI me-
dium supplemented with 5% FCS. The cells were then stimu-
lated for 0.5 h and 1 h with 100 U/ml IL-3 (Fig. 4A). To
examine endogenous levels of SOCS3, the lysates were immu-
noprecipitated with anti-SOCS3 and analyzed by Western blot,
while SOCS2 protein levels were determined from whole-cell
lysates (WCLs). Figure 4A (bottom) shows the expression of
SOCS2 following tetracycline withdrawal. SOCS3 was induced
0.5 h and still expressed 1 h after IL-3 stimulation in the
absence of SOCS2 (Fig. 4A, top, lanes 3 and 5), and SOCS3
protein levels were markedly reduced in the presence of
SOCS2 (lanes 4 and 6). The addition of the proteasome inhib-
itor MG132 restored SOCS3 protein levels (Fig. 4A, lanes 8
and 10). In Fig. 4B, a similar experiment was performed over
a longer time course. In this case, cells were stimulated for 1 h,
2 h, and 4 h in the presence (Fig. 4B, bottom) or absence (top)
of the proteasome inhibitors MG132 and LLnL. Again, in the
absence of SOCS2, SOCS3 was induced at 1 h following IL-3
stimulation (Fig. 4B, lane 2). This was sustained until 2 h (Fig.
4B, lane 3) and returned to background levels at 4 h (lane 4).
In contrast, when SOCS2 protein was expressed (Fig. 4B, lanes
5 to 8), the levels of endogenous SOCS3 were markedly de-
creased, being almost undetectable. This implies that the pres-
ence of SOCS2 causes the downregulation of endogenous
SOCS3. To further investigate the turnover of SOCS3 by
SOCS2, cells were also treated with the proteasome inhibitors
for 30 min prior to lysing (Fig. 4B, bottom). The lysates were
immunoprecipitated and immunoblotted as described above.
The addition of MG132 and LLnL increased SOCS3 levels,
such that expression was still detectable after 4 h, suggesting
that the transient expression was due to rapid degradation of
SOCS3 by the proteasome. In the presence of SOCS2 and
proteasome inhibitors, SOCS3 protein expression was rescued
(Fig. 4B, bottom, lanes 5 to 8). These results indicated that
SOCS2 accelerates the degradation of SOCS3 in a protea-
Expression of SOCS2 enhanced SOCS mRNA induced by
cytokines. If SOCS2 accelerates the turnover of SOCS3, the
prediction would be that there would be increased activity in
the signaling systems leading to more SOCS expression. To
investigate this, real-time PCR probes were designed to mea-
sure levels of mRNA for the SOCS genes and c-myc as a
control in Ba/F3-SOCS2 cells (Fig. 5A). Cells were stimulated
with IL-3 as described above, and mRNA was extracted and
analyzed using real-time PCR. The patterns of mRNA induc-
tion in the absence of SOCS2 mimicked the protein expression
discussed above. CIS, SOCS1, and SOCS3 mRNAs were in-
duced rapidly following stimulation and declined slightly at 180
min. However, in the presence of SOCS2, a stronger signal was
observed for all three SOCS family members, consistent with
the enhanced STAT activation detected. SOCS2 was detected
using two different sets of real-time PCR probes, one set which
detected both endogenous and exogenous SOCS2 and another
where the forward primer was located upstream of the coding
region and was therefore only able to detect endogenous
SOCS2. Expression of exogenous SOCS2 had little or no effect
on the endogenous levels of the message. When primers spe-
cific for c-myc were used, in the presence of SOCS2, levels of
c-myc mRNA were enhanced and prolonged, again consistent
with enhanced cytokine responses.
To substantiate this observation, SOCS mRNA levels were
analyzed by RT-PCR using 1 ?g of total RNA (Fig. 5B). Again,
the presence of SOCS2 did not diminish the cytokine-induced
mRNA levels of CIS, SOCS1, and SOCS3. SOCS2 did not
affect the level of SOCS mRNA at 30 or 60 min but maintained
expression of CIS, SOCS1, and SOCS3 at 180 min (Fig. 5B).
We further examined other IL-3-responsive genes including
Pim1, OSM, and BclxL and did not observe markedly en-
hanced expression of these genes in two separate experiments
(Fig. 5C). The enhanced mRNA expression observed in the
presence of SOCS2 supports the idea that SOCS2 can boost
signaling and sustain the activation of some STAT5-dependent
SOCS2 causes degradation of SOCS3 in a SOCS box-de-
pendent manner. Taken together, the data suggest that SOCS2
can restore JAK/STAT signaling via degradation of SOCS3.
We therefore wished to determine the mechanism by which
SOCS2 could affect SOCS3 protein levels. Since the SOCS box
motif is thought to interact with an E3 ligase complex and
target associated proteins for degradation (15, 27, 45), a mu-
tant SOCS2 construct was made in which the elongin B/C
FIG. 3. Effect of SOCS2 on cell proliferation. (A) Ba/F3-SOCS2
cells seeded at 1 ? 105cells/ml were cultured with tetracycline (no
SOCS2, black box) or without tetracycline (SOCS2, white box). Via-
bility was determined at 12-h intervals by trypan blue exclusion assay.
(B) Ba/F-SOCS2 cells were seeded at 1 ? 105cells/ml and grown in the
presence or absence of 4 ?g/ml tetracycline (bars are as described
above). At 24 h and 48 h, 10 ?l of MTT (0.5 mg/ml) was added to 100
?l of cell culture and incubated at 37°C for 2 h. The cells were
centrifuged (300 ? g for 3 min), and the supernatant was removed.
Dimethyl sulfoxide (200 ?l) was added and incubated for 10 min at
37°C. Plates were read at an optical density at 570 nm using a micro-
9120TANNAHILL ET AL.MOL. CELL. BIOL.
binding motif was deleted (SOCS2 dSB). We coexpressed
SOCS3 with the SOCS2 wild type or mutant in 293T cells.
When SOCS2 and SOCS3 were coexpressed, high levels of
SOCS2 targeted SOCS3 for degradation (Fig. 6A, top, lane 5)
with 5 ?g of SOCS2 plasmid almost completely blocking de-
tectable SOCS3 expression. However, transfection of up to 5
?g of the SOCS2 dSB mutation did not have this effect (Fig.
6A, top, lane 10), suggesting that SOCS2 targets SOCS3 for
degradation in a SOCS box-dependent manner.
The SOCS box region has been shown to bind to an E3 ligase
complex containing elongin B/C, Cullin5, and Rbx2 (26). We
therefore wished to determine whether this complex mediated
SOCS3 degradation. Interestingly, when SOCS3 was expressed
with elongin B/C, there was a significant reduction in SOCS3
protein levels compared to when SOCS3 was expressed alone
(Fig. 6B, last lane). In contrast, when SOCS2 was expressed
with elongin B/C, the level of SOCS2 protein detected was
elevated (Fig. 6B). This suggests that elongin B/C stabilized
SOCS2, whereas SOCS3 was less stable in the presence of the
elongins. This would hold true if SOCS2 could bind elongin
B/C and activate an E3 ligase complex that targets SOCS3 for
proteasomal degradation. To investigate this possibility,
SOCS2 or SOCS2 dSB, SOCS3, and elongin B/C were coex-
pressed in 293T cells (Fig. 6C). SOCS2, SOCS3, and elongin B
were all FLAG tagged, while elongin C was Myc tagged. Again,
when SOCS3 was coexpressed with SOCS2, we observed a
reduction in SOCS3 levels (Fig. 6C, top, lane 5). Moreover,
when SOCS3 was coexpressed with both SOCS2 and elongin
B/C, SOCS3 protein levels were much more markedly reduced
(Fig. 6C, top, lane 6) compared to when SOCS3 was expressed
alone (lane 3) or with SOCS2 (lane 5). This implied that
expression of SOCS2 resulted in the loss of SOCS3 protein and
that this effect was enhanced further in the presence of elongin
B/C. This reduction in SOCS3 levels was not observed when
FIG. 4. Effects of SOCS2 on SOCS3 protein expression. (A) Ba/F3 cells in which SOCS2 expression was controlled by the removal of
tetracycline were treated as described in the text. Cells were stimulated with 100 U/ml IL-3 for the indicated times. Lysates were immunopre-
cipitated (IP) and immunoblotted (IB) with a SOCS3 antibody (top). Alternatively, lysates were subjected to immunoblotting with M2-FLAG
antibody (bottom). (B) Ba/F3-SOCS2 cells were stimulated as described above but treated with the proteasome inhibitors MG132 (0.5 ?M) and
LLnL (0.5 ?M). Lysates were immunoprecipitated and immunoblotted with anti-SOCS3 (top). WCLs were immunoblotted with SOCS2 antibody.
VOL. 25, 2005 SOCS2 ACCELERATES SOCS3 DEGRADATION9121
SOCS3 was expressed with SOCS2 dSB (Fig 6C, top, lane 9
versus lane 11), further reinforcing the hypothesis that the loss of
SOCS3 is dependent on an intact SOCS box. When SOCS2 was
expressed with elongin B/C, SOCS2 protein levels were elevated
(Fig. 6C, top, lanes 6 and 7), strengthening the idea that elongin
B/C stabilized SOCS2. To verify that this degradation was pro-
teasome dependent, the cells were treated with the proteasome
inhibitors MG132 and LLnL. Figure 6C (bottom) shows that the
presence of these inhibitors rescued SOCS3 expression even in
the presence of SOCS2 (lane 5) and when expressed with both
SOCS2 and elongin B/C (lane 6). These data suggested that
SOCS2 targets SOCS3 for proteasomal degradation.
FIG. 5. SOCS2 enhances mRNA levels of CIS, SOCS1, and SOCS3. (A) Ba/F3-SOCS2 cells were incubated overnight in 2% FCS and
stimulated with 100 U/ml IL-3 as shown. mRNA levels were analyzed by real-time PCR using specific primers for CIS, SOCS1, SOCS2, SOCS3,
and c-myc. Data are expressed as relative mRNA levels. (B) RT-PCR was performed with 1 ?g of total RNA using primer pairs specific for CIS,
SOCS1, SOCS2, SOCS3, and GAPDH. The PCR products were resolved by 2% agarose gel electrophoresis and visualized with ethidium bromide
staining. (C) RT-PCR was performed with 1 ?g of total RNA using primer pairs specific for Pim-1, OSM, BCL-xL, SOCS2, and ?-actin. The PCR
products were resolved by 2% agarose gel electrophoresis and visualized with ethidium bromide staining. tet, tetracycline.
9122 TANNAHILL ET AL.MOL. CELL. BIOL.
SOCS2 interacts with SOCS3. Recently, SOCS2 has been
shown to bind elongin B/C (13), suggesting that it can form an
E3 ligase complex. We have shown that SOCS2 can target
SOCS3 for degradation via the proteasome and that this is
dependent on an intact elongin B/C binding site (Fig. 6C). In
order for SOCS3 to be degraded by SOCS2, we assumed that
SOCS2 and SOCS3 can associate. To examine this, we ana-
lyzed the interaction between SOCS2 and SOCS3 in a fusion
protein pull-down assay. As shown in Fig. 7A, His-tagged
SOCS2 fusion protein bound SOCS3 in vitro. To determine if
this interaction could occur endogenously, Ba/F3 SOCS2 cells
were grown in the presence and absence of tetracycline for
48 h. The cells were incubated in 2% FCS overnight and
stimulated with IL-3 as shown. Cell lysates were immunopre-
cipitated with anti-SOCS3 and immunoblotted for the pres-
ence of SOCS2. When SOCS2 is expressed with endogenous
FIG. 6. SOCS2 causes degradation of SOCS3 via its SOCS box.
(A) SOCS2 wild type (WT) or SOCS2 mutant in which the elongin C
binding region was mutated (SOCS2 dSB) was coexpressed with
SOCS3 as indicated. Lysates were immunoblotted (IB) with anti-
SOCS3 (top) or anti-SOCS2 (bottom). The first lane of each panel
represents 1 ?g of SOCS3 expressed alone, while the second lane
represents 1 ?g of SOCS2 expressed alone. Thereafter, the lanes
represent increasing amounts of SOCS2 expressed with 1 ?g of
SOCS3. (B) Five micrograms of FLAG-tagged SOCS2 and 2 ?g of
SOCS3, elongin B, and Myc-tagged elongin C cDNAs were transfected
into 293T cells. Lysates were immunoblotted with anti-FLAG or anti-
Myc, respectively. (C) Five micrograms of SOCS2 or SOCS2 dSB
FIG. 7. SOCS2 and SOCS3 interaction. (A) His-tagged SOCS2 fu-
sion protein (3 ?g) was precoupled to 50 ?l of 20% nickel-nitrilotri-
acetic acid beads in protein interaction buffer for 2 h at 4°C. The beads
were collected by centrifugation, and lysates from 293T cells tran-
siently transfected with 2 ?g empty vector (Ev) or FLAG-tagged
SOCS3 were added. Reaction mixtures were incubated for 2 to 4 h at
4°C in protein interaction buffer, washed, and analyzed by SDS-PAGE
as described in the text. Membranes were immunoblotted (IB) with
anti-FLAG (top) to detect SOCS3 and reprobed with anti-His to
detect His-tagged SOCS2 fusion protein (bottom). (B) Ba/F3-SOCS2
cells were grown in the presence and absence of tetracycline for 48 h.
Cells were incubated overnight in 2% FCS and stimulated with 100
U/ml IL-3 as shown. Proteasome inhibitors MG132 (0.5 ?M) and
LLnL (0.5 ?M) were added 1 h before lysing. Cell lysates were immu-
noprecipitated (IP) with SOCS3 antibody and immunoblotted with
anti-FLAG (top) or anti-SOCS3 (middle). SOCS2 expression is shown
in the bottom panel.
mutant and 2 ?g SOCS3 and elongin B/C were transfected into 293T
cells. Cells were treated with (bottom panels) or without (top panels)
the proteasome inhibitors MG132 (0.5 ?M) and LLnL (0.5 ?M). WB,
Western blot. Un, untransfected.
VOL. 25, 2005 SOCS2 ACCELERATES SOCS3 DEGRADATION9123
SOCS3, 1 h and 2 h after IL-3 stimulation, both proteins
coprecipitate when proteasome activity is blocked, suggesting
that an endogenous association between SOCS2 and SOCS3
can occur (Fig. 7B), which suggests that SOCS3 degradation is
being accelerated by SOCS2. Although the nature of this in-
teraction is yet to be determined, the findings demonstrate that
SOCS2 can form a complex with SOCS3 and suggest a mech-
anism which enables SOCS3 to be brought into contact with an
E3 ligase complex that ultimately results in its turnover by the
A clear picture has emerged in recent years showing that
many members of the SOCS family have an important role in
inhibition of the JAK/STAT pathway. The mechanisms by
which CIS, SOCS1, and SOCS3 function to block cytokine
signaling have been well documented (19). All three have been
shown to be induced rapidly and act in a feedback loop to
inhibit JAK/STAT activity by binding the JAKs, the receptor,
or both (6, 32, 39). However, although SOCS2 can inhibit GH
and PRL responses, it can also potentiate the signals induced
by many cytokines. In this study, we found that, in contrast to
other members of this family, SOCS2 appears to enhance cy-
tokine signaling rather than suppress it.
To date, the mechanism of action of SOCS2 is unclear. One
previous report suggested that SOCS2 may have a dual role
(12) since low concentrations of SOCS2 inhibited GH action
and higher concentrations of SOCS2 enhanced GH signaling.
Accordingly, both mice overexpressing SOCS2 and mice lack-
ing SOCS2 displayed a gigantism phenotype (31). This implies
that SOCS2 can have a positive and negative role in GH
signaling. It is therefore tempting to speculate that in other
systems, SOCS2 acts as an accelerator rather than an inhibitor
of cytokine signaling. Moreover, SOCS2-null mice show im-
proved responses to growth hormone, indicating an enhanced
effect on cytokine responses. However, higher levels of SOCS2
also block GH responses, suggesting that antagonism of other
SOCS may occur in these cells. Our observations support the
theory that expression of SOCS2 can enhance cytokine re-
sponses, most likely by driving degradation of other SOCS
We have demonstrated that both SOCS2 and SOCS3 are
cytokine-induced genes in human PBMCs but that the proteins
appear with different kinetics. SOCS3 protein was detected
rapidly (30 min following stimulation), while SOCS2 did not
appear until later. We also provide evidence that while SOCS3
inhibited both IL-2- and IL-3-induced tyrosine phosphoryla-
tion and proliferation, SOCS2 was able to enhance signaling.
Since SOCS3 protein expression was maintained in the pres-
ence of proteasome inhibitors and mRNA levels of SOCS3
were not reduced by SOCS2, it was evident that SOCS2 at least
partially enhanced signaling by reducing SOCS3 protein levels
via a proteasome-dependent mechanism. These observations
are further supported by the finding that IL-3-induced gene
expression was also enhanced when SOCS2 was highly ex-
pressed. It is possible that SOCS2 blocks the expression of
other SOCS family members, particularly since all SOCS pro-
teins compete for binding to the same E3 ligases. Indeed, our
preliminary data suggest that SOCS2 may also reduce SOCS1
expression (data not shown).
It is intriguing that cytokine-induced SOCS2 is expressed
much later in peripheral blood T cells. This suggests that
SOCS2 is not involved in the feedback loop that inhibits cyto-
kine signaling but may limit this inhibition. Moreover, the
finding that SOCS2 is still expressed 24 h after IL-2 treatment
suggests that it may be important to potentiate the prolifera-
tion of these rapidly dividing cells, perhaps by keeping the
expression of other SOCS at reduced levels. This is also true of
IL-3-induced SOCS2 in Ba/F3 cells (J. A. Johnston, unpub-
When expressed at high levels, SOCS2 has been demon-
strated to antagonize the inhibitory effects of SOCS1 on PRL
and GH signaling (5, 8, 33) by an unknown mechanism. CIS
levels are also lower in the presence of SOCS2, although the
effects are not as marked as for SOCS3 (data not shown), and
CIS is also induced later than SOCS1 or SOCS3 in response to
cytokine stimulation (3). Clearly, we have observed SOCS3
degradation in the presence of overexpressed SOCS2, but
since both proteins are induced by many ligands and appear to
be reciprocally regulated, the SOCS2-induced degradation of
SOCS3 would presumably function under normal physiological
conditions. This would provide a mechanism for eliminating
SOCS3 and the other SOCS family members and thus resen-
sitize cells for further cytokine-mediated responses.
The mechanism of action of SOCS2 remains unclear, al-
though several theories have been suggested. One theory holds
that high concentrations of SOCS2 may overcome the effect of
endogenous SOCS3 on GH signaling (8). SOCS3, unlike
SOCS2, contains a kinase-inhibitory region at its N terminus
which is thought to directly inhibit JAK activity (36). It has
been suggested that SOCS2 may compete with SOCS3 for
binding to the GH receptor, and the lack of a kinase-inhibitory
region on SOCS2 may result in the continuation of JAK activ-
ity and enhanced signaling (12). However, recently, Green-
halgh et al. reported that SOCS2 binds to the GH receptor at
Y487 and Y595, which are not classic immunoreceptor ty-
rosine-based inhibitory motifs, suggesting that SOCS3 will not
compete to bind these sites (13). Furthermore, the SH2 do-
mains of SOCS2 and SOCS3 differ significantly and are there-
fore likely to interact with different phosphotyrosine sequences
on different target molecules.
Since our data imply that in the presence of SOCS2, SOCS3
protein expression is downregulated, a more likely theory is
that SOCS2 may compete with SOCS3 for binding to the elon-
gin B/C complex. This interaction could occur via the BC box
region of the SOCS box. Other SOCS box-containing proteins,
including VHL (21), Muf1 (24), elongin A (1), and SOCS1
(15), are thought to be stabilized by this interaction. It has also
been shown that tyrosine phosphorylation in the SOCS box of
SOCS3 (14) and serine/threonine phosphorylation of SOCS1
(2) disrupt elongin binding. Therefore, SOCS2 may bind to or
compete with SOCS3 for elongin B/C, resulting in reduced
SOCS3 protein stability.
Another possibility is that SOCS2 may form part of an E3
ligase complex similar to that of VHL. The C-terminal domain
of the VHL protein is homologous to the SOCS box and
interacts with the elongin B/C complex which in turn binds the
Cullin family member Cul2 and RING finger protein Rbx1 to
9124 TANNAHILL ET AL.MOL. CELL. BIOL.
form an E3 ligase complex (25–27). Under normoxic condi-
tions, hypoxia-inducible factor 1? binds to the VHL E3 ligase
complex, resulting in ubiquitination and proteasomal degrada-
tion (20, 21). SOCS1 has been proposed to form part of an E3
ligase complex containing Cul5 and Rbx1 (24) to target asso-
ciated proteins for degradation. More recently, it has been
suggested that SOCS1 lacks a Cul5 binding site within the
SOCS box (26); however, it is plausible that other Cullin pro-
teins may interact with SOCS1.
As shown in Fig. 7, SOCS2 and SOCS3 can associate both in
vitro and in vivo, although the precise nature of this interaction
remains unknown. However, this study brings us a step closer
to understanding when the interaction between SOCS2 and
SOCS3 can occur. Our data imply that both proteins associate
upon cytokine stimulation when proteasome activity is
blocked. It is yet to be determined whether phosphorylation
plays a role in regulating the SOCS2-SOCS3 association. Since
we have observed association of these proteins, it is plausible
to suggest that SOCS2 may act as a linker which brings an E3
ligase complex into close proximity with SOCS3. This may be a
potential mechanism by which SOCS2 could result in the loss
of SOCS3 protein.
SOCS1 has been reported to regulate the half-life of VAV
(4) and the insulin receptor substrates IRS1 and IRS2 (35).
Also, SOCS1 inhibits the kinase activity of JAK2 (41) and the
TEL-JAK2 oncogene (9) in a phosphorylation-dependent
manner by inducing SOCS box-dependent proteasomal degra-
dation. SOCS1 therefore targets these substrates to the pro-
teasome for degradation. Also, SOCS1 and SOCS3 promoted
polyubiquitination and degradation of focal adhesion kinase in
a SOCS box-dependent manner which inhibited focal adhesion
kinase-dependent signaling events (30). Likewise, the SOCS
box has been implicated in the inhibition of granulocyte colo-
ny-stimulating factor signaling, suggesting a role for proteaso-
mal degradation mediated via SOCS1 and SOCS3 in down-
regulating granulocyte colony-stimulating factor responses
(42). This evidence suggests that the SOCS box is involved in
the proteasomal targeting of specific substrates, including per-
haps other SOCS. Therefore, SOCS2 may act as part of an E3
ligase and target SOCS3, and perhaps other SOCS, for ubiq-
uitination and degradation via the 26S proteasome.
Despite these observations, questions remain concerning
how these findings relate to immune homeostasis and disease.
SOCS3 deficiency leading to sustaining IL-6-induced STAT3
activation is thought to contribute to inflammatory diseases
such as rheumatoid arthritis, Crohn’s disease, and inflamma-
tory bowel disease (38). In comparison, sustained SOCS3 ex-
pression in T cells skews differentiation towards a Th2 re-
sponse which may lead to Th2-related allergy (37). It will be
interesting to establish if altered SOCS3 expression in these
instances is due to defects in the ability of SOCS2 to regulate
Blood packs were kindly donated by the Northern Ireland Blood
Transfusion Service. This work has been sponsored by the Wellcome
Trust (grant no. 070304/2/03/2) and the BBSRC (grant no. 81/C17863).
We thank G. Clarke for his continuing support and James Burrows,
Massimo Gadina, and Karim Dib for their helpful comments on the
1. Aso, T., D. Haque, R. J. Barstead, R. C. Conaway, and J. W. Conaway. 1996.
The inducible elongin A elongation activation domain: structure, function
and interaction with the elongin BC complex. EMBO J. 15:5557–5566.
2. Chen, X. P., J. A. Losman, S. Cowan, E. Donahue, S. Fay, B. Q. Vuong, M. C.
Nawijn, D. Capece, V. L. Cohan, and P. Rothman. 2002. Pim serine/threo-
nine kinases regulate the stability of Socs-1 protein. Proc. Natl. Acad. Sci.
3. Cohney, S. J., D. Sanden, N. A. Cacalano, A. Yoshimura, A. Mui, T. S.
Migone, and J. A. Johnston. 1999. SOCS-3 is tyrosine phosphorylated in
response to interleukin-2 and suppresses STAT5 phosphorylation and lym-
phocyte proliferation. Mol. Cell. Biol. 19:4980–4988.
4. De Sepulveda, P., S. Ilangumaran, and R. Rottapel. 2000. Suppressor of
cytokine signaling-1 inhibits VAV function through protein degradation.
J. Biol. Chem. 275:14005–14008.
5. Dif, F., E. Saunier, B. Demeneix, P. A. Kelly, and M. Edery. 2001. Cytokine-
inducible SH2-containing protein suppresses PRL signaling by binding the
PRL receptor. Endocrinology 142:5286–5293.
6. Elliott, J., and J. A. Johnston. 2004. SOCS: role in inflammation, allergy and
homeostasis. Trends Immunol. 25:434–440.
7. Endo, T. A., M. Masuhara, M. Yokouchi, R. Suzuki, H. Sakamoto, K. Mitsui,
A. Matsumoto, S. Tanimura, M. Ohtsubo, H. Misawa, T. Miyazaki, N.
Leonor, T. Taniguchi, T. Fujita, Y. Kanakura, S. Komiya, and A. Yoshimura.
1997. A new protein containing an SH2 domain that inhibits JAK kinases.
8. Favre, H., A. Benhamou, J. Finidori, P. A. Kelly, and M. Edery. 1999. Dual
effects of suppressor of cytokine signaling (SOCS-2) on growth hormone
signal transduction. FEBS Lett. 453:63–66.
9. Frantsve, J., J. Schwaller, D. W. Sternberg, J. Kutok, and D. G. Gilliland.
2001. Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic
cells through inhibition of JAK2 kinase activity and induction of proteasome-
mediated degradation. Mol. Cell. Biol. 21:3547–3557.
10. Fujimoto, M., and T. Naka. 2003. Regulation of cytokine signaling by SOCS
family molecules. Trends Immunol. 24:659–666.
11. Gadina, M., D. Hilton, J. A. Johnston, A. Morinobu, A. Lighvani, Y. J. Zhou,
R. Visconti, and J. J. O’Shea. 2001. Signaling by type I and II cytokine
receptors: ten years after. Curr. Opin. Immunol. 13:363–373.
12. Greenhalgh, C. J., P. Bertolino, S. L. Asa, D. Metcalf, J. E. Corbin, T. E.
Adams, H. W. Davey, N. A. Nicola, D. J. Hilton, and W. S. Alexander. 2002.
Growth enhancement in suppressor of cytokine signaling 2 (SOCS-2)-defi-
cient mice is dependent on signal transducer and activator of transcription 5b
(STAT5b). Mol. Endocrinol. 16:1394–1406.
13. Greenhalgh, C. J., E. Rico-Bautista, M. Lorentzon, A. L. Thaus, P. O.
Morgan, T. A. Willson, P. Zervoudakis, D. Metcalf, I. Street, N. A. Nicola,
A. D. Nash, L. J. Fabri, G. Norstedt, C. Ohlsson, A. Flores-Morales, W. S.
Alexander, and D. J. Hilton. 2005. SOCS2 negatively regulates growth hor-
mone action in vitro and in vivo. J. Clin. Investig. 115:397–406.
14. Haan, S., P. Ferguson, U. Sommer, M. Hiremath, D. W. McVicar, P. C.
Heinrich, J. A. Johnston, and N. A. Cacalano. 2003. Tyrosine phosphoryla-
tion disrupts elongin interaction and accelerates SOCS3 degradation. J. Biol.
15. Hanada, T., T. Yoshida, I. Kinjyo, S. Minoguchi, H. Yasukawa, S. Kato, H.
Mimata, Y. Nomura, Y. Seki, M. Kubo, and A. Yoshimura. 2001. A mutant
form of JAB/SOCS1 augments the cytokine-induced JAK/STAT pathway by
accelerating degradation of wild-type JAB/CIS family proteins through the
SOCS-box. J. Biol. Chem. 276:40746–40754.
16. Hibbert, L., and J. A. Johnston. 2001. Cytokine signalling and disease.
Expert Opin. Ther. Targets 5:641–653.
17. Hilton, D. J., R. T. Richardson, W. S. Alexander, E. M. Viney, T. A. Willson,
N. S. Sprigg, R. Starr, S. E. Nicholson, D. Metcalf, and N. A. Nicola. 1998.
Twenty proteins containing a C-terminal SOCS box form five structural
classes. Proc. Natl. Acad. Sci. USA 95:114–119.
18. Hoeflich, A., M. Wu, S. Mohan, J. Foll, R. Wanke, T. Froehlich, G. J. Arnold,
H. Lahm, H. J. Kolb, and E. Wolf. 1999. Overexpression of insulin-like
growth factor-binding protein-2 in transgenic mice reduces postnatal body
weight gain. Endocrinology 140:5488–5496.
19. Ihle, J. N. 1995. Cytokine receptor signalling. Nature 377:591–594.
20. Ivan, M. and W. G. Kaelin, Jr. 2001. The von Hippel-Lindau tumor sup-
pressor protein. Curr. Opin. Genet. Dev. 11:27–34.
21. Iwai, K., K. Yamanaka, T. Kamura, N. Minato, R. C. Conaway, J. W.
Conaway, R. D. Klausner, and A. Pause. 1999. Identification of the von
Hippel-Lindau tumor-suppressor protein as part of an active E3 ubiquitin
ligase complex. Proc. Natl. Acad. Sci. USA 96:12436–12441.
22. Johnston, J. A. 2003. Are SOCS suppressors, regulators, and degraders?
J. Leukoc. Biol. 75:743–748.
23. Kamizono, S., T. Hanada, H. Yasukawa, S. Minoguchi, R. Kato, M. Minogu-
chi, K. Hattori, S. Hatakeyama, M. Yada, S. Morita, T. Kitamura, H. Kato,
K. Nakayama, and A. Yoshimura. 2001. The SOCS box of SOCS-1 acceler-
ates ubiquitin-dependent proteolysis of TEL-JAK2. J. Biol. Chem.
24. Kamura, T., D. Burian, Q. Yan, S. L. Schmidt, W. S. Lane, E. Querido, P. E.
VOL. 25, 2005SOCS2 ACCELERATES SOCS3 DEGRADATION 9125
Branton, A. Shilatifard, R. C. Conaway, and J. W. Conaway. 2001. Muf1, a
novel elongin BC-interacting leucine-rich repeat protein that can assemble
with Cul5 and Rbx1 to reconstitute a ubiquitin ligase. J. Biol. Chem. 276:
25. Kamura, T., M. N. Conrad, Q. Yan, R. C. Conaway, and J. W. Conaway.
1999. The Rbx1 subunit of SCF and VHL E3 ubiquitin ligase activates Rub1
modification of cullins Cdc53 and Cul2. Genes Dev. 13:2928–2933.
26. Kamura, T., K. Maenaka, S. Kotoshiba, M. Matsumoto, D. Kohda, R. C.
Conaway, J. W. Conaway, and K. I. Nakayama. 2004. VHL-box and SOCS-
box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2
modules of ubiquitin ligases. Genes Dev. 18:3055–3065.
27. Kamura, T., S. Sato, D. Haque, L. Liu, W. G. Kaelin, Jr., R. C. Conaway, and
J. W. Conaway. 1998. The elongin BC complex interacts with the conserved
SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and
ankyrin repeat families. Genes Dev. 12:3872–3881.
28. Kopchick, J. J., L. L. Bellush, and K. T. Coschigano. 1999. Transgenic
models of growth hormone action. Annu. Rev. Nutr. 19:437–461.
29. Kubo, M., T. Hanada, and A. Yoshimura. 2003. Suppressors of cytokine
signaling and immunity. Nat. Immunol. 4:1169–1176.
30. Liu, E., J. F. Cote, and K. Vuori. 2003. Negative regulation of FAK signaling
by SOCS proteins. EMBO J. 22:5036–5046.
31. Metcalf, D., C. J. Greenhalgh, E. Viney, T. A. Willson, R. Starr, N. A. Nicola,
D. J. Hilton, and W. S. Alexander. 2000. Gigantism in mice lacking suppres-
sor of cytokine signalling-2. Nature 405:1069–1073.
32. Naka, T., M. Narazaki, M. Hirata, T. Matsumoto, S. Minamoto, A. Aono, N.
Nishimoto, T. Kajita, T. Taga, K. Yoshizaki, S. Akira, and T. Kishimoto.
1997. Structure and function of a new STAT-induced STAT inhibitor. Na-
33. Pezet, A., H. Favre, P. A. Kelly, and M. Edery. 1999. Inhibition and resto-
ration of prolactin signal transduction by suppressors of cytokine signaling.
J. Biol. Chem. 274:24497–24502.
34. Quaife, C. J., L. S. Mathews, C. A. Pinkert, R. E. Hammer, R. L. Brinster,
and R. D. Palmiter. 1989. Histopathology associated with elevated levels of
growth hormone and insulin-like growth factor I in transgenic mice. Endo-
35. Rui, L., M. Yuan, D. Frantz, S. Shoelson, and M. F. White. 2002. SOCS-1
and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of
IRS1 and IRS2. J. Biol. Chem. 277:42394–42398.
36. Sasaki, A., H. Yasukawa, A. Suzuki, S. Kamizono, T. Syoda, I. Kinjyo, M.
Sasaki, J. A. Johnston, and A. Yoshimura. 1999. Cytokine-inducible SH2
protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through
the N-terminal kinase inhibitory region as well as SH2 domain. Genes Cells
37. Seki, Y., H. Inoue, N. Nagata, K. Hayashi, S. Fukuyama, K. Matsumoto, O.
Komine, S. Hamano, K. Himeno, K. Inagaki-Ohara, N. Cacalano, A.
O’Garra, T. Oshida, H. Saito, J. A. Johnston, A. Yoshimura, and M. Kubo.
2003. SOCS-3 regulates onset and maintenance of T(H)2-mediated allergic
responses. Nat. Med. 9:1047–1054.
38. Shouda, T., T. Yoshida, T. Hanada, T. Wakioka, M. Oishi, K. Miyoshi, S.
Komiya, K. Kosai, Y. Hanakawa, K. Hashimoto, K. Nagata, and A. Yo-
shimura. 2001. Induction of the cytokine signal regulator SOCS3/CIS3 as a
therapeutic strategy for treating inflammatory arthritis. J. Clin. Investig.
39. Starr, R., T. A. Willson, E. M. Viney, L. J. Murray, J. R. Rayner, B. J.
Jenkins, T. J. Gonda, W. S. Alexander, D. Metcalf, N. A. Nicola, and D. J.
Hilton. 1997. A family of cytokine-inducible inhibitors of signalling. Nature
40. Tanimoto, K., Y. Makino, T. Pereira, and L. Poellinger. 2000. Mechanism of
regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau
tumor suppressor protein. EMBO J. 19:4298–4309.
41. Ungureanu, D., P. Saharinen, I. Junttila, D. J. Hilton, and O. Silvennoinen.
2002. Regulation of Jak2 through the ubiquitin-proteasome pathway involves
phosphorylation of Jak2 on Y1007 and interaction with SOCS-1. Mol. Cell.
42. van de Geijn, G. J., J. Gits, and I. P. Touw. 2004. Distinct activities of
suppressor of cytokine signaling (SOCS) proteins and involvement of the
SOCS box in controlling G-CSF signaling. J. Leukoc. Biol. 76:237–244.
43. Wilcox, A., K. D. Katsanakis, F. Bheda, and T. S. Pillay. 2004. Asb6, an
adipocyte-specific ankyrin and SOCS box protein, interacts with APS to
enable recruitment of elongins B and C to the insulin receptor signaling
complex. J. Biol. Chem. 279:38881–38888.
44. Yasukawa, H., A. Sasaki, and A. Yoshimura. 2000. Negative regulation of
cytokine signaling pathways. Annu. Rev. Immunol. 18:143–164.
45. Zhang, J. G., A. Farley, S. E. Nicholson, T. A. Willson, L. M. Zugaro, R. J.
Simpson, R. L. Moritz, D. Cary, R. Richardson, G. Hausmann, B. J. Kile,
S. B. Kent, W. S. Alexander, D. Metcalf, D. J. Hilton, N. A. Nicola, and M.
Baca. 1999. The conserved SOCS box motif in suppressors of cytokine
signaling binds to elongins B and C and may couple bound proteins to
proteasomal degradation. Proc. Natl. Acad. Sci. USA 96:2071–2076.
9126 TANNAHILL ET AL.MOL. CELL. BIOL.