The SOCS Box of SOCS-1 Accelerates
Ubiquitin-dependent Proteolysis of TEL-JAK2*
Shintaro
Kamizono
§,
Toshikatsu
Hanada
,
Hideo
Yasukawa
,
Shigeru
Minoguchi
,
Reiko
Kato
,
Mayu
Minoguchi
,
Kimihiko
Hattori¶,
Shigetsugu
Hatakeyama¶,
Masayoshi
Yada¶,
Sumiyo
Morita
,
Toshio
Kitamura
,
Hirohisa
Kato§,
Kei-ichi
Nakayama¶, and
Akihiko
Yoshimura
**
From the
Institute of Life Science, Kurume
University, Aikawa-machi 2432-3, Kurume 839-0861, the
§ Department of Pediatrics, Faculty of Medicine, Kurume
University, Kurume 830, the Departments of ¶ Molecular and
Cellular Biology and ** Immunology, Medical Institute of Bioregulation,
Kyushu University, Fukuoka 812-8582, and the
Department of
Hematopoietic Factors, Institute of Medical Science, University of
Tokyo, 108-8639, Japan
Received for publication, November 6, 2000, and in revised form, January 19, 2001
 |
ABSTRACT |
Fusion of the TEL gene on 12p13 to
the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia.
TEL-mediated oligomerization of JAK2 results in
constitutive activation of the tyrosine kinase (JH1) domain and confers
cytokine-independent proliferation on interleukin-3-dependent
Ba/F3 cells. Forced expression of the JAK inhibitor gene
SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells.
This suppression of TEL-JAK2 activity was dependent on SOCS
box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase
inhibition. Degradation of JAK2 depended on its phosphorylation and its
high affinity binding with SOCS1 through the kinase inhibitory region
and the SH2 domain. It has been demonstrated that von Hippel-Lindau
disease (VHL) tumor-suppressor gene product possesses the SOCS box that
forms a complex with Elongin B and C and Cullin-2, and it functions as
a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to
interact with Elongins; however, ubiquitin ligase activity has not been
demonstrated. We found that the SOCS box interacted with Cullin-2 and
promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of
dominant negative Cullin-2 suppressed SOCS1-dependent
TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3
ubiquitin-ligase-like activity of SOCS1 for activated JAK2 and may
provide a novel strategy for the suppression of oncogenic tyrosine kinases.
 |
INTRODUCTION |
Cytokines induce the activation of the JAK family tyrosine kinases
(JAKs)1 and the subsequent
recruitment of various signaling proteins to the receptor complex,
including the STAT family of transcription factors. Constitutive
activation of the JAK/STAT pathway has been found in many leukemic cell
lines, including cells transformed with Bcr-Abl (1, 2), as well as in
human T-cell lymphotrophic virus-1-transformed T cells (3, 4). A
constitutively activated form of STAT5 conferred factor-independent
growth on Ba/F3 cells (5), and that of STAT3 has also been shown to
function as an oncogene (6). Moreover, a constitutively activated JAK
kinase generated by chromosome translocation between the TEL
gene on 12p13 and the JAK2 gene on 9p24 has been shown to be associated with human leukemia (7, 8). TEL, a subset of the ETS family of
transcription factors, contains a conserved oligomerization domain,
known as the PNT domain, in the N-terminal region. Like other
TEL-tyrosine kinase fusion proteins such as the TEL-PDGF receptor
chain and TEL-Abl, the JAK2 tyrosine kinase domain is constitutively
activated by oligomerization mediated by the PNT domain. Stable
expression of TEL-JAK2 confers factor-independent growth on
IL-3-dependent Ba/F3 cells and induces myeloproliferative and T-cell lymphoproliferative diseases in mice (9).
The JAK/STAT pathway is regulated by several mechanisms, including
dephosphorylation by protein phosphatases and degradation by the
ubiquitin/proteasome system (see review; Yasukawa et
al.(10)). The CIS family (also referred to as the SOCS or SSI
family) has been shown to play an important role in regulating
cytokine signal transduction. CIS1, the first member of this family to
be cloned, suppresses STAT5 activation by binding to cytokine receptors
(11, 12). The second family member found, JAB/SOCS1/SSI-1, directly binds to the JAK2 kinase (JH1) domain, thereby inhibiting tyrosine kinase activity (13-15). Mutational analysis and biochemical
characterization revealed a novel type of inhibition of JAK2 tyrosine
kinase activity through the two independent binding sites of SOCS1/JAB:
the N-terminal kinase inhibitory region binds to the catalytic groove
of JH1, and the SH2 domain binds to the phosphorylated tyrosine residue Tyr-1007 in the activation loop (16, 17). Gene disruption studies have suggested that one of the major physiological functions of
SOCS1 is the negative regulation of the IFN
/STAT1 pathway (18,
19).
Six additional CIS/SOCS/SSI family members were cloned from a data base
search (20-22). In this family, the SH2 domain and the C-terminal
region of about 40 amino acids, referred to as the SOCS box, are
conserved. The data base search also revealed that a similar SOCS box
is present in several proteins containing ankyrin-like repeats,
Ras-like GTPases, or WD40 domains (20, 22). The SOCS box has been
implicated in protein stability or degradation of associated molecules,
because it was found to interact with the Elongin B and Elongin C
(Elongin B,C) complex, which may recruit Cullin-2 (Cul-2), Rbx1, and
the E2 ubiquitin-conjugating enzyme (23-25). Therefore, CIS family
members are hypothesized to function as E3-like ubiquitin-ligase
complexes against target molecules from an analogy with the von
Hippel-Lindau (VHL) tumor-suppressor gene product. However, no evidence
in support of this hypothesis has yet been reported. Kamura et
al. (23) reported that the protein levels of full-length JAK2 were
not affected by coexpression of SOCS1, and, rather, that coexpression
of Elongin B,C stabilized the SOCS1 protein. Furthermore, it has been
shown that the SOCS box is not essential for the inhibition of
cytokine-induced JAK/STAT activation by SOCS1 (16, 17, 23, 26).
Therefore, the role of the SOCS box of SOCS1 still remains to be elucidated.
To suppress the oncogenic potential of activated tyrosine kinases, we
introduced the SOCS1 gene into Ba/F3 cells transformed with TEL-JAK2
using a retrovirus system. Overexpression of SOCS1 could efficiently
suppress the transforming potential of TEL-JAK2. However, simple
inhibition of kinase activity by SOCS1 could not explain the
suppression of TEL-JAK2. We found that SOCS1 promoted ubiquitin-proteasome-dependent degradation of TEL-JAK2 and
full-length JAK2, and that this process required the C-terminal SOCS
box of SOCS1 as well as the phosphorylation of JAK2.
 |
EXPERIMENTAL PROCEDURES |
Cells and Transfection--
Murine IL-3-dependent
Ba/F3 cells were maintained in RPMI medium supplemented with 10% fetal
calf serum and 10% conditioned medium from WEHI-3B cells as a source
of IL-3. Ba/F3 cells were transformed with pCDNA3-TEL-JAK2 or
pCDNA3-Bcr-Abl as described previously (11). After selection with
G418 (1 mg/ml), cells that could grow without IL-3 were subsequently
selected. Transient transfection and the luciferase assay in 293 cells
have been described previously (20).
cDNA Construction--
Deletion, substitution, and chimeric
mutants were generated by standard PCR methods as described previously
(16, 27). Some of the mutants and wild-type SOCS1 were subcloned into a pMX-IRES-EGFP vector (28). For swapping of the SOCS box, the SOCS boxes
of SOCS1 (codon 167-212), CIS3/SOCS3 (codon 180-225), and CIS1 (codon
213-257) were interchanged by introducing an SplI site at
the joint. All constructs contained an N-terminal Myc- or
FLAG-tag (13). For TEL-JAK2 fusion, the human TEL (codon 1-162)
part was obtained by PCR and fused to the mouse JAK2 JH1 domain (codon
839-1127). This fusion gene corresponds to the TEL-JAK2 found in
B-cell lymphoblastic leukemia patients (9). Murine Cul-2 cDNA was
obtained by PCR from a brain cDNA library and cloned into a
pCDNA3 vector containing an N-terminal HA-tag. The R452C mutant was
created by site-directed mutagenesis.
Retrovirus Production and Infection--
Retroviruses were
produced by transient transfection of the PLAT-E packaging cell line
with cDNAs in pMX-IRES-EGFP (28). Forty-eight hours after
transfection, the culture supernatant was harvested and stored at
80 °C. BF/TEL-JAK or BF/Bcr-Abl cells (2 × 105
cells) were infected with appropriately diluted PLAT-E supernatants containing 10 µg/ml Polybrene for 24 h. After being washed,
cells were further cultured in an RPMI medium for an additional 24 or 48 h. Then, aliquots of cells (1 × 104) were
analyzed using a Coulter EPICS-XL flow cytometer. All experiments were
performed in the absence of IL-3.
In Vitro Kinase Assay--
An in vitro kinase assay
for TEL-JAK2 was performed as described previously (16). Briefly,
FLAG-tagged TEL-JAK2 expressed in 293 cells (3.0 µg/transfection)
grown in 10-cm dishes was immunoprecipitated with anti-FLAG antibody in
60 µl (50% v/v) of protein G-Sepharose. Then the resin was incubated
with 1 ml of cell extracts from 293 cells transiently expressing
wild-type SOCS1 (WT) or a deletion mutant lacking 40 amino acids at the
C terminus (dC40) at 4 °C for 1 h. After being washed twice
with kinase reaction buffer (50 mM Hepes-buffer, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 10 µM dithiothreitol,
and 10 µM Na3VO4), the beads were
resuspended in 20 µl of kinase reaction buffer containing the
substrate polypeptide, GST-EPOR cytoplasmic domain (GST-YY) (16) (0.1 mg/ml), and ATP (50 µM) and incubated at 30 °C for 5 min. Kinase activity was analyzed by immunoblotting of GST-YY with
anti-phosphotyrosine (4G10).
Immunoprecipitation and Western Blot
Analysis--
Immunoprecipitation and immunoblotting were performed as
described previously (11). Anti-JAK2 JH1 (
JAK2) rabbit polyclonal antibody, anti-Myc (
MYC) monoclonal and polyclonal antibodies, and
anti-phosphotyrosine (
PY, 4G10) antibodies have been described previously (16). For pulse-chase experiments, 293 cells (1 × 106) grown in 10-cm dishes were transfected with TEL-JAK2
(2.0 µg of plasmid) and WT or dC40 cDNA (0.02 µg). After
18 h, the cells were pulse-labeled with Tran35S-label
(ICN) at a concentration of 150 µCi/ml for 15 min and then scraped.
After being divided into four parts, cells were replated into 3.5-cm
dishes. Following the indicated chase periods, cells were lysed and
immunoprecipitated with anti-JAK2 antibody followed by protein
A-Sepharose, separated on SDS-polyacrylamide gel electrophoresis,
exposed, and quantified by a BAS-2000 imaging system (Fuji). To see the
effect of proteasome inhibitors, 293 cells transfected with TEL-JAK2
and WT-SOCS1 were treated with lactacystin or MG132 (25 µM each) for 30 min before labeling. The drugs were
maintained throughout the pulse-chase period. For the cycloheximide
treatment experiment, 293 cells (1 × 106) were
transfected with 2.0 µg of TEL-JAK2 plus WT or mutant SOCS1 (0.02 µg). Eighteen hours after transfection, cells were trypsinized and
divided into four parts. After a 5-h incubation period, cells were
attached to the dishes and then treated with 50 µg/ml cycloheximide for the indicated periods. The cell extracts were prepared and immunoblotted with anti-JAK2, anti-STAT5, and anti-Myc antibodies as
described (16). Band intensity was quantified by a densitometer as
described (16).
 |
RESULTS |
SOCS Box-dependent Suppression of TEL-JAK2 Transforming
Activity by SOCS1--
To determine whether the JAK inhibitor
SOCS1/JAB can suppress oncogenic tyrosine kinases, SOCS1 cDNA was
introduced into Ba/F3 cells transformed with either TEL-JAK2
(BF/TEL-JAK) or p210 Bcr-Abl (BF/Bcr-Abl) together with enhanced green
fluorescence protein (EGFP) using the bicistronic retrovirus vector
pMX-IRES-EGFP (28). Because the infected cells expressed both EGFP and
Myc-tagged SOCS1, the percentage of infected cells was determined as
the EGFP-positive rate by flow cytometry. The same virus was shown to
induce apoptosis of parental Ba/F3 cells in the presence of IL-3 (28).
As shown in Fig. 1 (A and
B), the population of wild-type SOCS1 (WT)-infected
BF/TEL-JAK cells decreased markedly, suggesting that WT-infected cells
disappeared with cell death. Indeed, WT-infected BF/TEL-JAK cells
underwent apoptosis characterized by DNA fragmentation (Fig.
1C, lane 2). Similar effects were observed even
in the presence of IL-3 (data not shown). WT did not affect the growth
of BF/Bcr-Abl cells (Fig. 1B), which is consistent with the
observation that SOCS1 did not inhibit Bcr-Abl tyrosine kinase activity
(data not shown). Thus, the inhibitory effect of SOCS1 was shown to be
specific to JAKs.

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Fig. 1.
Suppression of TEL-JAK2-dependent
growth of Ba/F3 cells by SOCS1. A, BF/TEL-JAK cells
were infected with retrovirus carrying IRES-EGFP alone
(Control) or wild-type SOCS1. The numbers of EGFP-positive
cells were scored by flow cytometry on the indicated day after
infection. Cells were cultured in the absence of IL-3. B and
C, BF/TEL-JAK cells were infected with a control virus
(open square and lane 1) or a retrovirus carrying
wild-type-SOCS1 (WT) (closed circle and
lane 2), or a dC40 mutant SOCS1 (dC40)
(closed square and lane 3). BF/Bcr-Abl cells were
also infected with a retrovirus carrying WT-SOCS1 (open
circle). The mean and standard error shown are from three
independent experiments. C, a DNA fragmentation assay was
performed 24 h after infection. Infection efficiency
(I.E.), which is the percentage of EGFP-positive cells, is
shown at the bottom. M, DNA size marker.
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We have shown that the N-terminal kinase inhibitory region and the SH2
domain, but not the C-terminal SOCS box, are essential for SOCS1 to
inhibit JAK kinase activity in vitro and in vivo (16). However, unexpectedly, the dC40 mutant lacking the entire SOCS
box could not suppress the growth of BF/TEL-JAK cells (Fig. 1,
B and C). To clarify the reason for this
discrepancy, we examined STAT5 tyrosine phosphorylation in BF/TEL-JAK
cells after infection (Fig. 2; infection
efficiency is listed as I.E.). Infection with WT and dC40,
but not with a control virus, resulted in a decrease in the tyrosine
phosphorylation of STAT5 (Fig. 2,
PY-STAT5). Reduction of STAT5 phosphorylation by WT was more profound than that by
dC40. Tyrosine phosphorylation of TEL-JAK2 was partially reduced by
dC40, suggesting that dC40 could reduce TEL-JAK2 kinase activity but
was not sufficient to induce apoptosis of BF/TEL-JAK cells at the
expression levels obtained by the retrovirus system. It should be noted
that the infection efficiency of the WT virus to BF/TEL-JAK2 judged by
flow cytometry was less than 55%, whereas those of dC40 and control
viruses were more than 75%, even though virus titers were similar when
assayed with NIH-3T3 cells. This is presumably because WT
virus-infected cells die rapidly after infection. Thus, the infection
efficiency of the WT virus will be underestimated. More drastically, we
noticed that the protein levels of TEL-JAK2 decreased in WT-infected
cells but not in dC40-infected cells (Fig. 2,
JAK2). WT-SOCS1 did not affect the protein
level of Bcr-Abl (Fig. 2,
Abl). We found a
70-80% decrease of TEL-JAK2 and PY-STAT5 levels in WT virus-infected
cells, whereas the infection efficiency was only 50%. This is probably
because of the underestimation of the infection efficiency of
WT-infected cells. These data suggest that kinase inhibition by SOCS1
is not sufficient to suppress the oncogenic potential of TEL-JAK2 and
that the C-terminal SOCS box is necessary for complete suppression of
the oncogenic potential of TEL-JAK2 by reducing the TEL-JAK2 protein
level.

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Fig. 2.
Expression levels of TEL-JAK2 after wild-type
and mutant SOCS1 infection. BF/TEL-JAK or BF/Bcr-Abl cells were
infected with retrovirus carrying the indicated cDNAs. One day
after infection, infection efficiency (I.E.) was determined
as the EGFP-positive fraction. Then the cells were lysed and
immunoblotted with the indicated antibodies. The phosphorylated forms
of TEL-JAK2 and Bcr-Abl detected with anti-PY
( PY) antibody were determined from their
molecular size. The intensity of the bands was quantified by a
densitometer and normalized with that of control virus-infected
cells.
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SOCS1 Promotes Proteasome-dependent Degradation of
TEL-JAK2--
We clarified the mechanism of reduction of the protein
level of TEL-JAK2 by coexpression of SOCS1 using a transient expression system in 293 cells. As shown in Fig.
3A, WT, but not dC40-SOCS1, also reduced the level of TEL-JAK2 in a dose-dependent
manner in 293 cells (Fig. 3A,
JAK2). Thus, SOCS1 reduced the TEL-JAK2 protein level SOCS-box-dependently not only in Ba/F3 cells
but also in 293 cells. Consequently, WT suppressed the
TEL-JAK2-mediated STAT activation ~50 times more efficiently than
dC40 (Fig. 3B). To confirm a similar kinase inhibitory
activity of WT and dC40, we performed an in vitro kinase
assay using the recombinant protein of the GST-tagged erythropoietin
receptor (EPOR) cytoplasmic domain as substrate (16). Consistently,
with the previous study using GST-JH1 as a constitutively activated
kinase, WT and dC40 could similarly suppress the in vitro
kinase activity of TEL-JAK2 (Fig. 3C, lanes 3 and
4). These results indicate that, although the SOCS box of
SOCS1 is not necessary for kinase inhibition, the inhibitory effect of
WT-SOCS1 was strongly enhanced by inducing degradation of TEL-JAK2
(Fig. 3, A and B).

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Fig. 3.
Effect of WT and dC40-SOCS1 on TEL-JAK2
stability in 293 cells and in vitro kinase
activity. 293 cells were transfected with pCDNA3 carrying
TEL-JAK2 (0.1 µg), pCDNA3 carrying Myc-tagged WT-SOCS1
(WT) or dC40-SOCS1 (dC40) (0.001, 0.01, 0.1, and
1.0 µg), -galactosidase (0.1 µg), and the APRE reporter
gene (0.5 µg) that can monitor STAT3 and STAT5 activity. The cell
lysate (30 µg of protein/lane) was prepared and subjected to
immunoblotting (A) with the indicated antibodies and a
luciferase assay (B). The values of -galactosidase
activity as an indicator of transfection efficiency are listed in
A. The membrane was reprobed with anti-STAT5
( STAT5) to show equal loading of the samples,
and the intensity of the bands was quantified by a densitometer. The
relative ratio (TJ/S ratio) of the band intensity of
TEL-JAK2 versus that of STAT5 is shown in A. In
C, FLAG-tagged TEL-JAK2 expressed in 293 cells was purified
by anti-FLAG antibody-conjugated protein G-Sepharose. After incubation
with 293 cell lysates containing WT- or dC40-SOCS1 for 1 h at
4 °C, the beads were reacted with GST-YY as a substrate in the
presence of 50 µM ATP for 5 min. The reaction mixture was
separated by SDS-polyacrylamide gel electrophoresis and immunoblotted
with PY and Myc antibodies. GST-YY was stained with Coomassie
Brilliant Blue (CBB).
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The half-life of TEL-JAK2 was examined in a metabolic pulse labeling
and chase experiment (Fig.
4A). The half-life of TEL-JAK2 was over 60 min, but coexpression of WT accelerated the decay of
TEL-JAK2, reducing its half-life to less than 30 min. dC40 did not
affect the half-life of TEL-JAK2. Accelerated degradation of TEL-JAK2
in the presence of WT, but not dC40, was also observed after cells were
treated with a protein synthesis inhibitor, cycloheximide (Fig.
4B). Normalized levels of TEL-JAK2 are shown as the TJ/S ratio against the levels of STAT5, which is a very stable protein (Fig.
4B).

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Fig. 4.
The SOCS box-dependent
proteasomal degradation of TEL-JAK2 by the coexpression of SOCS1.
A, pulse-chase experiment. 293 cells transfected with
TEL-JAK2 together with control vector (Vector), WT-SOCS1
(WT), or dC40-SOCS1 (dC40) were pulse-labeled
with [35S]methionine and cysteine for 15 min, divided
into four parts, and then chased for indicated periods. The TEL-JAK2
protein was immunoprecipitated with anti-JAK2. For proteasome inhibitor
treatment, 25 µM lactacystin (Lacta.) or MG132
was included in the medium throughout the pulse-chase periods. The
relative intensity of the bands quantified by a densitometer is shown.
B, cycloheximide treatment experiment. 293 cells were
transfected with TEL-JAK2 together with control vector
(Vector), WT, or dC40. After 18 h, cells were divided
into four parts and incubated for an additional 5 h. Then, the
cells were incubated with 50 µg/ml cycloheximide (CHX) for
the indicated periods and immunoblotted with JAK2 and STAT5
antibodies. The TJ/S ratio (see Fig. 3A legend) is listed.
C, 293 cells transfected with TEL-JAK2 (0.1 µg) and
WT-SOCS1 (lanes 1, 4, 0 µg; lanes 2,
5, 0.01 µg; lanes 3, 6, 0.1 µg)
were treated without (lanes 1-3) or with 25 µM lactacystin (lanes 4-6) for 6 h and
then immunoblotted with JAK2 and STAT5 antibodies.
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As shown in Fig. 4 (A and C), the rapid
degradation of TEL-JAK2 by coexpression of SOCS1 was significantly
delayed by treatment of the cells with two proteasome inhibitors,
lactacystin and MG132. These data suggest that SOCS1 promotes
proteasome-dependent degradation of TEL-JAK2.
Phosphorylation of the JH1 Domain Is Necessary for SOCS1-mediated
Degradation of JAK2--
Previously, Kamura et al. (20)
reported that SOCS1 overexpression did not affect wild-type JAK2
protein stability. We tried to resolve this discrepancy between
TEL-JAK2 and full-length JAK2. As shown in Fig.
5A, full-length JAK2 was much
less tyrosine-phosphorylated than TEL-JAK2 when expressed alone in 293 cells. Therefore, we suspect that the phosphorylation of the JH1 domain
is necessary for SOCS1-mediated degradation. Because glutathione
S-transferase (GST) is a dimer, the JH1 domain fused to GST
(GST-JH1) is another constitutively activated form of the JAK2 tyrosine
kinase domain (16). Like TEL-JAK2, GST-JH1 was markedly decreased in
its expression level when coexpressed with WT, but not with dC40 (Fig.
5B). Pulse-chase experiments revealed that WT-SOCS1 also
shortened the half-life of GST-JH1 (data not shown). Moreover, the
protein level of the phosphorylation-deficient mutant (FF) of GST-JH1
was not affected by SOCS1 (Fig. 5B). Therefore, reduction in
the GST-JH1 protein level by SOCS1 was dependent on tyrosine
phosphorylation or the activation of the JH1 domain.

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Fig. 5.
Phosphorylation of the JH1 domain is required
for SOCS1-induced degradation. A, 293 cells were
transfected with various concentrations of plasmids encoding either
full-length JAK2 (upper panel) or TEL-JAK2 (lower
panel). The cell lysate was immunoblotted with the indicated
antibodies. Plasmid concentrations are 0, 0.03, 0.1, 0.3, and 1.0 µg
from lanes 1 to 5. B, 293 cells were
transfected with 0.1 µg of GST-JH1 or GST-JH1/FF plasmids together
with increasing concentrations of WT-SOCS1 or dC40-SOCS1 (0, 0.01, 0.1, and 1.0 µg). The cell lysate was immunoblotted with the indicated
antibodies. The relative ratio (GJ/S ratio) of the band
intensity of GST-JH1 versus that of STAT5 is shown.
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Next, we examined whether SOCS1 promotes the degradation of activated
full-length JAK2. To achieve the activation of JAK2, JAK2 was fused to
gyrase B (Gyr-JAK2), and Gyr-JAK2 was dimerized by coumermycin (29). As
shown in Fig. 6A, coumermycin
treatment enhanced the tyrosine phosphorylation of Gyr-JAK2. Without
coumermycin, WT-SOCS1 did not affect the protein levels of JAK2 (Fig.
6B, left). However, WT, but not dC40, induced the
degradation of Gyr-JAK2 in the presence of coumermycin (Fig.
6B, Coum. (+),
JAK2).
These data suggest that the SOCS1-SOCS box can potentially induce the degradation of full-length JAK2 but that this process requires tyrosine
phosphorylation (or activation) of JAK2.

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Fig. 6.
SOCS1 accelerated degradation of full-length
JAK2 by forced dimerization. A, 293 cells transfected
with 0.1 µg of Gyrase-JAK2 fusion gene (Gyr-JAK2) were
treated with 0, 0.01, 0.1, 1, and 10 µM coumermycin for
1 h. The cell lysates were immunoprecipitated with JAK2, then
immunoblotted with JAK2 or PY antibodies. The asterisk
indicates the phosphorylated form. B, 293 cells transfected
with 0.1 µg of Gyr-JAK2 together with 0.1 µg of pCDNA3
(Vector), WT-SOCS1 (WT), and dC40-SOCS1
(dC40) were incubated with (+) or without ( ) 1 µM coumermycin in the presence of 50 µg/ml
cycloheximide for the indicated periods. Cell lysates were
immunoblotted with JAK2 and STAT5 antibodies. The relative ratio
(J/S ratio) of the band intensity of Gyr-JAK2
versus that of STAT5 is shown.
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The SOCS Box of SOCS1 Can Be Replaced with That of CIS1 but Not
That of SOCS3--
To examine the functional redundancy of the SH2
domain and the SOCS box for TEL-JAK2 degradation, we constructed
chimeric mutants among CIS1, CIS3/SOCS3, and SOCS1 (Fig.
7). The SH2 domain mutant R105E-SOCS1
exhibited a lesser effect on TEL-JAK2 protein stability. Thus, tight
binding of SOCS1 to the JH1 domain through the SH2 domain is necessary
for the degradation of TEL-JAK2. CIS1, which does not bind to JAK2, did
not induce TEL-JAK2 degradation, although CIS1 itself was unstable
compared with SOCS1 and SOCS3 (see
MYC blot). SOCS3, which also
suppresses JAK2 signaling (27), did not induce the degradation of
TEL-JAK2. As shown in Fig. 7 (A and B), the
mutant SOCS1 whose SOCS box was replaced with that of CIS1
(1/C) reduced the TEL-JAK2 level, whereas the mutant
replaced with the SOCS box of SOCS3 (1/3) did not. 1/C
induced the degradation of TEL-JAK2 more strongly than did WT-SOCS1.
This indicates that the SOCS box of SOCS1 can be replaced with that of
CIS1 but not with that of SOCS3. On the other hand, the SOCS3 mutant
whose SOCS box was replaced with that of SOCS1 (3/1) did not
affect TEL-JAK2 stability. SOCS3 and 3/1 bound to the JH1 domain, but their affinity was much lower than that of WT-SOCS1 or 1/C (27). All
these observations were confirmed when different amounts of wild-type
or mutant SOCS/CIS genes were expressed (Fig. 7B). These data suggest that the particular SOCS box and its tight binding or
proper orientation to the JH1 domain are necessary for the promotion of
the degradation of TEL-JAK2.

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Fig. 7.
The effect of SOCS box swapping on TEL-JAK2
degradation. A, schematic structures of SOCS1, CIS1,
and SOCS3 and their chimeric constructs are shown on the
left. 293 cells transfected with TEL-JAK2 and each construct
were incubated with 50 µg/ml cycloheximide for the indicated periods
and immunoblotted with JAK2, STAT5, and MYC antibodies. The
TJ/S ratio is listed. B, TEL-JAK2 (0.1 µg) and WT or
indicated mutant SOCS constructs (0, 0.01, or 0.1 µg of plasmids)
were transfected into 293 cells. Total cell lysates were blotted with
the indicated antibodies.
|
|
SOCS1-SOCS Box Promotes the Ubiquitination of TEL-JAK2, and
Dominant Negative Cul-2 Inhibits TEL-JAK2 Degradation--
Because
proteasome inhibitors suppressed SOCS1-mediated degradation of
TEL-JAK2, ubiquitination of TEL-JAK2 may be involved in the degradation
process. The SOCS box is similar to the BC box of VHL protein, which
interacts with the Elongin B,C complex. The VHL-Elongin B,C (VBC)
complex further recruits Cul-2 and Rbx-1 as subunits of ubiquitin
ligase. Rbx-1 has a RING-finger motif with which E2
ubiquitin-conjugating enzymes are suggested to interact. Kamura
et al. (23) and Zhang et al. (24) demonstrated
that the SOCS1-SOCS box binds to the Elongin B,C complex. Therefore, the SOCS box has been hypothesized to form a complex with Cul-2 and
Rbx-1 and function as an E3 ubiquitin ligase (25). However, neither the
existence of this complex nor the SOCS box-dependent ubiquitination of the target molecule has been demonstrated to date.
First, we examined the ubiquitination of TEL-JAK2 and GST-JH1 using
HA-ubiquitin. As shown in Fig. 8
(A and B), the ubiquitination of TEL-JAK2 and
GST-JH1 was markedly enhanced when they were coexpressed with WT-SOCS1,
whereas the dC40 mutant did not induce their ubiquitination. Next, we
examined the interaction between the SOCS box and Cul-2 (Fig.
9A). HA-tagged Cul-2 was
coprecipitated with WT, but not with dC40, in 293 cells. We also
confirmed the binding of the Elongin B,C complex with WT-SOCS1 (data
not shown). Thus, similarly to VHL, the SOCS1-SOCS box can interact
with the Elongin B,C complex and Cul-2. Overexpression of wild-type
Cul-2 (WT-CUL2) accelerated SOCS1-induced TEL-JAK2
degradation (Fig. 9, B and C), whereas SOCS1-induced degradation of TEL-JAK2 was almost completely blocked when mutant Cul-2 containing R452C substitution (R452C-CUL2)
was coexpressed (Fig. 9, B and C). R452 of Cul-2
corresponds to Arg-488 of Cdc53, and mutation of this residue may
disrupt the interaction with Rbx1 (30, 31). Thus, R452C-Cul-2
functioned as a dominant negative form of wild-type Cul-2. Taken
together, these observations suggest that the functional recruitment of
Elongin B,C and the Cul-2 complex through the SOCS box accelerated the
ubiquitination and proteasome-dependent degradation of
TEL-JAK2.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 8.
SOCS1 promotes the ubiquitination of
activated JAK2. 293 cells were transfected with 1 µg TEL-JAK2
(A) or GST-JH1 (B) and 0.5 µg of
HA-ubiquitin/pCDNA3 together with a 0.01-µg control vector
(lane 1) or plasmids carrying dC40 (lane 2) or
WT-SOCS1 (lane 3). To keep the levels of TEL-JAK2,
relatively low amounts of SOCS1 plasmid (0.01 µg) were used. The cell
extracts were precipitated with JAK2 (A) or GSH-Sepharose
(B) and then blotted with anti-HA
( HA) and GST antibodies. Total cell lysates
(TCL) were also blotted with the indicated antibodies.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9.
A dominant negative Cul-2 inhibits the
SOCS1-mediated degradation of TEL-JAK2. A, 293 cells
were transfected with HA-Cul-2 and FLAG-tagged WT-SOCS1 or dC40-SOCS1
and then immunoprecipitated with anti-FLAG
( FLAG) or control (unrelated monoclonal)
antibodies. The immunoprecipitates or total cell lysates were blotted
with the indicated antibodies. The asterisk shows a
nonspecific IgG band. B, 293 cells were transfected with
TEL-JAK2 and either wild-type Cul-2 (WT-CUL2) or R452C
mutant Cul-2 (R452C-CUL2) (0.5 µg) together with 0.01, 0.1, or 1.0 µg of WT-SOCS1 plasmids. The cell lysates were
immunoblotted with the indicated antibodies. The TJ/S ratio is listed.
C, 293 cells transfected with TEL-JAK2 and WT-SOCS1,
together with either WT-Cul-2 or R452C mutant Cul-2, were incubated
with 50 µg/ml cycloheximide for the indicated periods and
immunoblotted with JAK2 or STAT5 antibodies. The TJ/S ratio is
listed.
|
|
 |
DISCUSSION |
It has been demonstrated that the SOCS boxes of VHL and SOCS1
interact with the Elongin B,C complex. Although the VHL-SOCS box is
proposed to recruit Cul-2 and Rbx-1 and function as an E3 enzyme
subunit, the function of the SOCS box of the CIS/SOCS family has not
been resolved. In this paper, we demonstrate that the SOCS box of SOCS1
is critically involved in the
ubiquitin/proteasome-dependent degradation of TEL-JAK2 and
phosphorylated JAK2.
By analogy with the VHL complex, the SOCS boxes of SOCS1 and CIS1 were
suggested to be involved in the ubiquitin/proteasome-dependent degradation of JAK2 and the EPO receptor, respectively (25, 32).
Indeed, CIS1 itself is shown to be ubiquitinated and degraded very
rapidly (32). Because the tyrosine phosphorylation of the EPO receptor
in response to EPO was sustained by the treatment of cells with
proteasome inhibitors, we proposed the possibility that the
phosphorylated EPO receptor-CIS1 complex becomes a target of the
proteasome. Zhang et al. (24) also suggested that the SOCS
box of SOCS3 leads the SOCS3 protein to the degradation pathway, because SOCS3 degradation was blocked by proteasome inhibitors. In this
case, the SOCS box may be involved in ubiquitination of SOCS3 itself.
However, there is no direct evidence that SOCS box-containing proteins
have an E3-like ubiquitin-transfer activity with the target molecule.
Kamura et al. (23) reported that the Elongin B,C complex
increased the stability of SOCS1 and that SOCS1 overexpression did not
induce the degradation of JAK2.
It is notable that SOCS3 did not affect TEL-JAK2 degradation, because
SOCS3 has a SOCS box and can bind to JH1. The SOCS3-SOCS box was not
functional when fused to the SOCS1 N-terminal region and the SH2 domain
(1/3 mutant in Fig. 7). We found that SOCS3 lacking a SOCS
box is much more stable than wild-type SOCS3 in Ba/F3 cells (data not
shown). This suggests that the SOCS box of SOCS3 may not regulate the
protein levels of the target molecule but rather destabilize SOCS3
itself. Like CIS1, SOCS3 has been shown to be an unstable molecule that
is degraded by the ubiquitin/proteasome system (24). Therefore, the
SOCS box may be involved in inter- as well as intramolecular ubiquitin
transfer, depending on the structure of the SOCS box. Our experiments
do not exclude the possibility that SOCS-3 has another target besides
JAKs or cytokine receptors.
Using constitutively active forms of JAK2, we demonstrated that the
SOCS1-SOCS box could indeed induce ubiquitination and proteasome-dependent degradation of JAK2. Our study
indicated that the SOCS-box-dependent degradation of JAK2
requires the phosphorylation of JAK2 and strong interaction with SOCS1.
Confirming the result of Kamura et al. (23), SOCS1 did not
induce the degradation of full-length JAK2 when these two molecules
were simply expressed in 293 cells. This was probably due to the low
efficiency of the phosphorylation of JAK2 molecules (see Fig.
5A). Similarly to TEL-JAK2, phosphorylated full-length JAK2
by forced dimerization was degraded by coexpression of SOCS1. Thus,
acceleration of the degradation of SOCS1 is dependent on the activation
(or phosphorylation) of JAK2.
It is still not clear whether this accelerated degradation of
full-length JAK2 occurs under physiological conditions. Callus and
Mathey-Prevot (33) reported that proteasome-dependent
degradation is the major mechanism of the down-regulation of JAK2 after
stimulation with IL-3 in Ba/F3 cells. SOCS1 or other CIS/SOCS members
may be involved in this process. However, we and other researchers have
reported that the SOCS box of SOCS1 has no apparent role in the
suppression of cytokine-dependent signaling in 293 cells (16, 17). In these assays, SOCS1 was overexpressed by transient transfection before cytokine stimulation. We tried to see the effect of
SOCS1 on the degradation of activated JAK2 in response to IFN
using
embryonic fibroblasts from wild-type and SOCS1
/
mice. We could
confirm that IFN
-induced JAK2 phosphorylation was prolonged in
SOCS1
/
fibroblasts; however, we did not detect any decrease in the
protein level of JAK2 in either cell types. This is probably because
only a small fraction of JAK2 is phosphorylated in response to IFN
.
Further studies are necessary to address the role of the SOCS box on
JAK ubiquitination and degradation in physiological conditions.
After completion of this study, De Sepulveda et al. (34)
reported that SOCS-1 accelerated the ubiquitination and degradation of
Vav. Vav and SOCS1 form a protein complex through interactions between the Vav N-terminal regulatory region and the SH2 domain of
SOCS1 in a phosphotyrosine-independent manner. Thus, SOCS1 may induce
the degradation of Vav when expressed at very high levels. It has not
been reported whether or not the SOCS box of SOCS1 is necessary for the
ubiquitination of Vav. Therefore, the molecular mechanism of the
ubiquitination of Vav by SOCS1 is still unclear. Moreover, it has not
been demonstrated that such phosphorylation-independent interaction can
occur in physiological conditions. However, it would be interesting to
determine whether SOCS1 can induce ubiquitination and degradation of
phosphorylated signaling molecules other than JAKs. In future studies,
it will be possible to verify the physiological role of the SOCS box of
SOCS1 by introducing mutations in the SOCS box using a knock-in
strategy in mice.
SOCS-box-mediated ubiquitination and degradation of activated JAK2 are
reminiscent of c-Cbl-mediated ubiquitination and degradation of the
activated receptor tyrosine kinases (35-37). Other groups as well as
ours have shown that the c-Cbl RING-finger domain interacts with the E2
ubiquitin-conjugating enzyme, thereby accelerating the ubiquitination
of the epidermal growth factor receptor or the PDGF receptor with which
c-Cbl binds through its SH2 domain. Thus, ubiquitination and
proteasome-dependent degradation of activated tyrosine
kinases by a specific E3 complex may be a common mechanism in the
down-regulation of tyrosine kinases. In addition, we have reported that
c-Cbl suppressed EPO-induced STAT5 activation in collaboration with APS
(an adaptor containing PH and SH2 domains) (38). Because Cbl family
members possess the SH2 domain, multiple tyrosine phosphorylation
sites, and proline-rich motifs that interact with SH3 domains, they can
interact with many tyrosine-phosphorylated proteins as well as SH2- and
SH3-containing proteins. Therefore, Cbl could also be involved in
ubiquitin/proteasome-dependent degradation of activated
cytokine receptors and their downstream signal transducers. Our study
also provides a basis for the inhibition of oncogenic, constitutively
active tyrosine kinases by ubiquitination and degradation using the
SOCS box or the RING-finger domain.
 |
ACKNOWLEDGEMENTS |
We thank H. Ohgusu for excellent technical
assistance, Dr. G. Gilliland for critical reading of the manuscript,
Dr. T. Oda for p210 Bcr-Abl, and Dr. S. Watanabe for Gyr-JAK2.
 |
FOOTNOTES |
*
This work was supported by grants from the Ministry of
Education, Science, Sports, and Culture of Japan, the Fukuoka Cancer Research Foundation, the Mochida Memorial Foundation, Haraguchi Memorial foundation, Japan Research Foundation for Clinical
Pharmacology, and the Mitsubishi Foundation.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.:
81-942-37-6313; Fax: 81-92-642-6825; E-mail:
yakihiko@bioreg.kyushu-u.acjp.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010074200
 |
ABBREVIATIONS |
The abbreviations used are:
JAK, JAK family
tyrosine kinases;
STAT, signal transducers and activators of
transcription;
PDGF, platelet-derived growth factor;
IL-3, interleukin-3;
IFN
, interferon
;
Cul-2, Cullin-2;
VHL, von
Hippel-Lindau disease;
Elongin B, C, Elongin B and C complex;
PCR, polymerase chain reaction;
HA, hemagglutinin;
WT, wild type;
dC40, deletion mutant lacking 40 amino acids at the C terminus;
GST, glutathione S-transferase;
EPOR, erythropoietin receptor;
EGFP, expressed green fluorescence protein.
 |
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