Mechanism of Inhibition of Growth Hormone Receptor Signaling by Suppressor of Cytokine Signaling Proteins
Johnny A. Hansen,
Karen Lindberg,
Douglas J. Hilton,
Jens H. Nielsen and
Nils Billestrup
Hagedorn Research Institute (J.A.H., K.L., J.H.N., N.B.)
DK-2820 Gentofte, Denmark
The Walter and Eliza Hall Institute
for Medical Research (D.J.H.) Parkville, Victoria 3052,
Australia
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ABSTRACT
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In this study we have investigated the role
of suppressor of cytokine signaling (SOCS) proteins in GH
receptor-mediated signaling. GH-induced transcription was inhibited by
SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing
protein (CIS) had no effect. By using chimeric SOCS proteins it
was found that the ability of SOCS proteins to inhibit GH-mediated
transcription was located in the amino-terminal 4080 amino acids. In
SOCS-3, 46 amino acids C-terminal to the SH2 domain were required for
the inhibitory activity, while a truncated SOCS-1 having only 2 amino
acids C-terminal to the SH2 domain was able to inhibit GH-mediated
transcription. Both SOCS-1 and SOCS-3 were able to inhibit GH-induced
STAT5 (signal transducer and activator of transcription)
activation. SOCS-1 inhibited the tyrosine kinase activity of Janus
kinase 2 (JAK2) directly, while SOCS-3 only inhibited JAK2 when
stimulated by the GH receptor. All four SOCS proteins were able to bind
to a tyrosine-phosphorylated glutathione-S-transferase-GH
receptor fusion protein, and SOCS-3 required the same 46 C-terminal
amino acids for GH receptor binding as it did for inhibition of
GH-mediated transcription and STAT5 activation. These data suggest that
SOCS-1 and -3 can suppress GH-induced transcriptional activity,
presumably by inhibiting the kinase activity of JAK2 either directly in
the case of SOCS-1 or via binding to the tyrosine-phosphorylated GH
receptor in the case of SOCS-3.
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INTRODUCTION
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While much progress has been made toward understanding cytokine
receptor stimulation of the Janus kinase/signal transducer and
activator of transcription (JAK/STAT) pathway, much less is
known about how these signals are turned off. Recently a family of
proteins involved in the suppression of cytokine signaling has been
identified (1, 2, 3). The SOCS family of proteins consists of eight
members: CIS and SOCS-1 through 7. CIS was originally identified in
1995 as an immediate early gene induced by interleukin 3 (IL-3) and
erythropoietin (Epo) in BaF3 cells (4). CIS was found to be able to
bind to the phosphorylated Epo and IL-3 receptors and to suppress the
proliferation of hematopoietic cells (BaF3 and FDCP-1) in response to
IL-3 (4). SOCS-1 was subsequently identified as a factor capable of
inhibiting IL-6-induced differentiation of monocytic leukemic M1 cells
into macrophages (2), and at the same time a factor termed JAB was
cloned based on its ability to interact with the kinase domain of the
JAK2 kinase (5) and SSI-1 was cloned based on homology to the SH-2
domain of STAT3 (6). Analysis of the sequences of SOCS-1, JAB, and
SSI-1 revealed that these three factors were identical.
The induction of SOCS gene expression by cytokines has been
reported in a number of different cell types both in vitro
and in vivo. Stimulation of CIS and SOCS-1, -2, and -3 mRNA
expression in bone marrow cells by Epo, thrombopoietin,
granulocyte-colony stimulating factor (G-CSF), leukemia-inhibiting
factor (LIF), granulocyte macrophage-colony stimulating factor
(GM-CSF), IL-1, -2, -3, -4, -6, -7, -12, -13, and interferon-
(IFN-
) has been reported (2). In this study great variation in
inducibility was observed with some cytokines inducing the expression
of all four SOCS mRNA (GM-CSF and IFN
) while others induced only a
subset of the SOCS genes. In BaF3 cells, IL-2 and IL-3 induced the
expression of CIS (4, 7), and in M1 cells both IL-6 and LIF induced the
induction of SOCS-1 and CIS while no induction of SOCS-2 and -3 was
observed. In 3T3-F442A preadipocytes, GH induced a rapid and transient
expression of SOCS-3 and, to a lesser extent, SOCS-1 (8). In the
hypothalamus, leptin has been reported to induce the expression of
SOCS-3 mRNA, and increased levels of SOCS-3 mRNA were observed in the
hypothalamus of the obese lethal yellow (Ay/a) mouse, which
is known to exhibit leptin resistance (9).
SOCS-1 was originally identified by its ability to inhibit IL-6-induced
M1 cell differentiation (2) and by its binding to the kinase domain of
JAK2 (5), indicating an inhibitory action in IL-6 signaling at the
level of the JAK2 kinase. Subsequently it was demonstrated that SOCS-1
inhibited the intrinsic kinase activity of all four JAK
family members when overexpressed in COS or 293 cells (5). In
accordance with this observation, it was reported that SOCS-1 inhibited
IL-6-induced tyrosine phosphorylation of STAT3, the IL-6
receptor-signaling subunit gp130 and JAK2 (10). In contrast, it was
found that SOCS-3 and CIS were unable to inhibit the intrinsic kinase
activity of JAK2, and it was proposed that these SOCS proteins inhibit
cytokine signaling at a step distal to JAK activation. CIS has been
shown to interact directly with the phosphorylated Epo and GM-CSF
receptors, and it was suggested that this binding might prevent the
recruitment of STAT factors to the receptor/JAK complex.
The SOCS proteins can be divided into three domains: the N
terminus exhibiting little sequence identity among the SOCS proteins,
the centrally located SH2 domain, and the SOCS box located in the C
terminus (2). Recently it has been found that both the N terminus and
SH2 domain of SOCS-1 were required for suppression of IL-6 and LIF
signaling and inhibition of JAK activity (11). The SH2 domain and an
additional N-terminal 12 amino acids of SOCS-1 were found to be
required for interaction with tyrosine 1007 in the activation loop of
JAK2 and thus inhibition of kinase activity (12). The SOCS box of
SOCS-1 is not required for its inhibitory activity but rather seems to
be involved in protein stability (13), possibly by interacting with
elongins B and C (14).
GH preferentially induces the expression of SOCS-3 (8), and since it
has been observed that SOCS-3, in contrast to SOCS-1, could not inhibit
the intrinsic kinase activity of JAK2, we have in this study
investigated the mechanism by which SOCS-1 and SOCS-3 inhibit signaling
by the GH receptor. We have identified specific domains of SOCS-1 and
SOCS-3 involved in the suppression of GH-mediated transcription, STAT5
activation, and JAK2 activity, and binding to the
tyrosine-phosphorylated GH receptor.
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RESULTS
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The N-Terminal Domain of SOCS-1 and SOCS-3 Confers Specificity for
the Inhibition of GH-Induced Transcription
The effect of SOCS expression on GH-induced transcription
was analyzed by transient transfections of CHO cells with a
GH-responsive STAT5-dependent reporter. In cells not transfected with
SOCS expression plasmids, GH induced a 4-fold increase in reporter
activity (Fig. 1
). Coexpression of either
SOCS-1 or SOCS-3 inhibited GH-induced transcription in a dose-dependent
manner, while coexpression of CIS did not affect GH induction.
Expression of SOCS-2 resulted in a slightly higher induction by GH as
compared with control.

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Figure 1. Effect of SOCS on GH-Stimulated Transactivation of
the Spi 2.1 Promoter
CHO cells were transiently transfected with the Spi 2.1 promoter/CAT
construct, a GH receptor expression vector, together with the indicated
amount of SOCS expression vectors. After transfection, extracts were
prepared from cells cultured in the absence or presence of 20
nM GH. CAT activity was determined and normalized to
ß-galactosidase activity to control for transfection efficiency. The
level of CAT activity, in the absence of GH and SOCS expression
vectors, was given the value of 1. The results presented correspond to
the mean values of three independent experiments, with the error bars
representing the mean ± SD.
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To identify the domains of the SOCS proteins that impart the
specificity in the ability of SOCS-1 and SOCS-3 to inhibit GH-induced
transcription, we generated chimeric SOCS expression vectors by
swapping of the N-terminal domain and the SOCS box as shown in Fig. 2
, B and C. The construction of chimeric
SOCS proteins was facilitated by the introduction of specific
restriction sites between the N-terminal domain and the SH2 domain and
between the SH2 domain and the SOCS box. The activity of the SOCS
proteins containing these introduced restriction sites was found to be
identical to that of the wild-type SOCS proteins (Fig. 2A
vs. Fig. 1
). Analyzing the chimeric SOCS proteins revealed
that the SOCS box was interchangeable and did not confer specificity,
since SOCS proteins having the SOCS box of CIS and the N-terminal and
SH2 domains of SOCS-1 or SOCS-3 were able to inhibit GH-induced
transcription (Fig. 2B
). In contrast, the N-terminal domain was able to
confer specificity as shown by the inhibitory effect of chimeric SOCS
proteins having the N-terminal domain of SOCS-1 or SOCS-3 and the SH2
domain and the SOCS box from CIS (Fig. 2C
).

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Figure 2. The Role of SOCS Protein Domains in the Inhibition
of GH-Stimulated Transactivation of the Spi 2.1 Promoter
CHO cells were transiently transfected with the Spi 2.1 promoter/CAT
construct, together with plasmids expressing the GH receptor,
ß-galactosidase, alone or together with expression vectors (1 µg)
encoding mutated or chimeric SOCS proteins. The mutated and chimeric
SOCS proteins are illustrated schematically to the left
(A, B, and C). The black box represents the linker
domain. CAT activity was determined and normalized to the activity
observed in the absence of hGH. The results presented are the mean
values of three independent experiments, with the error bars
representing the mean ± SD.
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SOCS-3, but Not SOCS-1, Requires the Linker Domain between the SH2
Domain and the SOCS Box for Functional Activity
The functional role of the SOCS box and the linker domain between
the SOCS box and the SH2 domain was investigated by generating
truncated versions of SOCS-1 and SOCS-3 as shown schematically in Fig. 3
. The linker domain in SOCS-1 consists
of only 5 amino acids while that of SOCS-3 has 47 amino acids (Fig. 3C
). The activity of SOCS-1 was not altered by removing the SOCS box
and 3 amino acids of the linker domain, leaving only 2 amino acids
C-terminal to the SH2 domain, whereas in SOCS-3 46 amino acids of the
linker domain were required for function. Thus, in both SOCS proteins,
the SOCS box was dispensable, but SOCS-3 requires amino acids located
between positions 177 and 184.

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Figure 3. The Role of the C-Terminal Domain of SOCS-1 and
SOCS-3 in the Inhibition of GH-Stimulated Transactivation of the Spi
2.1 Promoter
CHO cells were transiently transfected with the Spi 2.1 promoter/CAT
construct, together with plasmids expressing the GH receptor,
ß-galactosidase, alone or together with expression vectors (1 µg)
encoding either the wild-type or truncated versions of SOCS-1 or SOCS-3
as illustrated schematically in panel A (SOCS-3) and panel B (SOCS-1).
CAT activity was determined and normalized to the activity observed in
the absence of GH. The results presented are the mean values of three
independent experiments, with the error bars representing the mean
± SD. C, The amino acid sequence of the region between the
SH2 domain and the SOCS-box in the murine SOCS-3 and SOCS-1 protein.
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SOCS-1 and SOCS-3 Inhibit STAT5 DNA Binding Activity
Since GH activation of reporter activity is mediated by STAT5, we
investigated the ability of SOCS proteins to inhibit GH-induced STAT5
activation by measuring the DNA binding activity of STAT5 by
electrophoretic mobility shift assay. Nuclear extracts from 293 cells
transfected with the GH receptor and STAT5, with or without SOCS
expression vectors, were analyzed using the STAT5 binding element from
the Spi 2.1 promoter. In the absence of SOCS expression or in cells
expressing CIS and SOCS-2, STAT5 DNA binding was observed (Fig. 4
). In cells transfected with SOCS-1 or
SOCS-3, no STAT5 DNA binding could be detected. Western blot analysis
using total cell extracts revealed that STAT5 was expressed at
comparable levels in all cell extracts (Fig. 4B
). The level of SOCS
protein expression was highest in cells transfected with CIS and lowest
in SOCS-3-transfected cells (Fig. 4C
).

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Figure 4. The Role of SOCS Proteins in the Activation of
STAT5 DNA Binding Activity Induced by GH
A, Electrophoretic mobility shift assay using the STAT5 binding element
from the Spi 2.1 promoter as a probe and nuclear extracts made from 293
cells transiently transfected with plasmids expressing the GH receptor
and HA-STAT5, alone (lanes 12) or together with an expression vector
encoding the indicated FLAG-SOCS protein (lanes 310). Serum-starved
cells were cultured in the absence or presence of 20 nM GH
for 5 min before nuclear extract preparation. B, Western blot analysis
was performed on total cell lysates using anti-HA antibodies to detect
STAT5; C, anti-FLAG antibodies were used to detect SOCS proteins. The
results shown are representative of three independent experiments.
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We also investigated the abilities of selected chimeric and truncated
SOCS proteins to inhibit STAT5 DNA binding (Figs. 5
and 6
).
It was demonstrated that the same chimeric SOCS proteins that could
inhibit GH-induced transcription also inhibited STAT5 activity.
Furthermore, we found that the region of the linker domain in SOCS-3
required for inhibition of transcription was also required for
inhibition of STAT5 activity.

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Figure 5. The Role of SOCS Domains in the Inhibition of
GH-Induced STAT5 Activation
A, Electrophoretic mobility shift assay using the STAT5 binding element
from Spi 2.1 promoter as a probe and nuclear extracts made from 293
cells transiently transfected with plasmids expressing the GH receptor
and HA-STAT5, alone (lanes 12) or together with an expression vector
encoding the indicated chimeric FLAG-SOCS proteins (lanes 310).
Serum-starved cells were cultured in the absence or presence of 20
nM GH for 5 min before nuclear extract preparation. B,
Western blot analysis was performed on total cell lysates using anti-HA
antibodies to detect STAT5; C, anti-FLAG antibodies were used to detect
SOCS proteins. The results shown are representative of three
independent experiments.
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Figure 6. The Role of the C-Terminal Part of SOCS-3 in the
Inhibition of GH-Induced STAT5 Activation
A, Electrophoretic mobility shift assay using the STAT5 binding element
from the Spi 2.1 promoter as a probe and nuclear extracts made from 293
cells transiently transfected with plasmids expressing the GH receptor
and HA-STAT5, alone (lanes 12) or together with an expression vector
encoding the indicated truncated FLAG-SOCS-3 proteins (lanes 312).
Serum-starved cells were cultured in the absence or presence of 20
nM GH for 5 min before nuclear extract preparation. B,
Western blot analysis was performed on total cell lysates using anti-HA
antibodies to detect STAT5; C, anti-FLAG antibodies were used to detect
SOCS proteins. The results shown are representative of three
independent experiments.
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SOCS-1 Inhibits the Intrinsic Activity of JAK2
Previous work has demonstrated that the interaction of SOCS-1 with
JAK kinases markedly reduces their kinase activity (5). To investigate
the possibility that SOCS-1 and SOCS-3 could influence STAT5 activation
by inhibition of the JAK2 kinase activity, JAK2 was transiently
expressed in 293 cells, alone or together with different SOCS-encoding
plasmids. Overexpression of the JAK2 protein in these cells resulted in
a constitutive phosphorylation of the kinase, presumably because the
high level of expression allows dimerization and
trans-phosphorylation to take place in the absence of GH and
GH receptor (Fig. 7
). By
immunoprecipitation of JAK2 followed by Western blot analysis using
antiphosphotyrosine antibodies, we observed that only SOCS-1 and not
SOCS-2, SOCS-3, or CIS was able to inhibit tyrosine phosphorylation of
the kinase (Fig. 7
).

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Figure 7. The Role of SOCS Proteins in JAK2 Activation
HEK 293 cells were transiently transfected with a JAK2- encoding
plasmid (1 µg) alone or together with a SOCS expression vector (10
µg). After transfection, cell lysates were used for
immunoprecipitation (IP) using JAK2 antibodies. A,
Immunoprecipitated JAK2 was separated by SDS-PAGE followed by Western
blot analysis using antiphosphotyrosine antibodies ( PY). B, The blot
shown in panel A was stripped and reprobed with anti-JAK2 antibodies.
C, Cell lysates were analyzed by Western blot analysis using anti-FLAG
antibodies.
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SOCS Proteins Bind to the Tyrosine-Phosphorylated GH Receptor
The fact that both SOCS-1 and SOCS-3 were found to inhibit
GH-induced transcription and STAT5 activation but that only SOCS-1 was
able to inhibit the intrinsic activity of JAK2, suggested to us that
SOCS-3 might require the presence of the GH receptor to exhibit its
inhibitory action on GH signaling. Previously, CIS has been
demonstrated to associate with tyrosine-phosphorylated IL-3ß and
Epo receptors (4). Therefore, we analyzed the ability of SOCS
proteins to bind tyrosine-phosphorylated
glutathione-S-transferase (GST)-GH receptor fusion protein
in a GST-binding assay. Human 293 cells were transiently transfected
with the individual SOCS-encoding plasmids, and cell lysates were used
for binding to GST-GH receptor fusion proteins. By this procedure, it
was demonstrated that all four SOCS proteins were able to associate
with GST-GH receptor 455638 fusion protein only when the fusion
protein was tyrosine phosphorylated (Fig. 8
). The amount of SOCS protein applied in
each reaction was evaluated by including a sample of cell lysate from
nontransfected cells and from each of the cell lysates used for the
binding reactions (lanes 79 and 1618).

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Figure 8. Binding of SOCS Proteins to Tyrosine-Phosphorylated
GST-GH Receptor 455638 Fusion Protein
Cell lysates made from 293 cells transiently transfected with
expression plasmids encoding FLAG-tagged SOCS protein were incubated
together with the indicated GST fusion protein coupled to
glutathione-Sepharose beads. The bound proteins were resolved by
SDS-PAGE and analyzed by Western blot analysis using anti-FLAG
antibodies. Cell lysates from either nontransfected cells (lanes 7 and
16) or from cells transiently transfected with the different FLAG-SOCS
encoding plasmids (lanes 8, 9, 17, and 18) were included for
comparison. The results shown are representative of three independent
experiments.
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We have previously shown that STAT5 can bind to three
phosphotyrosines in the GH receptor and that these three tyrosines
(534, 566, and 627) are required for GH activation of transcription and
STAT5 activation (15, 16). To test whether SOCS-3 could bind
to the same three phosphotyrosines as STAT5, we tested the ability of
specific phosphopeptides derived from the GH receptor sequence to
inhibit the binding of SOCS-3 to the tyrosine-phosphorylated GST-GHR
fusion protein. We found that a phosphopeptide containing tyrosine 487
was the most effective in competing for SOCS-3 binding. Phosphopeptides
containing tyrosines 534, 595, and 627 also competed for SOCS-3
binding, while phosphopeptide 566 only inhibited slightly (Fig. 9
). None of the nonphosphorylated
peptides affected SOCS-3 binding to the GST-GH receptor fusion
protein.

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Figure 9. Inhibition of SOCS-3 Binding to the
Tyrosine-Phosphorylated GST-GH Receptor 455638 Fusion Protein by
Phosphopeptides Derived from the GH Receptor
Cell lysates from 293 cells transiently transfected with the
FLAG-SOCS-3 encoding plasmid were incubated together with
tyrosine-phosphorylated GST-GH receptor 455638 fusion protein coupled
to glutathione-Sepharose beads in the presence of the indicated peptide
in a concentration of 300 µM. Precipitated proteins were
subjected to SDS-PAGE and analyzed by Western blot analysis using
FLAG antibodies. The results shown are representative of three
independent experiments.
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To test whether SOCS-3 could directly compete for STAT5 binding
to the tyrosine-phosphorylated GST-GH receptor fusion protein, we
included increasing amounts of SOCS proteins in a GST-GH receptor STAT5
binding assay (Fig. 10
). From this
experiment it was observed that none of the SOCS proteins could inhibit
STAT5 binding to the receptor, even though an approximately 50-to
250-fold molar excess of SOCS protein compared with STAT5 was used.
This finding suggests that SOCS-3 does not inhibit STAT5 binding to the
GH receptor and that the inhibition of STAT5 by SOCS-3 cannot be
explained by competition for binding sites within the GH receptor.

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Figure 10. The Effect of SOCS Proteins on STAT5 Binding to
the Tyrosine-Phosphorylated GST-GH Receptor 455638 Fusion Protein
A, Cell lysates from STAT5-transfected 293 cells were incubated
together with the GST-GH receptor 455638 TKX1 fusion protein coupled
to glutathione-Sepharose beads. Increasing amounts (50- and 200-fold
molar excess) of cell lysate from 293 cells transiently transfected
with the indicated SOCS-encoding plasmid was included in the reaction.
Precipitated proteins were resolved by SDS-PAGE and analyzed by Western
blot analysis using anti-HA antibodies. Lanes 8 and 9 contain cell
lysates from nontransfected or STAT5-transfected 293 cells,
respectively. B, Cell lysates from either nontransfected cells (lane
10) or cells transiently transfected with the indicated SOCS expression
plasmid are included for comparison.
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To analyze the functional domain of SOCS-3 involved in the inhibition
of GH-induced transcription and STAT5 activation, we used GST-GH
receptor binding to examine the truncated forms of SOCS-3. The same
region of the linker domain that was required for inhibition of
GH-induced transcription and STAT5 activation was also required for
binding to the tyrosine-phosphorylated GST-GH receptor fusion
protein (Fig. 11
). These
observations indicate that SOCS-3 binding to the GH receptor plays an
important role in the mechanism by which it suppresses GH-mediated
signaling.

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Figure 11. Binding of Truncated SOCS-3 to the
Tyrosine-Phosphorylated GST-GH Receptor 455638 Fusion Protein
Cell lysates made from 293 cells transiently transfected with different
expression plasmids encoding either wild-type or truncated versions of
FLAG-tagged SOCS-3 protein were incubated together with the
tyrosine-phosphorylated GST-GH receptor 455638 fusion protein coupled
to glutathione-Sepharose beads. The proteins were resolved by SDS-PAGE
and analyzed by Western blot analysis using anti-FLAG antibodies.
Lysates from either nontransfected cells (lane 6) or from cells
transiently transfected with the different expression plasmids encoding
either wild-type or truncated versions of FLAG-tagged SOCS-3 protein
(lanes 711) are included.
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SOCS-3 Inhibits JAK2 Activity by a GH Receptor-Dependent
Mechanism
To investigate the hypothesis that SOCS-3 can inhibit GH
signaling by inhibiting JAK2 kinase activity in a GH receptor-dependent
manner, we transiently transfected 293 cells with JAK2 alone or
together with a SOCS-3 encoding plasmid in combination with an
increasing amount of a GH receptor-encoding plasmid. In contrast to the
experiment with overexpression of the JAK2 protein in the 293 cells,
which resulted in a constitutive tyrosine phosphorylation of the kinase
(Fig. 7
), a reduced amount of JAK2-encoding plasmid was used in this
experiment, resulting in a GH-inducible and GH receptor-dependent
tyrosine phosphorylation of JAK2. By immunoprecipitation of JAK2
followed by Western blot analysis using antiphosphotyrosine antibodies,
we observed that SOCS-3 was able to inhibit GH-induced tyrosine
phosphorylation of JAK2 (Fig. 12
).
Inhibition of this tyrosine phosphorylation of the kinase was observed
using 3 µg GH receptor expression plasmid, and further inhibition of
the GH-induced tyrosine phosphorylation of JAK2 was observed using
10 µg GH receptor expression plasmid. This finding suggests that
SOCS-3 can inhibit JAK2 tyrosine phosphorylation by a mechanism
dependent on the GH receptor.

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Figure 12. Inhibition of GH-Stimulated JAK2 by SOCS-3
HEK 293 cells were transiently transfected with JAK2 (50 ng) and SOCS-3
(20 µg) and the indicated amount of GH receptor. After transfection,
cells were cultured in the absence or presence of 20 nM GH
for 5 min. Cell lysates were used for immunoprecipitation (IP) using
anti-JAK2 antibodies. A, Immunoprecipitates were analyzed by SDS-PAGE
followed by Western blot analysis using antiphosphotyrosine antibodies
( -PY). B, An identical amount of immunoprecipitate was analyzed by
Westen blotting using anti-JAK2 antibodies. C, Cell lysates were
analyzed by SDS-PAGE followed by Western blot analysis using anti-FLAG
antibodies.
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DISCUSSION
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We have previously shown that SOCS-3 is induced transiently by GH
both in vivo and in vitro, while the expression
of SOCS-1, SOCS-2, and CIS is induced only marginally by GH (8). In
this study we examine the mechanism by which SOCS proteins inhibit
GH-induced transcription of the STAT5-responsive Spi 2.1 promoter. Both
SOCS-1 and SOCS-3 were found to inhibit GH-induced transcription of the
Spi 2.1 promoter. The generation of chimeric SOCS proteins showed that
the N-terminal domain dictates the specificity by which SOCS proteins
inhibit GH-mediated signaling. Previously, the N-terminal domain has
been found to be essential for SOCS-1 inhibition of IL-6 signaling and
the ability to inhibit JAK activity (13), and the N-terminal domains of
SOCS-1 and SOCS-3 were found to be functionally interchangeable for
inhibition of IL-6 and LIF signaling (11). However, only the SH2 domain
of SOCS-1 was found to be required for binding to JAK. Together these
data indicate that the N-terminal domain is required for the inhibitory
action of SOCS proteins on JAK activity and that some degree of
specificity is encoded within this domain since the N-terminal domains
of SOCS-1 and SOCS-3 are able to inhibit GH signaling when placed in
conjunction with the SH2 domain and SOCS box from CIS. In agreement
with this it was recently reported that SOCS-1 and, to a minor extent,
SOCS-3 were the only SOCS proteins that interacted directly with the
kinase domain of JAK2 both in vitro and in vivo
(10) and that amino acids N-terminal to the SH2 domain were required
for the interaction with JAK2 (12).
The C-terminal 40-amino acid region of the SOCS proteins has been
termed the SOCS box, and this domain exhibits approximately 50%
identity among the SOCS family members. The functional role of this
domain is not known as it has been reported to be not required for
inhibition of IL-6-induced transcription or JAK kinase activation. In
this study we found that the SOCS box is not important for inhibition
of GH-induced transcription by SOCS-1 and SOCS-3. However, the linker
region between the SH2 domain and the SOCS box of SOCS-3 was required
for SOCS-3 to inhibit GH signaling. This linker region is considerably
longer in SOCS-3 compared with SOCS-1 (Fig. 3
). Inhibitory activity was
observed when only two amino acids of the linker region were present in
SOCS-1, whereas almost the entire linker region (46 amino acids) was
required for SOCS-3 inhibition of GH-induced transcription. The same 46
amino acids were also required for inhibition of GH-induced activation
of STAT5.
Previous studies have shown that CIS is able to bind to the
tyrosine-phosphorylated erythropoietin and IL-3 receptors (4), and it
was suggested that CIS binding to these receptors reduces the
interaction between STAT5 and the receptor, resulting in inhibition of
STAT5 activation. Using GST-GH receptor fusion proteins, we could show
that all four SOCS proteins were able to bind specifically to the
tyrosine-phosphorylated GH receptor. It was also demonstrated that
binding of SOCS-3 to the GST-GH receptor fusion protein was inhibited
by a phosphopeptide containing tyrosine 487, whereas phosphopeptides
containing either tyrosine 534, 595, or 627 were less inhibitory and
phosphopeptide 566 inhibited only slightly. Since we previously
identified three of these tyrosine residues (Y534, Y566, and Y627) as
STAT5 binding sites (15, 16) in the GH receptor, we tested whether
SOCS-3 could compete with STAT5 for binding to the GH receptor.
However, no competition could be observed, suggesting that the
inhibitory mechanism of SOCS-3 in GH signaling was not by competing for
STAT5 binding sites in the GH receptor. The fact that Y566 did not bind
SOCS-3 but is able to bind STAT5 might explain the lack of competition
between these two factors. The observation that all four SOCS members
are able to bind to the GH receptor also suggests that binding to the
receptor is not sufficient for inhibition of GH signaling. The role of
SOCS binding to the GH receptor is not known at present; however, we
found that the same region of the linker domain in SOCS-3 that was
required for inhibition of GH-induced signaling was also required for
binding to the GH receptor. This finding was surprising since the SH2
domain is believed to be able to bind phosphotyrosines by itself, but
in the case of SOCS-3 an additional 46 amino acids C-terminal to the
SH2 domain seem to be required for this binding.
Thus, these data in combination with the observations that only
SOCS-1 was able to inhibit tyrosine phosphorylation of overexpressed
JAK2, whereas SOCS-3 was able to inhibit GH-stimulated tyrosine
phosphorylation of JAK2 only in the presence of the GH receptor,
indicate that SOCS-1 and SOCS-3 inhibit GH signaling by two different
mechanisms. It is tempting to suggest that SOCS-1 directly binds
to the JAK2 kinase and thereby inhibits the kinase activity, whereas
SOCS-3 only inhibits JAK2 kinase activity after binding to the GH
receptor. This hypothesis furthermore predicts that two functional
domains are present in the SOCS proteins: one, which includes the SH2
domain, is involved in binding of the SOCS protein to phosphotyrosines
in cytokine receptors or in JAK kinases, and the other, which includes
the N-terminal domain, is involved in the actual inhibition of kinase
activity. Interestingly, a similar difference in the mechanism by which
GH activates STAT1 and STAT5 was previously reported (17). STAT1 is
activated by a mechanism that does not require GH receptor tyrosine
phosphorylation (18), whereas STAT5 activation by GH is dependent upon
phosphorylation of at least one of three tyrosines in the intracellular
domain of the GH receptor (16, 19).
The physiological role of SOCS proteins in GH signaling is not
known at present; however, since SOCS-3 is the major SOCS protein
induced by GH both in vitro and in vivo, this
factor is presumed to be the main regulator of GH signaling. The
transient nature of SOCS-3 mRNA induction by GH and the relative short
half-life of SOCS-3 protein suggests that SOCS-3 acts in a classical
negative feed-back loop, suppressing GH signaling for a limited time
period. Interestingly GH is secreted in a pulsatile manner in most
species with a frequency of 3 to 4 h between each peak. This time
period is in good agreement with the time required for SOCS-3 levels to
return to basal levels after a GH pulse, and the role of SOCS-3 might
be to protect against overstimulation by GH or alternatively to
restrict the time in which a cell is responsive to GH stimulation.
 |
MATERIALS AND METHODS
|
---|
Plasmids
Flag epitope-tagged SOCS-1, SOCS-2, SOCS-3, and CIS expression
vectors were constructed in the pEF-BOS plasmid as described previously
(2). The rat GH receptor expression vector pLM 108 was constructed as
described (20). The hemagglutinin-epitope-tagged STAT5 expression
vector was obtained from Dr. Groner (Georg-Speyer-Haus, Institute for
Biomedical Research, Frankfurt, Germany) (21), and the JAK2
expression vector was obtained from Dr. Schlessinger (New York
University, New York).
Cell Culture
Chinese hamster ovary (CHO) cells were cultured in Hams
F-12 medium (Life Technologies, Inc., Gaithersburg, MD)
supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml
streptomycin. Cells were transfected at 90% confluency in 60-mm cell
culture dishes. Twenty-four hours before transfection, cells were
washed twice and incubated with 3 ml serum free GC3 medium (1:1 mixture
of DMEM and Hams F-12 (Life Technologies, Inc.),
adjusted to pH 7.1 with 7.5% NaHCO3 solution, supplemented
with 10 µg/ml transferrin, 160 mU/ml insulin, 2 mM
L-glutamine, 2 mM nonessential amino acids, 100
U/ml penicillin, and 100 µg/ml streptomycin). Cells were transiently
transfected by calcium phosphate precipitation as described previously
(15). Each dish was transfected with 0.011 µg SOCS expression
vector, 3 µg pCH110 ß-galactosidase expression vector, 1.5 µg Spi
2.1/chloramphenicol acetyltransferase (CAT) plasmid, and 1.5 µg GH
receptor expression plasmid, and cultured in the absence or presence of
20 nM hGH (Novo Nordisk, Bagsvaerd,
Denmark) overnight. CAT assay was performed on total cellular
extracts as described (22) using ß-galactosidase as an internal
control. Human kidney 293 cells were cultured in DMEM supplemented with
10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 2 mM L-glutamine. Cells were
seeded in 100-mm cell culture dishes with 10 ml culture medium and
transiently transfected the next day (50% confluency) by calcium
phosphate precipitation.
Peptides
Synthetic peptides were purchased from Affinity Research
Products Ltd. (Mamhead, UK) either nonphosphorylated or tyrosine
phosphorylated. Five 13 amino acid long peptides derived from the GH
receptor were synthesized; LANIDFYAQVSDI (peptide Y487), FIMDNAYFCEADA
(peptide Y534), FNQEDIYITTESL (peptide Y566), EMPVPDYTSIHIV (peptide
Y595), and FLSSCGYVSTDQL (peptide Y627). The peptides were purified by
HPLC and their composition verified by mass spectrometry.
GST-GHR Binding Assay
Cell extracts were isolated by addition of lysis buffer (50
mM HEPES, pH 7.2, 250 mM NaCl, 10% glycerol, 2
mM EDTA, 2 mM EGTA, 0.1% NP-40, 1
mM 4-(2-aminoethyl) benzene sulfonyl fluoride
(AEBSF), 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml
herbimycin, and 1 mM sodium orthovanadate) to the cells
followed by 20 min incubation on ice. After centrifugation at
10,000 x g for 10 min at 4 C, the supernatants were
used for GST binding assays. Varying amounts (indicated in each
individual experiment) of the extracts were added to 50 µl (50%)
glutathione-Sepharose 4B beads to which 25 µg of GST fusion protein
were bound and incubated for 1214 h at 4 C with rotation (when
competition with GH receptor peptides was performed, the peptide was
added before the cell lysate). The Sepharose pellets were then washed
five times with ice-cold lysis buffer and analyzed by Western blot
analysis.
GST Fusion Proteins
The cDNA encoding the membrane-proximal region or the
carboxy-terminal region of the rat GH receptor was amplified by PCR
from the GH receptor plasmid pLM 108. PCR products were ligated into
the GST fusion vector pGEX-5X-3 (Pharmacia Biotech,
Piscataway, NJ). The resulting plasmids were sequenced to verify the
fidelity of PCR and to confirm proper, in-frame cloning. Induction and
affinity purification of GST proteins and GST-GH receptor fusion
proteins were performed as recommended by the manufacturer
(Pharmacia Biotech). In addition, GST proteins and
tyrosine-phosphorylated GST-GH receptor fusion proteins were
induced and purified from the Escherichia coli TKX1 strain
that harbors a plasmid-encoded inducible tyrosine kinase gene as
recommended by the manufacturer (Stratagene, La Jolla,
CA).
Immunoprecipitation
Protein G-Sepharose (Pharmacia Biotech) and 110
µl of the respective antibody were added to the cell lysate (prepared
in IP buffer: 50 mM Tris/HCl, pH 7.5, 0.1 M
Triton X-100, 137 mM NaCl, 2 mM EGTA, 1
mM AEBSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1
mM sodium orthovanadate), and samples were incubated 1214
h at 4 C. Samples were washed three times with IP buffer, and the
pellets were then washed five times with ice-cold lysis buffer and
analyzed by SDS-PAGE and Western blot analysis.
Nuclear Extracts and Electrophoretic Mobility Shift Assay
(EMSA)
Cells were cultured with or without GH (20 nM) for 5
min, washed twice with ice-cold PBS, and lysed in buffer A (20
mM HEPES, pH 7.9, 10 mM KCl, 1 mM
MgCl2, 1 mM EDTA, 1 mM
dithiothreitol, 0.5 mM AEBSF, 1 mM sodium
orthovanadate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 20%
glycerol) containing 0.5% Triton X-100. After 5 min of incubation on
ice, the nuclei were collected by centrifugation at 2500 x
g for 7 min at 4 C. The nuclei were resuspended in 5 volumes
of a hypertonic buffer (buffer A containing 400 mM NaCl)
and incubated on a rocking platform for 30 min at 4 C. The supernatant
was collected after centrifugation at 20,000 x g for
30 min at 4 C. The double-stranded Spi 2.1 GLE1 oligonucleotide
(5'-agctATGTTCTGAGAAAATC-3' and 5'-agctGATTTTCTCAGAACAT-3') was
32P-labeled in a fill-in reaction using
[
-32P]dCTP and DNA polymerase (Klenow fragment).
Approximately 20 fmol probe were used per reaction with 10 µg nuclear
extract in EMSA buffer (100 mM HEPES, pH 7.9, 10
mM NaCl, 1 mM MgCl2, 1
mM EDTA, 10% glycerol) containing 0.1 µg/µl
double-stranded poly dI/dC (polydeoxyinosinic-deoxycytidylic acid).
EMSA reactions were preincubated for 30 min at 30 C before separation
on a 5% polyacrylamide gel containing 2% glycerol and 0.25% TBE (25
mM Tris/HCl, 25 mM boric acid, and 0.25
mM EDTA, pH 7.9). The gel was dried and exposed to x-ray
film.
Construction of SOCS Mutants
SOCS chimera expression vectors were generated by introduction
of ClaI sites (between the N-terminal domain and the SH2
domain) and NotI sites (between the SH2 domain and the
SOCS-box) in the various SOCS-encoding plasmids using the Quickchange
site-directed mutagenesis kit (Stratagene). The plasmids
were digested with XbaI/ClaI, and cDNA fragments
encoding the following amino acid sequences were purified: CIS (179
and 80257); SOCS-1 (177 and 78212); and SOCS-3 (143 and
44225). The plasmids were also digested with
XbaI/NotI, and cDNA fragments encoding the
following amino acid sequences were purified: CIS (1210 and
211257); SOCS-1 (1174 and 175212); and SOCS-3 (1184 and
185225). The various SOCS domain swap mutants were generated by
cross-ligation of appropriate fragments, e.g.
(fragments 1, 3, and 3) encoding SOCS-1 (177) fused to SOCS-3
(44225), etc. The different SOCS mutants encoding truncated forms of
SOCS-1 and SOCS-3 were generated by introducing stop codons using the
Quickchange site-directed mutagenesis kit
(Stratagene).
Western Blot Analysis
Proteins were resolved by SDS-PAGE (4% stacking gel, 7.5%,
10%, or 12% running gel) and transferred by electroblotting to
ECL nitrocellulose membranes (Amersham Pharmacia Biotech,
Arlington Heights, IL). Membranes were blocked for 1 h in TBST
buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl,
and 0.1% Tween 20) containing 5% nonfat dry milk. Primary antibody
diluted in TBST was added, and the blot was incubated for 1 h at
room temperature. After three successive 20-min washes with TBST, the
secondary antibody was added and membranes were incubated for 1
additional hour, and the proteins were visualized by the ECL detection
system according to the manufacturers instructions (Amersham Pharmacia Biotech).
 |
ACKNOWLEDGMENTS
|
---|
We thank Jannie Rosendahl Christensen for technical assistance
and Dr. Erica Nishimura for critical review of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Nils Billestrup, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark.
Johnny A. Hansen and Karen Lindberg are supported by the Danish
Research Academy. Part of this work was supported by The National
Health and Medical Research Council, Canberra, Australia, The NIH,
Bethesda, Maryland (Grant CA-22556), and the Australian Federal
Governments Cooperative Research Centre Program.
Received for publication April 29, 1999.
Revision received July 16, 1999.
Accepted for publication July 20, 1999.
 |
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