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* Bayer Pharma Research Center, Wuppertal, Germany;
Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany;
Novartis Pharma, Basel, Switzerland; and
Department of Microbiology and Immunobiology, Queen’s University of Belfast, Belfast, North Ireland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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G-CSF has been shown to specifically activate Janus kinase (JAK)1, JAK2, and TYK2 as well as the transcription factors STAT1, STAT3, and STAT5 (12, 13, 14, 15, 16). STAT3 activation has been implicated in G-CSF-mediated differentiation (17), whereas STAT5 seems to be involved in G-CSF-mediated proliferation and survival (18). Recent reports have revealed that several proteins containing Src-homology 2 (SH2) domains are recruited to the G-CSFR in an activation-dependent manner, thereby modulating G-CSFR signaling (19, 20, 21).
Suppressor of cytokine signaling-3 (SOCS-3), alternatively referred to as cytokine-inducible SH2-containing protein-3 (CIS-3), belongs to the SOCS family of proteins which have been shown to be induced by a number of cytokines and negatively regulate signal transduction in a classical feedback loop (21, 22, 23, 24). SOCS-proteins share a central SH2 domain and a C-terminal motif called the SOCS box (25, 26, 27) which is thought to be involved in protein degradation via the ubiquitin-proteasome pathway (28, 29, 30). Originally, SOCS-1 and SOCS-3 were shown to bind to JAK and inhibit their kinase activity (22, 23, 24). We and others have recently reported that SOCS-3 exerts part of its inhibitory action on IL-6 signal transduction by binding to pY759 within the signal transducing receptor subunit gp130 (31, 32). Both IL-6 and gp130 reveal similarities with G-CSF and the G-CSFR, respectively, and are thought to be evolutionary related. Thus, it is important to determine whether there is a similar inhibitory mechanism mediated by SOCS-3.
In this study, we show that SOCS-3 is induced by G-CSF in myeloid cells and inhibits G-CSFR-mediated signal transduction. Most importantly, we find that SOCS-3 is recruited to the G-CSFR in a phosphorylation-dependent manner. Furthermore, we identify phosphotyrosine (pY) 729 as being the major binding site for SOCS-3 on the G-CSFR and show direct binding of SOCS-3 to this pY motif.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biotinylated peptides were purchased from Jerini Biotools (Berlin, Germany). The respective sequences of the phosphorylated and unphosphorylated G-CSFR peptides used in this study were: PTLVQT(pY)VLQGDP (pY704), TSDQVL(pY)GQLLGS (pY729), SPGPGH(pY)LRCDST (pY744), TPSPKS(pY)ENLWFQ (pY764), PTLVQTYVLQGDP (Y704), TSDQVLYGQLLGS (Y729), SPGPGHYLRCDST (Y744), and TPSPKSYENLWFQ (Y764). Cell culture medium, fetal calf serum, and other media supplements were obtained from Life Technologies (Rockville, MD). Restriction enzymes were exclusively from NEB (Frankfurt, Germany). Zeocin was purchased from Invitrogen (Karlsruhe, Germany).
Cells and culture conditions
Human embryonic kidney (HEK293/CRL-1573) cells were obtained from American Type Culture Collection (Manassas, VA). PG13 (33) packaging cells were used for experiments with the agreement of H. Hanenberg, University of Düsseldorf, (Düsseldorf, Germany). U937 cells were obtained from American Type Culture Collection. HEK293 and PG13 packaging cells were grown in DMEM. U937 cells were cultured in RPMI medium. All media were supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C, 5% CO2. For stimulation, cells were treated with 50 ng/ml human rG-CSF (Amgen, Thousand Oaks, CA) and harvested at the time points indicated. Transduced cells were additionally cultured with 10 µg/ml zeocin.
Plasmids
The retroviral vector pMSCVneo (Clontech Laboratories, Heidelberg, Germany) was digested with EcoRI. Then, a PCR-amplified fragment containing the multiple cloning site (MCS) of BlueScriptII KS+ (Stratagene, Heidelberg, Germany) was digested with ApoI/EcoRI and ligated into pMSCVneo. Primers flanking the MCS of BlueScriptII KS+ were 5'-TTTAAATTTGAATTTGAGCTCCACCGCGGTGGCGGCCGC-3' and 5'-TTTAAATTTTAGTGGATCCCCCGGGCTGCAGGAATTC-3', respectively. pMSCVneo was then digested with BglII/AccI and a PCR-amplified fragment comprising a internal ribosome entry site-enhanced GFP-zeocin sequence was ligated BamHI/AccI into the MSCV backbone. PCR primers for the IRES-EGFP-ZEO fragment were as follows: 5'-TTTAAATTTGTCGACTCAGTCCTGCTCCTCGGCCACGAAGT-3' and 5'-TTTAAATTTGGATCCAATATTTAAATACCATGGCAATTGGAT-3'. This construct was named pMYZ404. Human SOCS-3 cDNA was amplified from EST no. 725896 (Research Genetics, Huntsville, AL) and cloned into pMYZ404 (pMYZ404-SOCS-3). Human SOCS-1 was PCR-amplified from a human lymph node cDNA library (Invitrogen, Karlsruhe, Germany) and subcloned into pcDNA 4.1 HisMax (Invitrogen). The myc-tagged SOCS-1 construct was kindly provided by Dr. A. Yoshimura (Kyushu University, Fukuoka, Japan). Human G-CSFR and STAT response element-luciferase cDNAs were a kind gift from Dr. A. Wilmen (Bayer, Wuppertal, Germany). The STAT response element consisted of six identical repeats of the STAT1, STAT3, STAT4, and STAT5 DNA binding motif TTCnnnGAA (34), spaced by 13 nucleotides, respectively. Y704F and Y729F G-CSFR mutants were generated using the QuikChange mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Mutagenesis primers for the Y704F as well as the Y729F mutant were as follows: 5'-CCCCACTCTGGTCCAGACCTTTGTGCTCCAGGGGGACCC-3' (Y704 sense), 5'-GGGTCCCCCTGGAGCACAAAGGTCTGGACCAGAGTGGGG-3' (Y704F antisense), 5'-GGCACCAGCGATCAGGTCCTTTTTGGGCAGCTGCTGGGCAGCCCC-3' (Y729F sense), and 5'-GGGGCTGCCCAGCAGCTGCCCAAAAAGGACCTGATCGCTGGTGCC-3' (Y729F antisense).
Transfection and retroviral transduction of HEK293 cells
HEK293 cells were transfected with 5 µg of human G-CSFR and the luciferase reporter construct under the control of the STAT response element cDNA using the lipofectamine transfection reagent (Life Technologies); 48 h after transfection, single clone screening was performed. After 3 wk, a number of clones stably carrying the cDNAs were expanded. The clones that responded best to G-CSF stimulation were taken for the experiments. For transient expression of SOCS-1 and SOCS-3, HEK293 cells were transfected with the indicated amount of cDNA using the fuGENE6 (Roche, Mannheim, Germany) transfection reagent.
Stable PG13 producer cells containing pMYZ404 and pMYZ404-SOCS-3 were grown to 100% confluency. Then, medium was exchanged and 1 day later retroviral supernatant was collected, filtered, and stored at -70°C until used. HEK293/G-CSFR/STAT/luciferase cells (1 x 105) were seeded into six-well plates, incubated overnight, and then infected with the retroviral supernatants in the presence of 5 µg/ml protamine. After 24 h of infection, media were replaced and after 48 h of cultivation, cells were selected with 50 µg/ml zeocin. The transduction efficiency was assessed by fluorescence microscopy. After 2 wk of selection, the cells were analyzed and used for experiments.
RT-PCR analysis of human polymorphonuclear neutrophils (PMN) and U937 cells for SOCS-3 expression
Blood samples were layered onto Ficoll-Hypaque, and PMN were isolated by centrifugation and hypotonic lysis of erythrocytes. Cells were rested in serum-free medium for 1 h (PMN) or 2 h (U937 cell line), and then stimulated with 50 ng/ml G-CSF for the times indicated. Total RNA was isolated from PMN (5 x 106 cells/RT-PCR) or cultured U937 (10 x 106 cells/RT-PCR) cells using the RNA STAT-60 method (Biogenesis, Poole, U.K.) following the manufacturer’s instructions. RT-PCR was performed using 1 µg of total cell mRNA using the OneStep RT-PCR kit from Qiagen (Hilden, Germany). PCR amplification was performed using primer pairs specific for human CIS (upstream primer, 5'-GATCTGCTGTGCATAGCCAA-3'; downstream primer, 5'-ACAAAGGGCTGCACCAGTTT-3'), SOCS-1 (upstream primer, 5'-GAGAGCTTCGACTGCCTCTT-3'; downstream primer, 5'-AGGTAGGAGGTGCGAGTTCA-3'), SOCS-2 (upstream primer, 5'-GATAAGCGGACAGGTCCAGA-3'; downstream primer, 5'-AAGAAGGCAAGGCATTCTGA-3'), SOCS-3 (upstream primer, 5'-CTCAAGACCTTCAGCTCCAA-3'; downstream primer, 5'-TTCTCATAGGAGTCCAGGTG-3'), and human GAPDH as a control (upstream primer, 5'-TGATGACATCAAGAAGGTGG-3'; downstream primer, 5'-TTACTCCTTGGAGGCCATGT-3'), the predicted products for CIS, SOCS-1, SOCS-2, SOCS-3, and GAPDH being 445, 562, 500, 554, and 244 bp, respectively. The PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining.
STAT reporter gene assays and STAT activation in total cell lysates
For reporter gene assays, HEK293/G-CSFR/STAT/luciferase cells were seeded on a 96-well microtiter plate at a density of 1 x 103 cells/well. Cells were incubated overnight, stimulated with 0.2 pg/ml to 1 µg/ml human rG-CSF and luciferase activity was measured after 6 h of stimulation.
To study the effect of SOCS-1 and SOCS-3 on STAT-3 activation, HEK293/G-CSFR/STAT/luciferase were transiently transfected with SOCS-3 or myc-tagged SOCS-1 constructs and stimulated with 50 ng/ml G-CSF for 10 min. Total cell lysates were prepared and analyzed by Western blot analysis. To examine the extent of STAT3 phosphorylation, the blot was detected with a specific phospho-STAT3 Ab (cell signaling) and then redetected with a specific STAT3 Ab (C20; Santa Cruz Biotechnology, Heidelberg, Germany). Expression of SOCS-1 and SOCS-3 was detected with a myc-Ab (9E10; Research Diagnostics, Flanders, NJ) or a SOCS-3 Ab (M20; Santa Cruz Biotechnology), respectively.
Peptide and coimmunoprecipitation assays
Approximately 0.15 µmol of the biotinylated peptides were immobilized by incubation with 2.5 mg streptavidin (SA) Sepharose (Amersham Pharmacia Biotech, Freiburg, Germany). For SOCS-1 and SOCS-3 precipitation, cells were lysed in 500 µl lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 0.1 mM EDTA) supplemented with NaF (50 mM), pepstatin A (2 µg/ml), leupeptin (5 µg/ml), aprotinin (5 µg/ml), PMSF (1 mM), and Na3VO4 (1 mM). Equal amounts of cellular protein and expressed SOCS proteins in each sample were obtained by mixing the total cell lysates before the precipitation experiment. SOCS-1 and SOCS-3 were precipitated by incubating total cell lysates with immobilized peptides at 4°C overnight. Precipitates were washed three times with 500 µl lysis buffer. The precipitated proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Stratagene) using a semidry electroblotting apparatus. Polyclonal rabbit anti-SOCS-3 was generated by using amino acids 5–21 of the human SOCS-3 protein. SOCS-1 was detected using a penta-His Ab (Qiagen). A polyclonal goat anti-rabbit HRP-conjugated secondary Ab (Santa Cruz Biotechnology) was used to visualize the immunoreactive bands by ECL techniques.
For immunoprecipitation experiments, cells were lysed as described above. Cell lysates were incubated with either G-CSFR (Research Diagnostics) or SOCS-3 Ab, resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Stratagene). The phosphorylation status was verified using the 4G10 pY Ab (Upstate Biotechnology, Lake Placid, NY). Bands were visualized as described above.
To coimmunoprecipitate endogenous G-CSFR with endogenous SOCS-3, U937 cells were rested in serum-free medium for 4 h and then stimulated with 50 ng/ml G-CSF for the times indicated. Proteasome inhibitor MG132 (4 µM) and 100 nM Na3VO4 were added 10 min before stimulation. The cells were lysed in PBS containing 0.5% Nonidet P-40 supplemented with NaF (50 mM), leupeptin (5 µg/ml), aprotinin (5 µg/ml), PMSF (1 mM), and Na3VO4 (1 mM) and SOCS-3 was precipitated with SOCS-3 Ab. Precipitated proteins were resolved by SDS-PAGE and visualized by Western bot techniques using Abs to SOCS-3 and G-CSFR.
Expression of SOCS-3 in bacteria
SOCS-3 was expressed as a thioredoxin fusion protein in BL21
(DE3) Escherichia coli (Stratagene). Bacteria were grown in
Luria-Bertani medium containing 100 µg/ml ampicillin at 37°C
to an A600 of 1.0 and then induced with 1
mM isopropyl-1-thio-
-galactopyranoside. Cells were harvested after
3 h of expression, resuspended in 50 mM Tris-HCl, pH 8.0, 10%
glycerol, and lysed by sonication. SOCS-3 was purified on a HiTrap
chelating column (Amersham Pharmacia Biotech) with Nickel-IDA as the
matrix. Native eluted SOCS-3 was dialyzed into 50 mM Tris-HCl, 10 mM
DTT, pH 8.5, and purified to homogeneity by anion exchange
chromatography on a MonoQ column (Amersham Pharmacia Biotech). For
biosensor measurements, the protein was dialyzed against 50 mM
Tris-HCl, pH 8.0, 10 mM DTT, 0.05%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.
Purity of the recombinant protein was monitored by SDS-PAGE.
Biosensor analysis
Biotinylated peptides were loaded on a SA-coated Biosensor chip (Biacore, Freiburg, Germany). The amount of loaded peptide was 80 ± 4 fmol/mm2 chip surface which corresponds to 141 ± 5 response units. Before loading of the sensor chip with peptide, the surface was washed three times for 30 sec with 1 M NaCl in 50 mM NaOH. Peptides (100 ng/ml) were loaded onto the chip until 150 response units. Protein-peptide interactions were measured by injection of serial dilutions of SOCS-3 over the chip surface at a flow rate of 20 µl/min for 1 min. Before injection of SOCS protein, the sensor chip was flushed with BSA (0.1 mg/ml) at a flow rate of 20 µl/min for 1 min. For measurement of the KD value, the flow rate was enhanced to 100 µl/min to obtain a higher resolution of kinetics. For this type of experiment, SOCS-3 was injected for 4 min, dissociation time was 5 min, regeneration of the chip between the measurements in all experiments performed was done at 20 µl/min with 1 M NaCl in 50 mM NaOH for 30 s. Binding curves were analyzed by using the BiaEvaluation software 3.0.1 (Biacore). To correct for nonspecific binding events, an empty sensor surface without peptide was analyzed in parallel during protein injection. Additionally, thioredoxin was injected at high concentrations (3.5 µM) to rule out nonspecific interactions of the fusion protein of SOCS-3. Curves were plotted with subtracted nonspecific binding. Determination of the dissociation constants was done by Scatchard analysis (35).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of SOCS-3 in G-CSF signaling, we first
analyzed SOCS-3 mRNA induction in the human myeloid precursor cell line
U937 that endogenously expresses the G-CSFR. SOCS-3 mRNA induction in
these cells was monitored by RT-PCR. Fig. 1
A shows that although SOCS-3
mRNA is already constitutively expressed in U937 cells, stimulation
with G-CSF leads to an immediate increase in SOCS-3 mRNA with the
expression peaking around 45 min after stimulation. The mRNA levels
then gradually decline and return to nearly basal levels within 3
h. In parallel, we checked for the induction of other members of the
SOCS family, namely CIS, SOCS-1, and SOCS-2. As shown in Fig. 1A, SOCS-1 is also induced by G-CSF, with mRNA levels
peaking around 60 min after stimulation. CIS and SOCS-2 mRNA are not
regulated by G-CSF. To further investigate whether G-CSF also induces
SOCS-3 in freshly isolated PMN, the PMN fraction of healthy donors was
stimulated with G-CSF. We observed that G-CSF also strongly induces
SOCS-3 mRNA in these primary cells with similar induction kinetics as
in U937 cells. As seen in U937 cells, G-CSF also induced SOCS-1 mRNA in
neutrophils (Fig. 1B). SOCS-2 and CIS mRNA was not found to
be regulated by G-CSF in these cells (data not shown).
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As we and others have observed that SOCS-3 acts on IL-6 signal
transduction by binding to pY759 of gp130 (31, 32) and as
the G-CSFR is closely related to gp130, it was important to determine
whether there is a similar inhibitory mechanism mediated by SOCS-3 in
the case of the G-CSFR. We first investigated the effect of SOCS-3 on
G-CSF-induced signal transduction. For this purpose, HEK293 cells were
stably transfected with cDNAs encoding G-CSFR and a STAT-luciferase
response element responsive to STAT1, STAT3, and STAT5 and then
retrovirally transduced with a construct coding either for green
fluorescent protein (GFP) or SOCS-3. Cells were stimulated with G-CSF
and luciferase activity was measured after 6 h. The dose-response
curves in Fig. 2A clearly
demonstrate the inhibitory effect of SOCS-3 on the STAT response
causing a depression of the maximal response to G-CSF by 74%. The
control experiment with cells transduced with a vector containing GFP
alone gave a similar dose-response curve as the mock-transduced cells
and the same maximal response was maintained.
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B shows that SOCS-1, like SOCS-3, impairs G-CSF-induced
STAT3 phosphorylation. We also found SOCS-1 to inhibit the
STAT-mediated induction of gene expression after G-CSF in a reporter
gene assay (data not shown). SOCS-3 is recruited to the G-CSF receptor upon G-CSF stimulation
SOCS-3 was initially shown to inhibit JAK/STAT signaling by
binding to and inhibiting JAK (22, 23, 24). As we and others
have found that SOCS-3 exerts at least part of its inhibitory action
through binding to cytokine receptor pY motifs (31, 32, 36, 37, 38, 39), we investigated whether SOCS-3 is also recruited to the
G-CSFR. To study possible interactions of SOCS-3 with the G-CSFR,
coimmunoprecipitation experiments were performed in HEK293 cells stably
transfected with G-CSFR and SOCS-3 or G-CSFR alone. Fig. 3, A and B, shows
that both proteins can be readily coimmunoprecipitated. Cell lysates
from stimulated and nonstimulated cells either transduced with human
SOCS-3 or nontransduced were incubated with an Ab recognizing epitopes
on the human G-CSFR. As shown in Fig. 3A, SOCS-3 associates
with the G-CSFR in a stimulation-dependent manner. Phosphorylation of
the G-CSFR was verified by probing the membrane with an anti-pY Ab.
Similarly, Fig. 3B shows the coimmunoprecipitation of the
G-CSFR with SOCS-3. Cell lysates of HEK/G-CSFR or HEK/G-CSFR/SOCS-3
cells were precipitated with an Ab raised against SOCS-3 and probed for
tyrosine phosphorylation, G-CSFR, and SOCS-3. Coprecipitated G-CSFR
could only be detected in cells stimulated with G-CSF whereas no G-CSFR
was detected in nonstimulated cells as well as HEK293/G-CSFR cell
lysates lacking SOCS-3. These data confirm the stimulation-dependent
association of SOCS-3 with the G-CSFR.
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C shows that endogenous
G-CSFR can be coprecipitated with endogenous SOCS-3 in a
G-CSF-dependent manner. SOCS-3 protein expression can already be
detected in nonstimulated U937 cells which coincides with the
observation that SOCS-3 mRNA is constitutively expressed in these cells
(Fig. 1A). After stimulation with G-CSF the SOCS-3 protein
levels gradually increase (Fig. 3C). SOCS-3 directly binds to a pY peptide corresponding to tyrosine Y729 of the G-CSFR
To identify the binding site for SOCS-3 on the G-CSFR, peptide
precipitation assays were performed with phosphorylated and
nonphosphorylated biotinylated peptides comprising the four tyrosine
motifs Y704, Y729, Y744, and Y764 of the human G-CSFR. The G-CSFR
peptides were incubated with cell lysates from HEK293 cells expressing
the G-CSFR and SOCS-3 (Fig. 4A). The peptides were
precipitated with SA Sepharose and coprecipitated SOCS-3 was visualized
by immunoblotting. Fig. 4A shows that SOCS-3 preferentially
binds to a peptide containing pY729. Small amounts of SOCS-3 were also
coprecipitated with the peptide encompassing pY704.
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B shows that purified SOCS-3 was
readily coprecipitated with the pY729 peptide, showing direct
binding to this motif. Nonphosphorylated peptides did not show
any interaction with SOCS-3. The direct interaction of SOCS-3 to
specific G-CSFR-peptides thus is pY-dependent and
sequence-specific.
Peptide precipitation assays performed with cells overexpressing SOCS-1
showed no interaction with the peptides of the G-CSFR (Fig. 4C)
SOCS-3 binds to pY729 within the G-CSFR
To assess the relevance of SOCS-3 binding to the phospho-Y729 and
-Y704 motifs within the G-CSFR, we generated G-CSFR mutants where
either Y704 or Y729 were substituted with phenylalanine. HEK293 cells
which have been transduced with SOCS-3 or a control vector were then
transiently transfected with the mutated receptors. As shown in Fig. 5, SOCS-3 was coimmunoprecipitated with
the G-CSFR in a stimulation-dependent manner in cells carrying the
mutated receptor G-CSFR/Y704F. However, immunoprecipitation of the
mutant receptor G-CSFR/Y729F failed to coprecipitate any SOCS-3
protein.
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To determine the importance of SOCS-3 recruitment to pY729 for
G-CSF-induced signal transduction, we performed reporter gene assays in
HEK293 cells that were stably transfected with cDNAs encoding the
G-CSFR point mutants Y704F or Y729F and a luciferase reporter construct
responsive to STAT1, STAT3, and STAT5. The cells were retrovirally
transduced with a construct coding either for GFP or SOCS-3. Cells were
stimulated with G-CSF and luciferase activity was measured after 6
h. Reporter assays in cells carrying a Y704F mutant showed similar
suppression of the luciferase signal (Fig. 6A) as observed with the
wild-type receptor (shown in Fig. 2). The suppressive effect of SOCS-3
could be overridden by mutating tyrosine Y729 to phenylalanine (Fig. 6B). The control experiment with cells transduced with a
vector containing GFP alone gave similar dose-response curves as the
mock-transduced cells and the same maximal response was maintained.
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Next, the affinity of the interaction between SOCS-3 and the
G-CSFR was determined. Phosphorylated and nonphosphorylated G-CSFR
peptides were immobilized on SA chips and the interaction with purified
rSOCS-3 was measured by means of surface plasmon resonance (SPR). Fig. 7, A and C, shows
the sensograms of the two phosphopeptides pY729 and pY704 which
interact with SOCS-3. KD was
calculated by Scatchard analysis (Fig. 7, B and
D). The SPR data (Table I)
revealed a calculated KD of 2.8 µM
for pY729 and 6.8 µM for pY704. The phosphopeptides pY744 and pY764
revealed KD values higher than 30
µM. Nonphosphorylated peptides which served as negative controls
showed no interaction with SOCS-3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). With the exception
of SOCS-3 our data differ from a previous report where G-CSF was
reported to induce CIS and SOCS-2, but not SOCS-1 mRNA expression, in
bone marrow cells (22). In contrast, induction of SOCS-1
mRNA following treatment with G-CSF has been shown in the murine
myeloid cell line NFS-60 (23). This differential induction
of SOCS protein members by G-CSF is very interesting and needs further
investigation.
STAT1, STAT3, and STAT5 have been shown to be activated by G-CSF
(16, 18, 50). Although STAT3 has been linked to myeloid
cell differentiation (17, 51), STAT5 seems to be involved
in G-CSF-dependent cell proliferation (18). Because we
found a potent induction of SOCS-3 mRNA in U937 cells as well as in
primary neutrophils upon G-CSF stimulation, we examined the impact of
SOCS-3 on G-CSF-induced STAT DNA binding by means of a STAT-luciferase
assay. In these experiments, we observed a significant
(p < 0001) reduction in luciferase activity
suggesting that SOCS-3 acts as a feedback inhibitor of G-CSF-mediated
signal transduction (Fig. 2A). A dose-response analysis
revealed suppression of the maximal response in SOCS-3-expressing cells
by 74%. In addition, we demonstrate that SOCS-1 also negatively
regulates G-CSFR-mediated signal transduction (Fig. 2B).
Like SOCS-1, SOCS-3 was initially shown to inhibit JAK/STAT signaling
by binding to and inhibiting JAK (22, 23, 24). We and others
have recently shown that SOCS-3 not only interacts with JAK, but also
exerts at least part of its inhibitory effect by binding to the
phosphorylated IL-6-type cytokine receptor subunit gp130 (31, 32). Because the G-CSFR shares 
45% homology with gp130 and
these two receptors are structurally related to a great extent, it was
important to determine whether there is a similar inhibitory mechanism
mediated by SOCS-3. We performed coimmunoprecipitation experiments and
could show that SOCS-3 interacts with the G-CSFR in a
stimulation-dependent manner (Fig. 3). Furthermore, we were able to
coimmunoprecipitate endogenous G-CSFR with endogenous SOCS-3 in a
G-CSF-dependent manner, indicating that the interaction is of
physiological relevance (Fig. 3C). This, together with other
recent data (36, 37, 38, 39, 52), strongly suggests that
recruitment of SOCS-3 to cytokine receptor pY motifs is required for
SOCS-3 to negatively regulate cytokine signaling.
To identify the recruitment site for SOCS-3 on the G-CSFR, we performed
a peptide precipitation assay with phosphorylated and nonphosphorylated
biotinylated peptides comprising one of the four tyrosine motifs of the
cytoplasmic part of the G-CSFR. We found that pY729 was the major
binding site for SOCS-3 and determined a
KD of 2.8 µM for this interaction.
In addition, we found pY704 to interact with SOCS-3, the calculated
KD for this interaction being around
6.8 µM. For comparative reasons, KD
values of the SOCS-3/G-CSFR peptide interaction were set into relation
with dissociation constants of known interaction partners of SOCS-3. We
similarly performed SPR measurements with SOCS-3 and a biotinylated
gp130 peptide comprising the binding motif for SOCS-3, namely pY759. In
these experiments, a KD of 210 nM
was obtained.4 The determined affinity is in the
same range of the affinity measured in a recent study reporting a
KD value of 42 nM for this interaction
(32). This difference may be explained by the use of
peptides corresponding to the amino acid sequence of human gp130 (pY759
motif (56), amino acid sequence: A-TSSTVQpYSTVVHSG) vs murine gp130
(pY757 motif, Ref. 32 , amino acid sequence:
acetyl-STASTVEpYSTVVHSG) and, in contrast, by differences in the
experimental conditions used in both studies. The G-CSFR peptide
binds SOCS-3 with 13-fold less avidity than the pY759 motif of
gp130.
In comparison, SOCS-1, which we also found to be induced by G-CSF, did
not interact with G-CSFR-derived phosphopeptides (Fig. 4C).
This is in agreement with the established inhibitory mechanism of
SOCS-1 through direct binding to the kinases of the Janus family
(23, 24, 53) and emphasizes the difference in the
inhibitory mechanisms of SOCS-1 and SOCS-3.
The alignment of the peptide sequence around the tyrosine motifs Y729
and Y704 of the G-CSFR with known SOCS-3 recruiting motifs (Table II) (31, 32, 37, 38, 39, 52)
shows a significant sequence homology in the case of the motif pY729
whereas the amino acid sequence around pY704 shares no homology with
the proposed consensus sequence h-X-pY-h/S/T-X-L/V-h-h (with h =
hydrophobic) optimal for SOCS-3 recruitment (52).
Subsequent coimmunoprecipitation experiments, as well as reporter gene
assays with either wild-type G-CSFR or the Y704F and Y729F mutants,
were performed to clarify the role of the two tyrosine motifs in regard
to the SOCS-3 action in the context of the full-length G-CSFR and the
cellular environment. We observed a complete loss of SOCS-3 binding
upon mutation of Y729, strongly suggesting that pY729 is the
physiological binding site for SOCS-3 and that pY704 does not play a
significant role in recruiting SOCS-3 in the context of the full-length
G-CSFR (Fig. 5). Reporter gene assays performed with the wild-type
receptor, as well as with Y704F and Y729F receptor, mutants revealed
that mutation of Y704 has no effect on the suppression of
G-CSF-mediated STAT activation by SOCS-3. In contrast, substitution of
Y729 to phenylalanine resulted in luciferase activities similar to
those found in control or mock-transduced cells. These data, together
with the data from our coimmunoprecipitation experiments, strongly
suggest that Y729 of the G-CSFR is essential for the inhibitory effect
of SOCS-3 on G-CSFR-mediated signal transduction.
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Taken together, we have shown that SOCS-3 is strongly and rapidly induced by G-CSF in myeloid cells and in turn potently inhibits G-CSFR-mediated signal transduction. We provide evidence that the inhibitory action of SOCS-3 on G-CSF signaling involves the direct binding of SOCS-3 to the activated G-CSFR and we identify pY729 as being the recruitment site for SOCS-3. To evaluate the implications of SOCS-3 recruitment to the receptor motif pY729 on the various signaling components involved in G-CSF signal transduction, further studies are necessary and are currently under investigation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter C. Heinrich, Department of Biochemistry, Medical School, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany. E-mail address: heinrich{at}rwth-aachen.de
3 Abbreviations used in this paper: SCN, severe congenital neutropenia; JAK, Janus kinase; SH2, Src-homology 2; SOCS, suppressor of cytokine signaling; CIS-3, cytokine-inducible SH2-containing protein-3; HEK, human embryonic kidney; MCS, 3-methylcholanthrene; PMN, polymorphonuclear neutrophil; GFP, green fluorescent protein; pY, phosphotyrosine; SA, streptavidin; SPR, surface plasmon resonance.
Received for publication November 26, 2001. Accepted for publication May 30, 2002.
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in NFS-60 cells. Growth Factors 13:251.[Medline]
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