From the Centre de Génétique
Moléculaire et Cellulaire, Unité Mixte de Recherche CNRS
5534, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne,
France, ¶ INSERM U119, 13009 Marseille, France, and the ** Ontario
Cancer Institute, Princess Margaret Hospital, Toronto, Ontario M5G
2M9, Canada
Received for publication, March 1, 2001, and in revised form, April 4, 2001
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ABSTRACT |
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Macrophage colony-stimulating factor receptor
(M-CSF-R) is a tyrosine kinase that regulates proliferation,
differentiation, and cell survival during monocytic lineage
development. Upon activation, M-CSF-R dimerizes and autophosphorylates
on specific tyrosines, creating binding sites for several cytoplasmic
SH2-containing signaling molecules that relay and modulate the M-CSF
signal. Here we show that M-CSF-R interacts with suppressor of cytokine signaling 1 (Socs1), a negative regulator of various cytokine and
growth factor signaling pathways. Using the yeast two-hybrid system,
in vitro glutathione S-transferase-M-CSF-R
pull-down, and in vivo coimmunoprecipitation experiments,
we demonstrated a direct interaction between the SH2 domain of Socs1
and phosphorylated tyrosines 697 or 721 of the M-CSF-R kinase insert
region. Moreover, Socs1 is tyrosine-phosphorylated in response to
M-CSF. Ectopic expression of Socs1 in FDC-P1/MAC and EML
hematopoietic cell lines decreased their growth rates in the presence
of limiting concentrations of M-CSF. However, Socs1 expression did not
totally suppress long term cell growth in the presence of saturating
M-CSF concentrations, in contrast to other cytokines such as stem cell
factor and interleukin 3. Taken together, these results suggest that
Socs1 is an M-CSF-R-binding partner involved in negative regulation of
proliferation signaling and that it differentially affects cytokine
receptor signals.
Hematopoiesis is mainly under the control of soluble and
membrane-bound factors acting through specific transmembrane receptors (1). These receptors transduce intracellular messages by recruiting and
activating a given set of cytoplasmic molecules that initiate specific
signaling pathways, which then spread throughout the compartments of
the cell, modifying its activity (2). What results from setting off
these multiple biochemical signals is a global cellular response:
proliferation, differentiation, activation, and/or survival. It is
therefore important to determine the identity, stoichiometry, and
function of the molecules present in the initial signaling complex
formed in concert with the growth factor receptor.
Macrophage colony-stimulating factor
(M-CSF1; also called CSF-1)
is a key regulator of monocytic lineage development. In bone marrow,
M-CSF stimulates proliferation and differentiation of committed
progenitors, leading to the production of blood monocytes and tissue
macrophages (3, 4). Osteopetrotic op/op mutant mice lacking functional
M-CSF show impaired monocyte development and deficiency in osteoclasts
and macrophages and can be cured by M-CSF injection (5). All the
biological effects of M-CSF are mediated by a single receptor, encoded
by the protooncogene c-fms, which is expressed on the
surface of cells undergoing monocytic development (6, 7). Ectopic
expression of the M-CSF receptor (M-CSF-R or Fms) in different
hematopoietic cell lines enables M-CSF-dependent
proliferation and monocyte/macrophage differentiation (8-10). Fms is a
member of class III of the receptor tyrosine kinase family,
which includes Kit, Flt3, and the The suppressor of cytokine signaling 1 (Socs1) protein, also called
SSI-1 or JAB, is a member of the Socs family, composed of eight
molecular adaptors that negatively regulate cytokine signaling
pathways (19-24). Analysis of mice with homozygous inactivation of the
Socs1 gene confirmed its role as a negative growth signal regulator.
Socs1 Socs1 is also a modular multidomain protein that can interact with
other signaling proteins. Its N-terminal region contains diproline
sites that interact with the SH3-containing proteins Nck, Grb2, the p85
subunit of phosphatidylinositol 3-kinase, Fyn, and Itk kinases. The
Socs1 SH2 domain binds to the N-terminal region of the guanine
nucleotide exchange factor Vav in a phosphotyrosine-independent manner.
The C-terminal Socs box, common to all Socs family members, interacts
with B and C elongins (19, 28).
We have previously shown that Socs1 also binds to the Kit and Flt3
receptor tyrosine kinases. The interaction requires Socs1 SH2 domain
and phosphorylation of the receptors on as yet unknown tyrosine
autophosphorylation sites (19). Although it does not inhibit Kit
tyrosine kinase activity, ectopic expression of Socs1 strongly
suppresses the proliferative signal of the Kit receptor, suggesting
that negative regulation involves not only Janus kinase inhibition but
also other mechanisms as yet unknown (19). In the present study, we
investigated the interaction between Socs1 and M-CSF-R to 1) identify
the receptor site(s) of the interaction as a model for other receptor
tyrosine kinases and 2) determine whether Socs1 has the same effect on
two closely related receptor tyrosine kinases, Kit and Fms. We show
that Socs1 directly associates with tyrosine-phosphorylated M-CSF
receptor upon M-CSF binding. In vitro and in vivo
experiments demonstrated that this interaction involves the SH2 domain
of Socs1 and tyrosines 697 or 721 of the Fms kinase insert (KI) domain.
Ectopic expression of Socs1 in two different hematopoietic cell lines
inhibited M-CSF-dependent proliferation but only in the
presence of limiting concentrations of M-CSF. This result suggests that
Socs1 could act as a general negative regulator of receptor tyrosine
kinase proliferation signals, but its effects may be quantitatively
different depending on the type of receptor.
Yeast Two-hybrid System--
As previously described (17), the
LexA-Fms wild-type (WT) or K614A baits contain the entire cytoplasmic
domain (amino acids 536-976) of murine Fms. Mutation of lysine 614, located in the ATP binding site, into alanine (K614A) results in a
kinase-inactive Fms protein. The YRN974 yeast strain containing an
integrated LexA-Operator-green fluorescent protein (GFP)
cassette (29) was transformed with two plasmids: pBTM116, encoding
LexA-Fms WT or K614A, and pVP16, either empty (as a negative control)
or containing the Socs1 partial cDNA referred to here as clone 99, which was isolated in a previous two-hybrid screen using LexA-c-Kit as
a bait (19). Three individual clones were isolated on selective agar
plates and expanded in similar selective liquid medium. Exponentially growing cultures were then analyzed for fluorescence intensity using a
Becton Dickinson FACScalibur flow cytometer. Plasmids, selective media,
and the transformation protocol have been described previously
(30).
Growth Factors, Cell Cultures, and Infections--
The source of
M-CSF was a conditioned medium of Sf9 insect cells expressing
recombinant murine M-CSF from a baculovirus vector (31).
X63-interleukin 3 (IL-3) cell-conditioned medium (IL3-CM) was used as a
source of IL-3 (32). The source of stem cell factor (SCF) was a
conditioned medium from BHK/MKL cells (BHK/MKL-CM) (33). M1
cells (34, 35) and their derivatives were maintained in Iscove's
modified Dulbecco's medium (IMDM; Life Technologies, Inc.)
supplemented with 10% fetal bovine serum (FBS, Dutcher). Either wild
type (WT) or mutated on tyrosines 697, 706, and 721 (YTF), Fms was
introduced into M1 cells by retroviral infection; infected cells were
selected for high cell surface Fms expression by fluorescence-activated
cell sorting (FACS) after staining with polyclonal anti-Fms antibody as
previously described (36). FDC-P1/MAC cells (37) and their
derivatives were maintained in IMDM, 10% FBS supplemented
either with M-CSF (1000 units/ml) or 1% IL3-CM. EML-Fms cells (38) and
their derivatives were maintained in IMDM, 20% horse serum (Roche
Molecular Biochemicals) supplemented with M-CSF (1600 units/ml).
M1-Fms, FDC-P1/MAC, and EML-Fms cells were infected by coculture with
virus-producing GP+E-86 packaging cell lines expressing either empty
pMiev or pMiev-hemagglutinin (HA)-Socs1 retroviral vectors (19).
Nonadherent infected cells were then sorted for GFP expression by
FACS.
Proliferation Assays--
For short term proliferation assay,
FD-MAC/Miev and FD-MAC/Socs1 cells were washed twice with IMDM and
seeded at 2 × 104 cells/ml in IMDM, 10% FBS
supplemented or not with various concentrations of M-CSF or 1% IL3-CM.
Cultures (100 µl) were set up in duplicate in 96-well microtiter
plates and incubated at 37 °C and 5% CO2 in a
humidified atmosphere for 4 days. Proliferation was then monitored by
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (39). For clonal assay, washed FD-MAC/Miev and FD-MAC/Socs1 cells were plated in IMDM supplemented with 1%
methylcellulose (Flucka, Buchs, Switzerland), 10% FBS, and growth
factors as specified in Fig. 4E. Cultures (500 µl)
were performed in 24-well culture plates, and colonies were scored
after 7 days of culture. For growth curves, EML-Fms/Miev and
EML-Fms/Socs1 cells were washed twice with IMDM and seeded at 5 × 104 cells/ml in IMDM, 20% horse serum supplemented or not
with M-CSF (200 or 1600 units/ml) or SCF (10% BHK/MKL-CM). Viable cell
number was determined every 2 days, and cultures were split and fed.
Antibodies--
Monoclonal antibodies to murine Grb2 (G16720)
and the p85 subunit of phosphatidylinositol 3-kinase (P13020) were from Transduction Laboratories. Polyclonal rabbit antisera to the murine Fms
cytoplasmic domain (4599B), to the murine Shc, and to the murine Socs1
(99B2) have been previously described (17, 19, 36). Monoclonal
antibodies anti-phosphotyrosine and anti-HA were hybridoma supernatants
of 4G10 and 12C5 clones, respectively.
Cell Stimulation, Immunoprecipitation, and Western
Blot--
Cells were washed in phosphate-buffered saline and
resuspended in IMDM supplemented with 1% FBS for 5 h at 37 °C.
Starved cells were then resuspended in phosphate-buffered saline, 1%
FBS containing 2 mM Na3VO4 and then
stimulated or not with M-CSF (15,000 units/ml) for different times at
37 °C or for 1 h in ice. Cells were lysed in ice-cold lysis
buffer (15) containing a protease inhibitor mixture (Roche Molecular
Biochemicals, number 1836170) and phosphatase inhibitor mixture II
(Sigma, number P5726). Equalized cell lysates were mixed for 18 h
with 5 µl of anti-Socs1 antiserum and 15 µl of protein G coupled to
agarose beads (Amersham Pharmacia Biotech). The beads were
washed three times with 1 ml of lysis buffer, and bound proteins were
released by boiling for 5 min in Laemmli buffer. Proteins from cell
lysates and immunoprecipitates were separated on SDS-polyacrylamide
gels, transferred to nitrocellulose membrane, and blotted with various
antibodies as previously described (15). Antibody binding was
visualized using horseradish peroxidase-conjugated secondary antibodies
(Sigma) and enhanced chemiluminescence reagent (ECL+, Amersham
Pharmacia Biotech).
Binding Assay Using Phosphorylated Glutathione S-Transferase
(GST)-Fms KI Fusion Proteins--
The entire KI region of WT and
mutant murine Fms (amino acids 668-746) was cloned in pGEX1 (Amersham
Pharmacia Biotech) and produced as tyrosine-phosphorylated GST-KI
fusion proteins as previously described (15). For the binding assay,
12.5 µg of immobilized fusion proteins were mixed for 18 h at
4 °C with 750 µg of cell lysates of GP+E-86 cells expressing
either WT or mutated (R105K) HA epitope-tagged Socs1 (19). The beads
were washed three times with 1 ml of lysis buffer, and bound proteins
were released by boiling for 5 min in Laemmli buffer. Total cell
lysates and bound proteins were run on a 12% polyacrylamide gel,
blotted, and probed with anti-HA antibody to detect Socs1 protein and
anti-Grb2 antibody as a positive control.
Interaction of M-CSF-R with Socs1 in the Yeast
Two-hybrid System--
In a yeast two-hybrid screen using LexA-Kit as
a bait (19), we isolated a VP16-cDNA clone (clone 99) that
encompasses the N-terminal and SH2 domain sequences of Socs1 (Fig.
1A). We then investigated the
interaction of clone 99 with the LexA-Fms cytoplasmic domain fusion
protein, which is able to autophosphorylate in yeast and to interact
with specific partners (17). The interaction was analyzed by flow
cytometry using the YRN974 yeast strain in which the reporter gene
encoded the GFP (29). As shown in Fig. 1B, yeast clones
expressing either LexA-Fms wild type (WT) or LexA-Fms
kinase-dead (KD) as bait, together with VP16, show only autofluorescence (thin lines). Coexpression of LexA-Fms WT
and VP16-clone 99 (Fig. 1B, thick lines)
increased the fluorescence intensity (left panel), whereas
coexpression of LexA-Fms kinase-dead and VP16-clone 99 had no effect
(right panel). These results suggested that one or more
M-CSF-R tyrosine phosphorylation sites directly interact with
Socs1.
Socs1 SH2 Domain Interacts with M-CSF-R Phosphotyrosine 697 and
721--
To confirm the specific interaction between Socs1 and
Fms observed in yeast and to determine which Fms autophosphorylation sites are concerned, we investigated Socs1 interaction with Fms in vitro. GST fusion proteins containing either the WT Fms
KI or KI mutated at each different autophosphorylation site (Tyr-697, Tyr-706, Tyr-721) were expressed in the Epicurian TKX1 bacteria strain that enables phosphorylation of tyrosine residues. The phosphorylated fusion proteins immobilized on glutathione-Sepharose were mixed with lysates of GP+E-86 cells expressing HA epitope-tagged Socs1. The presence of Socs1 (and Grb2 as a control) among precipitated proteins was examined by Western blotting using the relevant antibodies (Fig. 2A). Socs1 specifically
associated with GST-Fms KI WT but not with GST alone, as was also the
case for the positive control Grb2. As expected, mutation of tyrosine
697 abolished Fms interaction with Grb2 adaptor (14). Although mutation
of all three tyrosines abolished Socs1 interaction, none of the single
mutations had an effect on Socs1 binding (Fig. 2A),
suggesting that Socs1 is able to bind to two or three Fms
phosphotyrosines.
Using the three combinations of double mutants, we found that mutation
of both tyrosines 697 and 721 was the only combination that totally
abolished Socs1 interaction with Fms (Fig. 2B). However, tyrosines 697 and 721 are binding sites of Grb2 and p85, respectively, both molecules that are able to bind to the diproline motifs in the
Socs1 N-terminal region (19). To verify that the interaction observed
in vitro between Socs1 and Fms was direct and not mediated by p85 and Grb2, we used a mutant of the phosphotyrosine-binding pocket
of the Socs1 SH2 domain (R105K) (19) in the binding assay with GST-Fms
KI WT. As shown in Fig. 2C, the R105K mutation totally abrogated Socs1 binding to Fms KI, demonstrating that the interaction is mediated through the Socs1 SH2 domain.
M-CSF-R Interacts with Socs1 and Induces Its Tyrosine
Phosphorylation in Vivo--
To examine the interaction between
M-CSF-R and Socs1 in vivo, we expressed HA-Socs1 in the
hematopoietic cell line M1 (34) using the pMiev-HA-Socs1-GFP retrovirus
expressing both HA epitope-tagged Socs1 and the GFP protein from a
single bicistronic mRNA (19). Because M1 cells do not normally
express M-CSF-R (35), WT or triple mutant YTF (Phe-697, Phe-706,
Phe-721) Fms was ectopically expressed in these cells by
retroviral infections. M1/Fms WT or YTF cells expressing both Fms and
Socs1 were either unstimulated or stimulated by M-CSF. Analysis of
total cell lysates showed that both cell lines expressed similar
amounts of Fms and Socs1 (Fig.
3A, bottom panels)
and that both receptors were activated by M-CSF, because they induced
similar protein tyrosine phosphorylation patterns (Fig. 3A,
top panel), except for some bands including the receptor
itself, as previously described (36). In addition, Socs1 expression did
not modify the overall tyrosine phosphorylation patterns in response to
M-CSF (data not shown). After immunoprecipitation with anti-Socs1
antibody, Western blot analysis showed that phosphorylated WT Fms but
not YTF Fms coimmunoprecipitated with Socs1 in an
M-CSF-dependent manner (Fig. 3B).
Some Fms-binding partners, such as p85 and phospholipase C Socs1 Negatively Regulates the M-CSF-R Proliferation
Signal--
Socs1 was isolated by its ability to suppress macrophage
differentiation of leukemic myeloblast M1 cells induced by
leukemia inhibitory factor or IL-6 by suppressing gp130 and STAT3
phosphorylation (21). As previously described (40), M-CSF also induced
macrophage differentiation in M1-Fms cells, and we did not observe any
significant effect of Socs1 expression on this process. The effect of
Socs1 on the M-CSF-dependent proliferation signal could not
be investigated in M1-Fms cells because of their factor independence
and their differentiation in response to M-CSF. As a model, we then
expressed Socs1 in the FDC-P1/MAC factor-dependent
hematopoietic cell line that expresses endogenous M-CSF-R and can grow
in medium supplemented either with IL-3 or M-CSF (37). Cells were
infected by coculture with virus-producing GP+E-86 cells expressing
either empty pMiev or pMiev-HA-Socs1 retroviral vectors in the presence
of saturating concentrations of IL-3 or M-CSF (1000 units/ml) and then
sorted for GFP expression by FACS. When cells were infected in the
presence of IL-3, we were able to isolate GFP-positive cells only from the control Miev population but not from Socs1-expressing cells. This
result was expected, because Socs1 is a strong negative regulator of
Janus kinases, which are necessary for the IL-3 signal.
Moreover, we have observed in several cell systems that Socs1
overexpression did not allow cells to proliferate and survive in
response to IL-3 (data not shown), in accordance with previous reports
that IL-3 activation of luciferase reporter genes is suppressed by Socs1 (22, 24). On the contrary, when cells were infected in the
presence of M-CSF, we were able to select cells with high GFP
expression from both populations, referred to here as FD-MAC/Miev and
FD-MAC/Socs1 (Fig. 4A). As
with M1-Fms cells, Western blot analysis demonstrated the
expression of Socs1 (Fig. 4B), its interaction with
phosphorylated Fms, and its tyrosine phosphorylation in response to
M-CSF in FD-MAC/Socs1 cells (Fig. 4C).
The effect of Socs1 on growth rate was then examined using a short term
proliferation assay in the presence of various M-CSF concentrations
(Fig. 4D). At saturating concentrations of M-CSF (>500
units/ml), there was no significant growth rate effect of Socs1,
although FD-MAC/Socs1 cells grew slightly more slowly than FD-MAC/Miev
control cells. However, in the presence of limiting M-CSF
concentrations (between 30 and 125 units/ml), there was a 50% decrease
in the growth of FD-MAC/Socs1 cells. This effect was confirmed in a
clonal assay, where FD-MAC/Socs1 cells produced fewer and smaller
colonies as compared with the FD-MAC/Miev cells in the presence of 125 units/ml M-CSF (Fig. 4E). These results suggest that Socs1
may act as a negative regulator of the M-CSF-R proliferation signal in
FDC-P1/MAC cells, but only in the presence of limiting concentrations
of M-CSF.
To compare the effect of Socs1 on Kit and Fms signaling in the same
cellular background, we expressed Socs1 in EML-Fms cells (38). Because
we had previously shown that Socs1 suppresses SCF-dependent
proliferation in EML cells (19), Socs1 was retrovirally transduced in
EML-Fms cells maintained in the presence of a saturating concentration
of M-CSF (1600 units/ml). EML-Fms/Socs1 and control EML-Fms/Miev cells
were selected for high GFP expression (Fig. 5A), and Socs1 protein
expression was verified by Western blotting (Fig. 5B). Cells
were washed free of growth factors and then cultivated in the presence
of low (200 units/ml) or high (1600 units/ml) concentrations of M-CSF
or in the presence of a saturating concentration of SCF (10%
BHK/MKL-CM) (Fig. 5C). As expected, Socs1 expression totally
inhibited SCF-dependent proliferation and had no effect on
M-CSF-dependent proliferation in the presence of 1600 units/ml M-CSF. However, when limiting concentrations of M-CSF (200 units/ml) were present, Socs1 was then able to block proliferation
totally, and more than 90% of the EML-Fms/Socs1 cells died after a
week of culture. This result confirmed that, as in FDC-P1/MAC cells, Socs1 negatively regulates the M-CSF-R proliferation signal only at
limiting M-CSF concentrations, suggesting that Socs1 has differential inhibitory effects on Kit and Fms signaling.
In the present study, we have characterized the in
vitro and in vivo interaction between Socs1 and
Fms/M-CSF-R. In vivo interaction between Socs proteins and
several receptor tyrosine kinases has been previously reported;
Socs1 binds to both Kit and Flt3 (19), Socs2 binds to the insulin-like
growth factor receptor (41), and Socs3 binds to the insulin receptor
(42). Until now, the receptor autophosphorylation sites involved in
these interactions remained to be determined. Here we show that the SH2
domain of Socs1 binds to two phosphotyrosines located in the Fms kinase insert. These two sites are tyrosines 697 and 721, which are the Fms
binding site for the Grb2 adapter and the p85 subunit of
phosphatidylinositol 3-kinase, respectively. Interestingly, the Socs1
N-terminal region contains diproline motifs that recognize the SH3
domains of Grb2 and p85 (19), suggesting that Socs1 binding may not
decrease, but on the contrary extend, the M-CSF-R signaling repertoire. Because these two sites are the first Socs1 binding sites to be described on receptors, it would be interesting to test whether Grb2
and/or p85 sites are also docking sites for Socs1 in other receptor
tyrosine kinases. We have also shown that Socs1 was rapidly and
transiently tyrosine-phosphorylated in response to M-CSF, as described
for other Fms-interacting molecules (17). Socs2 and Socs3 tyrosine
phosphorylation has recently been described in response to insulin-like
growth factor and IL-2, respectively (43, 44). The precise role and
importance of Socs tyrosine phosphorylation in regulating its function
is yet unknown. Concerning Socs1, it would be interesting to locate the
phosphorylation site(s) and to determine whether tyrosine
phosphorylation also occurs in response to other signals and whether it
affects such Socs1 functions as Janus kinase inhibition.
The biological relevance of the interaction between Socs1 and M-CSF-R
is supported by the fact that Socs1 expression resulted in a decreased
M-CSF-dependent proliferation of two different cell lines,
including the FDC-P1/MAC cells that express endogenous M-CSF-R and are
a physiological model for Fms signaling (37). Socs1 protein is unstable
in cells, and Socs1 synthesis is strongly repressed at the translation
level (45). Indeed, endogenous Socs1 protein has so far been described
only in mouse T lymphocytes after immunoprecipitation (45). For this
reason, we expressed Socs1 ectopically to study its functional
interactions in vivo. Socs1 promoter expression has been
detected in vivo in cells of the monocyte/macrophage lineage
(27), suggesting that both M-CSF-R and Socs1 are coexpressed during
monocytic development. Interestingly, Socs1 negatively regulated
M-CSF-R proliferation signal only in the presence of nearly
physiological limiting concentrations of M-CSF (46).
Altogether, our data suggest that Socs1 may act as a negative regulator
of the M-CSF-R proliferation signal; there are different ways in which
Socs1 could be involved in Fms signaling. First, M-CSF-R activates the
Janus kinase/signal transducer and activator of transcription pathway
in myeloid cells (47); Socs1 recruitment by M-CSF-R could then
specifically inhibit this pathway. Socs1 may also link the receptor to
the ubiquitin machinery, as demonstrated for Vav (28), which could
occur through the interaction of the Socs box with ubiquitin ligase
complexes. The present study also clearly shows that Socs1 does not
equally affect proliferation signals of different receptors. Socs1
strongly suppressed IL-3 and SCF receptor signals, whereas its effects
on M-CSF-R were less intense. In that respect, Socs1 offers an example
of how a signaling molecule can differentially modify receptor
signaling and thereby control the balance between self-renewal and
commitment to differentiation. Finally, our experiments demonstrated
that it is possible to obtain stable expression of Socs1 in IL-3- or SCF-dependent cell lines as long as they are maintained in
the presence of high concentrations of M-CSF. Such cells represent valuable models for studying the early effects of Socs1 on other receptor signals after shifting the cells from M-CSF to other growth
factor (e.g. IL-3 or SCF)-containing medium. In conclusion, our work provides the first evidence for a connection between M-CSF-R
and Socs1, and we are now investigating the role of other members of
the Socs family during monocyte development as controlled by
M-CSF-R.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
platelet-derived growth
factor receptors (11). Ligand binding induces receptor dimerization and
trans-autophosphorylation in specific tyrosines of the cytoplasmic
domain, creating binding sites for src homology 2 (SH2)-containing
proteins (12). Known Fms partners include Src family members (13), Grb2
and Mona adaptors (14, 15), the p85 subunit of phosphatidylinositol
3-kinase (16), phospholipase C
2 (17), and FMIP (18). Some of
these interactions are difficult to detect in vivo, being
transient or expressed only at one particular stage of monocyte
development, and have been revealed only by means of sensitive
techniques such as yeast two-hybrid screen (15, 17, 18).
/
mice died within 21 days of birth, with a complex fatal
neonatal disease, due to a defect in the negative regulation of
interferon
signaling (25-27). Socs1 binds to all Janus kinase
family members and inhibits their tyrosine kinase activity, which is
needed for the activation of signal transducer and activator of
transcription (STAT). Because the Janus kinase pathway is also
essential for signaling by many cytokine receptors devoid of intrinsic
kinase activity, Socs1 may be a general negative regulator of signaling
through these receptors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction between Fms cytoplasmic domain
and Socs1 in the yeast two-hybrid system. A, schematic
representation of the Socs1 protein. P represents a PxxP
diproline motif. The region encompassed by the clone 99 is shown.
B, flow cytometry analysis of Fms/Socs1 interaction using
the yeast strain YRN974 carrying an integrated LexA-Operator-GFP
cassette. Data are representative of three independent experiments.
KD, kinase-dead.
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Fig. 2.
In vitro interaction between Fms
KI domain and Socs1 SH2 domain. GST-Fms KI fusion proteins were
produced as phosphoproteins in Epicurian cells, immobilized on
glutathione-Sepharose, and incubated with lysates of GP+E-86 cells
expressing either WT or mutated (R105K) HA epitope-tagged Socs1. Total
cell lysate and bound proteins were run on a 12% polyacrylamide gel,
blotted, and probed with anti-HA antibody for the detection of Socs1
protein and with anti-Grb2 antibody as a positive control. A
and B, effects of single, double, or triple tyrosine
(Y) to phenylalanine (F) mutations in the Fms KI
domain. C, effect of arginine (R) 105 to lysine
(K) mutation in the phosphotyrosine-binding pocket of Socs1
SH2 domain.
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Fig. 3.
Socs1 interacts with Fms and becomes
tyrosine-phosphorylated in M1/Fms/Socs1 cells stimulated with
M-CSF. A and B, M1 cells overexpressing HA
epitope-tagged Socs1 and either WT or a triple mutant (YTF) M-CSF-R
mutated on tyrosines 697, 706, and 721 were stimulated (+)
or not ( ) at 37 °C for 1.5 min by M-CSF. Cell lysates
were immunoprecipitated (IP) with anti-Socs1 antiserum.
Total cell lysates (A) and immunoprecipitates (B)
were run on 7.5 or 12% polyacrylamide gels, blotted, and probed with
anti-phosphotyrosine (PY), anti-Fms, or anti-HA antibodies.
C and D, M1 cells overexpressing HA
epitope-tagged Socs1 and wild-type M-CSF-R (Fms) were stimulated or not
at 37 °C for the indicated times by M-CSF. Cell lysates were
immunoprecipitated with anti-Socs1 antiserum. Total cell lysates
(C) and immunoprecipitates (D) were run on 7.5 or
12% polyacrylamide gels, blotted, and probed with anti-phosphotyrosine
or anti-HA antibodies.
2,
phosphorylate in response to M-CSF-R activation (17). Socs1 tyrosine
phosphorylation was then examined after M-CSF stimulation at 37 °C
for various times. M-CSF stimulation of M1/Fms WT/Socs1 cells induced
very rapid protein tyrosine phosphorylation, which then decreased after
2.5 min (Fig. 3C). Weak but significant tyrosine phosphorylation of Socs1 was detected as early as 30 s, reaching a
maximum at 1 min and decreasing between 2.5 and 5 min (Fig. 3D), demonstrating that Socs1 was rapidly and transiently
tyrosine-phosphorylated in response to M-CSF.
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Fig. 4.
Socs1 expression in myeloid FDC-P1/MAC cells
decreases M-CSF-dependent proliferation. FD/MAC cells
were infected by either empty pMiev or pMiev-HA-Socs1 retroviral
vectors in the presence of M-CSF (1000 units/ml) and sorted for GFP
expression by FACS (A). HA epitope-tagged Socs1 expression
was checked by Western blot analysis in FD-MAC/Socs1 cells as
compared with control FD-MAC/Miev cells. Equal gel loading was verified
by blotting with anti-SHC antibody (B). Cells were
stimulated or not by M-CSF for 1 h in ice and then lysed, and
Socs1 was immunoprecipitated; immunoprecipitates were run on 7.5 or
12% polyacrylamide gels, blotted, and probed with anti-phosphotyrosine
(PY) or anti-HA antibodies (C). 2 × 103 cells were seeded in 100 µl of IMDM, 10% FBS
supplemented or not with various concentrations of M-CSF or 1% of
IL3-CM. Proliferation was then monitored after 4 days by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
(D). 100 cells in 500 µl were plated in IMDM supplemented
with 1% methylcellulose, 10% FBS, and growth factors as specified,
and colonies were scored after 7 days of culture (E). Data
represent mean values ± S.E. of three independent
experiments (D and E).
View larger version (24K):
[in a new window]
Fig. 5.
Socs1 expression in EML-Fms cells abolishes
its growth in response to a limiting M-CSF concentration. EML-Fms
cells were infected by either empty pMiev or pMiev-HA-Socs1 retroviral
vectors in the presence of M-CSF (1600 units/ml) and sorted for GFP
expression by FACS (A). HA epitope-tagged Socs1 expression
was checked by Western blot analysis in EML-Fms/Socs1 cells as
compared with control EML-Fms/Miev cells. Equal gel loading was checked
by blotting with anti-SHC antibody (B). Cells were washed
twice with IMDM and seeded at 5 × 104 cells/ml in 1 ml of IMDM, 20% horse serum containing no growth factor, M-CSF (200 or
1600 units/ml), or SCF (1% of BHK/MKL-CM). The viable cell number was
determined every 2 days, and cultures were split and fed. Data
represent mean values of two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. R. Niedenthal and T. Tamura for the generous gift of the YRN974 yeast strain and Dr. Larry Rohrschneider for providing valuable material and critically discussing the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from the CNRS, INSERM, Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer, Fondation pour la Recherche Médicale, the Medical Research Council of Canada, and the National Cancer Institute of Canada.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.: 33 4 72 43 10 51; Fax: 33 4 72 44 05 55; E-mail: bourette@biomserv.univ-lyon1.fr.
Recipient of postdoctoral fellowships from the Medical
Research Council of Canada and La Ligue Nationale contre le Cancer.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M101878200
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ABBREVIATIONS |
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The abbreviations used are: M-CSF, macrophage colony-stimulating factor; M-CSF-R, M-CSF receptor; SH, src homology; Socs1, suppressor of cytokine signaling 1; KI, kinase insert; WT, wild type; GFP, green fluorescent protein; IL, interleukin; CM, conditioned medium; SCF, stem cell factor; BHK, baby hamster kidney; IMDM, Iscove's modified Dulbecco's medium; FBS, fetal bovine serum; FACS, fluorescence-activated cell sorting; HA, hemagglutinin; GST, glutathione S-transferase.
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