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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 113-124
By
From the Institute of Hematology, Erasmus University, Rotterdam; and
the Department of Hematology, Dr Daniel den Hoed Cancer Center,
Rotterdam, The Netherlands.
The granulocyte colony-stimulating factor receptor
(G-CSF-R) activates multiple STAT proteins. Although the
membrane-proximal cytoplasmic region of the G-CSF-R is necessary and
sufficient for activation of STAT1 and STAT5, activation of STAT3
requires the membrane distal region that contains four tyrosines.
Although one of these (Y704) has previously been shown to be involved
in STAT3 activation from a truncated G-CSF-R derived from a patient with severe chronic neutropenia (SCN), this tyrosine is not required for STAT3 activation by the full-length G-CSF-R. To investigate possible alternative mechanisms of STAT3 activation, we generated a
series of Ba/F3 cell transfectants expressing the wild-type G-CSF-R or
mutant receptors that either completely lack tyrosines or retain just
one of the four cytoplasmic tyrosines of the G-CSF-R. We show that, at
saturating G-CSF concentrations, STAT3 activation from the full-length
G-CSF-R is efficiently mediated by the C-terminal domain in a manner
independent of receptor tyrosines. In contrast, at low G-CSF
concentrations, Y704 and Y744 of the G-CSF-R play a major role in STAT3
activation. Both tyrosine-dependent and -independent mechanisms of
STAT3 activation are sensitive to the Jak2 inhibitor AG-490, follow
similar kinetics, and lead to transactivation of a STAT3 reporter
construct, indicating functional equivalence. STAT3 activation is also
impaired, particularly at nonsaturating G-CSF concentrations, in bone
marrow cells from mice expressing a truncated G-CSF-R
(gcsfr-
GRANULOCYTE COLONY-STIMULATING
factor (G-CSF) plays a crucial role in the regulation
of granulopoiesis by stimulating the proliferation, survival, and
maturation of myeloid progenitor cells.1-4 The various
biological effects of G-CSF are mediated through a receptor (G-CSF-R)
of the hematopoietin receptor superfamily, which forms homo-oligomeric
complexes upon ligand binding.5,6 The cytoplasmic domain of
the human G-CSF-R contains four conserved tyrosine residues (Y704,
Y729, Y744, and Y764) that serve as potential docking sites for Src
homology 2 (SH2) domains of signaling proteins.7 Deletion
studies have shown that the membrane-proximal cytoplasmic region of the
G-CSF-R, lacking all four tyrosines, is indispensable for transduction
of growth signals, whereas the carboxy-terminal region, including three
tyrosines, is involved in the induction of neutrophilic
maturation.8-10
We have previously identified mutations in the G-CSF-R that delete the
carboxy-terminal domain in approximately 20% to 25% of cases of
severe congenital neutropenia (SCN) and an even higher proportion of
cases of acute myeloid leukemia (AML) preceded by SCN, indicating a
positive role for this region in modulating G-CSF signaling in primary
cells.11-13 When expressed in myeloid cells these truncated
receptors transduce a strong growth signal but are defective in
maturation signaling.12 We recently generated mice with a
similar mutation in the gcsfr gene (gcsfr- Like other hematopoietin receptors, the G-CSF-R lacks intrinsic
tyrosine kinase activity but activates cytoplasmic tyrosine kinases.2,5 Signal transduction pathways that involve
activation of Janus tyrosine kinases (Jaks), Jak1, Jak2, and Tyk2, and
signal transducer and activator of transcription (STAT) proteins,
STAT1, STAT3, and STAT5, have been linked to the
G-CSF-R.15-22 Jaks associate with the membrane-proximal
cytoplasmic region of the G-CSF-R and become activated upon
ligand-induced receptor homo-oligomerization.18,19,22,23 Jak activation leads to tyrosine phosphorylation of a conserved tyrosine residue in the C-terminus of the STAT proteins. Subsequently, STAT proteins form stable homodimers and heterodimers by interactions between the SH2 domain of one STAT protein and the phosphotyrosine of
another STAT protein before translocation to the nucleus, where they
influence transcription of target genes by binding to specific regulatory sequences.24 A crucial question regards how the
STATs are recruited to the receptor/Jak complexes to mediate their
activation. It has been proposed that receptor-associated Jaks
phosphorylate specific STATs via their recruitment to particular
cytoplasmic domains of each receptor. For example, interleukin-4
(IL-4)-induced activation of STAT6 is mediated by tyrosines 578 and
606 of IL-4-R.25 Similarly, multiple tyrosine residues in
the cytoplasmic domain of gp130 and LIF receptor mediate STAT3
activation,26 whereas tyrosine 440 in the cytoplasmic
domain of the interferon- The membrane-proximal region of the G-CSF-R, containing the conserved
box 1 and box 2 subdomains, is sufficient for G-CSF-induced activation
of STAT1 and STAT5, but not for STAT3.18,20 Importantly, activation of STAT3 has recently been linked to G-CSF-mediated differentiation.29 Determining the mechanisms of STAT3
activation is vital, therefore, for understanding how the G-CSF-R
controls the processes of proliferation and differentiation. In this
study, we show that different mechanisms of STAT3 activation operate depending on ligand concentration. At saturating levels of G-CSF, STAT3
activation is effectively mediated by a mechanism involving the
C-terminal region of the G-CSF-R, which does not require the presence
of phosphorylated receptor tyrosine residues to provide docking sites.
In contrast, at low G-CSF concentrations, tyrosine-dependent docking
via Y704 and Y744 plays a major role in the activation of STAT3. Both
tyrosine-dependent and -independent mechanisms of STAT3 activation
require Jak2 activity, follow similar kinetics, and result in
transcriptionally active STAT3 complexes. In addition, we show reduced
STAT3 activation from truncated G-CSF-R derived from SCN patients, even
at saturating G-CSF concentrations. Moreover, there is an altered dose
response in STAT3 compared with STAT5, which exacerbates this reduced
activation, such that at lower G-CSF concentrations the STAT3
deficiency is amplified compared with other G-CSF signals. These
findings reveal an unexpected heterogeneity in the signaling function
of the G-CSF-R under different receptor saturation conditions that
typically occur in vivo during basal granulopoiesis (low G-CSF) or
during "emergency" conditions, such as bacterial infections (high
G-CSF), and suggest that decreased STAT3 activation may contribute to
the defective maturation signaling from truncated G-CSF receptors
derived from patients with SCN.
Cells and cell culture.
The IL-3-dependent murine pro-B-cell line Ba/F330 and a
subline of the IL-3-dependent murine myeloid cell line
32Dcl3,31 called 32D.cl8.6, were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 10 ng of
murine IL-3 per mL at 37°C and 5% CO2. Mice containing
the gcsfr- Site-directed mutagenesis.
The pLNCX expression clones of human G-CSF-R wild-type (WT), the
deletion mutant mDA (d715), the single tyrosine-to-phenylalanine (Y
Transfections and analysis.
For stable transfections, Ba/F3 and 32D cells were electroporated with
10 µg PvuI-digested pLCNX clones, using a
Progenetor (Hoefer Scientific Instruments, San Francisco,
CA) apparatus set at 230 V, 100 µF, and 1 second. After 24 hours of
incubation, cells were selected with G418 (GIBCO-BRL, Breda, the
Netherlands) at a concentration of 1.2 and 0.8 mg/mL, respectively.
Multiple clones were expanded for further analysis. To determine
G-CSF-R expression levels, cells were incubated at 4°C for 60 minutes sequentially with 10 µg/mL of biotinylated mouse anti-human
G-CSF-R monoclonal antibody LMM741 (PharMingen, San Diego, CA), 5 µg/mL of phycoerythrin (PE)-conjugated streptavidin, 5 µg/mL of
biotinylated anti-streptavidin antibody, and finally 2 µg/mL of
PE-conjugated streptavidin, with washing between each antibody step.
Samples were analyzed by flow cytometry using a FACScan (Becton
Dickinson, San Jose, CA). At least three independently derived cell
lines of each construct with homogeneous receptor expression, as
determined by equivalent mean fluorescence following
fluorescence-activated cell sorter (FACS) analysis, were selected for
further study. For transient transfections, Ba/F3 cells were
electroporated with 50 µg of a STAT3(m67)-luciferase construct
described previously33 at 260 V, 490 µF, and 1 second.
After 18 hours of recovery on IL-3, cells were washed and divided into
two portions, which were incubated separately for a further 8 hours
with either IL-3 or G-CSF, before harvesting for luciferase assays.
Preparation of cell lysates, immunoprecipitation, and Western
blotting.
Cells were deprived of serum and factors for 4 hours at 37°C in
RPMI 1640 medium at a density of 4 × 106 per mL and
then stimulated with either RPMI 1640 medium alone or in the presence
of 100 ng/mL human G-CSF. At different time points, 10 volumes of
ice-cold phosphate-buffered saline (PBS) supplemented with 10 µmol/L
Na3VO4 were added. Subsequently, cells were
pelleted and lysed by incubation for 30 minutes at 4°C in lysis
buffer (20 mmol/L Tris-HCl pH 8.0, 137 mmol/L NaCl, 10 mmol/L EDTA, 100 mmol/L NaF, 1% Nonidet P-40, 10% glycerol, 2 mmol/L Na3VO4, 1 mmol/L Pefabloc SC, 50 µg/mL
aprotinin, 50 µg/mL leupeptin, 50 µg/mL bacitracin, and 50 µg/mL
iodoacetamide). Insoluble materials were removed by centrifugation at
4°C for 15 minutes at 15,000g. Immunoprecipitations were
performed on the clarified cell lysates by incubation overnight at
4°C with anti-STAT3 antibodies (sc-482; Santa Cruz Biotechnology
Inc, Santa Cruz, CA). Protein A-Sepharose beads (Pharmacia, Uppsala,
Sweden) were then added for 1 hour at 4°C. After washing the beads
with ice-cold lysis buffer 4 times and once with PBS, bound proteins
were eluted by boiling for 5 minutes in sodium dodecyl sulphate (SDS)
sample buffer. Following SDS-polyacrylamide gel electrophoresis
(SDS-PAGE), proteins were transferred onto nitrocellulose (0.2 µm;
Schleicher & Schuell, Dassel, Germany). Filters were blocked by
incubation in TBST (10 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 0.05%
[vol/vol] Tween-20) containing 0.6% (wt/vol) bovine serum albumin
(BSA). Antibodies used for Western blotting were anti-phosphotyrosine
antibody 4G10 (Upstate Biotechnology Inc, Lake Placid, NY),
anti-G-CSF-R (sc-693; Santa Cruz), and anti-STAT3 described above, and
were diluted in TBST containing 0.6% (wt/vol) BSA. After washing with
TBST, immune complexes were detected with horseradish
peroxidase-conjugated species-specific antiserum (DAKO, Glostrup,
Denmark), followed by enhanced chemiluminescence reaction (DuPont,
Boston, MA). In some instances, membranes were stripped in 62.5 mmol/L
Tris-HCl pH 6.7, 2% SDS, and 100 mmol/L Preparation of nuclear extracts.
Cells were stimulated as described above then pelleted and resuspended
in ice-cold hypotonic buffer (20 mmol/L HEPES pH 7.8, 20 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L
Na4P2O7, 1 mmol/L DTT, 1 mmol/L
EDTA, 1 mmol/L EGTA, 0.2% Tween-20, 0.125 µmol/L okadaic acid, 1 mmol/L Pefabloc SC, 50 µg/mL aprotinin, 50 µg/mL leupeptin, 50 µg/mL bacitracin, and 50 µg/mL iodoacetamide).34 Cells
were vortexed for 10 seconds and the nuclei pelleted by centrifugation at 15,000g for 30 seconds. Nuclear extracts were prepared by
resuspension of the nuclei in high-salt buffer (hypotonic buffer with
420 mmol/L NaCl and 20% glycerol) and extraction of proteins by
rocking for 30 minutes at 4°C. Insoluble materials were removed by
centrifugation at 4°C for 15 minutes at 15,000g. For the
Jak2 inhibition studies, cells were preincubated with the specific
inhibitor AG-49035 at 50 µmol/L for 1 hour before
stimulation.
Electrophoretic mobility shift assay (EMSA).
Nuclear extracts of approximately 0.4 to 0.5 × 106
cells were incubated for 20 minutes at room temperature with 0.2 ng of
32P-labeled double-stranded oligonucleotide (5 to 10 × 103 cpm) and 2 µg of poly(dI-dC) in 20 µL of
binding buffer (13 mmol/L HEPES, pH 7.8, 80 mmol/L NaCl, 3 mmol/L NaF,
3 mmol/L NaMoO4, 1 mmol/L DTT, 0.15 mmol/L EDTA, 0.15 mmol/L EGTA, and 8% glycerol).36 The oligonucleotide
probes used in this study were m67 (5 Luciferase assays.
IL-3 and G-CS-treated cells were lysed in a minimal volume of
luciferase lysis buffer (25 mmol/L Tris phosphate pH 7.8, 15% glycerol, 1% Triton X-100, 1 mmol/L DTT, 8 mmol/L MgCl2)
and, after removal of cell debris by centrifugation, the supernatant was assayed on a Biocounter M2500 luminometer (Lumac, Landgraaf, The
Netherlands) using an equal volume of luciferin solution (1 mmol/L
luciferin, 1 mmol/L adenosine triphosphate [ATP], 8 mmol/L MgCl2) as a substrate. Because IL-3 activates equivalent
levels of STAT3 in each of the clones analyzed (data not shown),
luciferase activity in IL-3-treated cells was taken as an internal
control. To match for variations in the transfection efficiency of the reporter construct, the G-CSF-mediated effects on the reporter constructs are expressed as the ratio of G-CSF-mediated
transactivation to IL-3-mediated transactivation.
In vitro binding analysis.
The cytoplasmic domain of the human G-CSF-R and the SH2 domain of STAT3
were cloned into pET-15b (Novagen, Madison, WI) and pGEX-2TK
(Pharmacia), respectively, using standard PCR protocols to amplify the
appropriate coding regions. For the production of
tyrosine-phosphorylated G-CSF-R cytoplasmic domain, the pET clone was
introduced into the Escherichia coli strain TKB1 (Stratagene, La Jolla, CA), which contains an inducible tyrosine kinase, and fusion
protein produced according to the manufacturer's instructions. For
production of GST and GST-STAT3(SH2), plasmids were transformed into
XL-1 Blue (Stratagene), with proteins expressed and purified on
glutathione-Sepharose 4B beads as described.39 Equivalent amounts of bead-bound GST or GST-STAT3(SH2) were incubated with tyrosine-phosphorylated G-CSF-R cytoplasmic domain in TBST containing 10% glycerol and 1 mmol/L DTT at 4°C for 1 hour, before extensive washing with the same buffer. To detect bound G-CSF-R, beads were boiled in SDS-sample buffer and subjected to Western blot analysis with
anti-G-CSF-R antibody, as described earlier.
Construction and expression of different forms of the human G-CSF-R.
Ba/F3 cells have provided a convenient model for the study of specific
signaling pathways from the G-CSF-R.15,19,20,23,32 To
investigate the mechanisms of STAT3 activation via the G-CSF-R, a
series of triple Y
G-CSF-induced activation of STAT3 can occur in the complete absence
of tyrosines in the cytoplasmic domain of the G-CSF-R.
Tyrosine 704 of the G-CSF-R was assumed to be the major docking site
for STAT3 because it fits the consensus STAT3 activation sequence,
YxxQ.26 However, while Y704 is essential for STAT3 activation by the truncated form of the receptor,20
substitution of this or other tyrosines in the full-length G-CSF-R had
little effect on STAT3 activation, suggesting the involvement of other mechanisms.20 To investigate this further, we compared
STAT3 activation from the WT G-CSF-R with the mutants mDA, truncated at
position 715; mDAF, truncated at position 715 with a Y704F mutation;
and mO, which represents the full-length receptor "null mutant"
with no tyrosines (Fig 1A). As described previously,20 the
WT G-CSF-R activates a large amount of STAT3 homodimer (upper band) as
well as STAT3:STAT1 heterodimer and some STAT1 homodimer (Fig 2A). Interestingly, in this direct
comparison, the truncation mutant mDA also activates the same
complexes, although clearly at a reduced level, further suggesting
STAT3 activation pathway(s) from the receptor C-terminus. Mutant mDAF,
on the other hand, only induces very minor activation of STAT3,
probably via indirect mechanisms involving STAT1.20
Strikingly, mutant mO activated STAT3 almost to the same level as the
WT receptor. This indicates that although Y704 is essential for STAT3
activation from truncated receptors, receptor tyrosines are not
required for robust STAT3 activation from the full-length receptor.
Analysis of STAT3 tyrosine phosphorylation in response to G-CSF showed
a similar reduction from truncated receptors (Fig 2B). This confirms
that the WT receptor activates more STAT3 than truncated forms, and
rules out differences in nuclear transport or binding affinity as a
cause of the decreased STAT3 binding observed by EMSA.
Tyrosine-dependent STAT3 activation from the G-CSF-R predominates at
low G-CSF concentrations.
Given the already strong activation of STAT3 from mO, we then
investigated what additional, and perhaps more subtle, role the G-CSF-R
tyrosine residues may play in STAT3 activation from the full-length
G-CSF-R. Therefore, we compared STAT3 activation by the WT G-CSF-R, the
triple mutants, and the null mutant (Fig 3A). At 100 ng/mL G-CSF (~4 to 5 nmol/L), a concentration
approximately 10-fold above the receptor Kd,5
WT G-CSF-R induced the highest level of STAT3 tyrosine phosphorylation,
followed by mA and mC, which activated approximately 80% of this
level. Compared with WT G-CSF-R, activation of STAT3 by mB, mD, and mO
was reduced to approximately 60%. Analysis of STAT3 activation at this
G-CSF concentration by EMSA produced similar results (Fig 3B).
Strikingly, however, at a concentration of 1 ng/mL G-CSF, which is
approximately 10-fold below the Kd of the G-CSF-R, these
differences become much more pronounced. At this level of receptor
saturation, STAT3 activation by mutants mB, mD, and mO was several fold
below that of the WT G-CSF-R, whereas mA and mC showed an intermediate
level. Serial dilution of the nuclear extracts from cells stimulated at
100 ng/mL G-CSF confirmed that the EMSA was in the linear range using
extracts stimulated with this ligand concentration (Fig 3C and D),
ruling out probe depletion in the EMSA as the cause of the difference
between stimulation with high and low G-CSF concentrations.
STAT3 docks directly to Y704 and Y744 of the G-CSF-R.
To determine if the activation mediated by Y704 and Y744 is due to
direct binding of STAT3, as reported for other cytokine receptors,26,27,40,41 we added phosphopeptides spanning
each of the four cytoplasmic tyrosines of the murine G-CSF-R to STAT binding reactions. If the SH2 domain of STAT3 has specificity for the
phosphotyrosine-containing sequence, then the equivalent peptide should
be able to disrupt preformed STAT3 dimers, leading to a reduction in
binding by EMSA. The addition of phosphopeptides covering either Y703
or Y743 of the murine G-CSF-R, equivalent to Y704 or Y744 of the human
receptor, respectively, successfully competed for STAT3 dimer
formation, as determined by DNA binding (Fig 4A). This suggests direct docking of
STAT3 to these phosphorylated tyrosines on the activated receptor. The
Y743 peptide competed less efficiently for STAT3 binding than Y703,
indicating a possible difference in relative affinity for STAT3 (Fig
4B). However, unlike the Y703 peptide, which contains the same YxxQ
motif found in the human G-CSF-R, the Y743 peptide contains a YxxS
motif rather than the YxxC sequence found in the equivalent position of
the human receptor, so whether the same difference in affinity occurs with the human receptor is as yet unclear. To confirm that direct binding occurs, we examined interactions between the isolated STAT3 SH2
domain and tyrosine-phosphorylated G-CSF-R cytoplasmic domain in vitro
(Fig 4C). Binding of the G-CSF-R was observed with the GST-STAT3(SH2),
but not to GST alone. Interestingly, only specific forms of the
tyrosine-phosphorylated G-CSF-R cytoplasmic domain bound, presumably
because only these forms contain Y704 and/or Y744 in a
tyrosine-phosphorylated form. No specific binding was observed if a
non-tyrosine-phosphorylated G-CSF-R was used as a probe (data not
shown).
Tyrosine-dependent and -independent routes of STAT3 activation are
functionally equivalent.
Because mDA almost exclusively uses a receptor tyrosine-dependent route
of STAT3 activation (via Y704), whereas mO relies solely on a receptor
tyrosine-independent route, comparison of mDA and mO provides an
opportunity to separately investigate these different routes of STAT3
activation from the G-CSF-R. To analyze whether STAT3 activation in
both cases depends on Jak activity, we repeated the EMSA with cells
pretreated with the specific Jak2 inhibitor AG-490.35
Activation of STAT3 by both mDA and mO was sensitive to the Jak2
inhibitor (Fig 5A), indicating that Jak2 is
required for tyrosine-dependent and -independent activation routes.
Furthermore, time-course studies showed that, although mDA and mO
activated STAT3 at slightly different levels, time kinetics of
activation were similar (Fig 5B). Finally, to determine if the STAT3
complexes activated by the alternate routes had comparable transactivating properties, we investigated the ability of the different mutants to activate transcription of a STAT3-luciferase reporter construct.33 Eighteen hours after transfection,
cells were incubated with G-CSF or IL-3 for 8 hours and lysates assayed for luciferase activity (Fig 5C). Clones expressing the WT G-CSF-R, or
the mDA and mO mutants, all activated the reporter to an extent comparable to their STAT3 DNA binding as analyzed by EMSA.
Reduced STAT3 activation from truncated G-CSF receptors found in
severe chronic neutropenia.
We have recently generated mice with a mutation in the gcsfr
gene (gcsfr-
The data presented here reveal multiple mechanisms of STAT3 activation
by the G-CSF-R. Firstly, we have shown that activation from the
full-length receptor at saturating ligand concentrations is effectively
mediated by the C-terminus in a non-tyrosine-dependent manner.
Presumably this is achieved by docking through an intermediate molecule
associated with the C-terminal domain of the G-CSF-R. Secondly,
activation of STAT3 can also occur via Y704 or Y744, which appears to
be the major mechanism of activation at low G-CSF concentrations. In
contrast, Y704 alone is the major mechanism for a C-terminal truncated
mutant of the G-CSF-R found in patients with severe congenital
neutropenia.11 Finally, previous studies have shown that
STAT3 can also be activated to a small extent indirectly, via
heterodimer formation with STAT1 or STAT5.20,21
We thank M. Parren-van Amelsvoort and S. Oomen for excellent technical
advice and assistance, M. von Lindern and T. de Koning-Ward for
critical reading of the manuscript, A. Yoshimura and T. Hirano for
plasmids, A. Levitzki for the Jak2 inhibitor, and K. van Rooyen for
exquisite graphical work.
Submitted March 16, 1998;
accepted September 1, 1998.
Address reprint requests to Alister C. Ward, PhD, Institute of
Hematology, Erasmus University (Room H Ee 1314), P.O. Box 1738, 3000 DR
Rotterdam, The Netherlands; e-mail: ward{at}hema.fgg.eur.nl.
1.
Nicola NA:
Hemopoietic cell growth factors and their receptors.
Annu Rev Biochem
58:45, 1989[Medline]
[Order article via Infotrieve]
2.
Demetri GD, Griffin JD:
Granulocyte colony-stimulating factor and its receptor.
Blood
78:2791, 1991
3.
Lieschke GJ, Grail D, Hodgson G, Metcalf D, Stanley E, Cheers C, Fowler KJ, Basu S, Zhan YF, Dunn AR:
Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.
Blood
84:1737, 1994
4.
Liu F, Wu HY, Wesselschmidt R, Kornaga T, Link DC:
Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice.
Immunity
5:491, 1996[Medline]
[Order article via Infotrieve]
5.
Fukunaga R, Ishizaka Ikeda E, Seto Y, Nagata S:
Expression cloning of a receptor for murine granulocyte colony-stimulating factor.
Cell
61:341, 1990[Medline]
[Order article via Infotrieve]
6.
De Vos AM, Ultsch M, Kossiakoff AA:
Human growth hormone and extracellular domain of its receptor: Crystal structure of the complex.
Science
255:306, 1992
7.
Moran MF, Koch CA, Anderson D, Ellis C, England L, Martin GS, Pawson T:
Src homology region 2 domains direct protein-protein interactions in signal transduction.
Proc Natl Acad Sci USA
87:8622, 1990
8.
Dong F, van Buitenen C, Pouwels K, Hoefsloot LH, Lowenberg B, Touw IP:
Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation.
Mol Cell Biol
13:7774, 1993
9.
Fukunaga R, Ishizaka-Ikeda E, Nagata S:
Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor.
Cell
74:1079, 1993[Medline]
[Order article via Infotrieve]
10.
Ziegler SF, Bird TA, Morella KK, Mosley B, Gearing DP, Baumann H:
Distinct regions of the human granulocyte colony-stimulating factor receptor cytoplasmic domain are required for proliferation and gene induction.
Mol Cell Biol
13:2384, 1993
11.
Dong F, Hoefsloot LH, Schelen AM, Broeders CA, Meijer Y, Veerman AJ, Touw IP, Lowenberg B:
Identification of a nonsense mutation in the granulocyte-colony-stimulating factor receptor in severe congenital neutropenia.
Proc Natl Acad Sci USA
91:4480, 1994
12.
Dong F, Brynes RK, Tidow N, Welte K, Lowenberg B, Touw IP:
Mutations in the gene for the granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe c |
Top
Abstract
Introduction
715). These findings suggest that G-CSF-induced STAT3 activation during basal granulopoiesis (low G-CSF)
and "emergency" granulopoiesis (high G-CSF) are differentially controlled. In addition, the data establish the importance of the
G-CSF-R C-terminus in STAT3 activation in primary cells, which has
implications for understanding why truncated G-CSF-R derived from SCN
patients are defective in maturation signaling.
715 mice) that showed a basal neutropenia.
(IFN-
chain is required
for STAT1 phosphorylation and activation, presumably through specific
interaction with the SH2 domain of STAT1.
F) substitution mutants Y704F, Y729F, Y744F, and Y764F, and the combined mutant mDAF (d715-Y704F) have been described
previously.
-CCTGGGCTTGTGGGGCTGC), GRFR11
(5
-mercaptoethanol at
50°C for 30 minutes, reblocked, washed, and reprobed. For signal
quantification, Western blots were scanned with an ArcusII Color
Scanner (Agfa, The Hague, the Netherlands) and the data processed using
ImageQuant Software (Molecular Dynamics, Sunnyvale, CA).
) or with 100 ng/mL G-CSF (+).
Nuclear extracts were prepared and incubated with
32P-labeled double-stranded m67 oligonucleotide. (B) STAT3
immunoprecipitation from lysates from Ba/F3 cells expressing WT or
mutant G-CSF-R proteins. Serum- and growth factor-starved cells were
incubated for 10 minutes at 37°C without factor (
