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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 15-24
RAPID COMMUNICATION
Identification of a Novel Stat3 Recruitment and Activation Motif Within
the Granulocyte Colony-Stimulating Factor Receptor
By
Arup Chakraborty,
Kevin F. Dyer,
Michael Cascio,
Timothy A. Mietzner, and
David J. Tweardy
From the Division of Infectious Diseases, the Departments of Medicine
and the Department of Molecular Genetics and Biochemistry, University
of Pittsburgh School of Medicine and the University of Pittsburgh
Cancer Institute, 200 Lothrop St, Pittsburgh, PA.
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ABSTRACT |
Stat3 is essential for early embryonic development and for myeloid
differentiation induced by the cytokines granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6). Two isoforms of Stat3 have
been identified,  (p92) and  (p83), which have distinct transcriptional and biological functions. Activation of both Stat3 and Stat3 requires the distal cytoplasmic domain of the G-CSFR, which contains four Tyr at positions 704, 729, 744, and 764. The studies reported here were undertaken to determine which, if any, of
these tyrosine residues participated in Stat3/ recruitment and
activation. We showed that Stat3 and Stat3 were affinity purified
using phosphopeptides containing Y704 and Y744 but not by
nonphosphorylated peptide analogues or by phosphopeptides containing Y729 and Y764. Complementary results were obtained in studies examining
the ability of these peptides to destabilize and inhibit DNA binding of
activated Stat3. Both Y704 and Y744 contributed to optimal activation
of Stat3/ in M1 murine myeloid leukemia cells containing
wild-type and Y-to-F mutant G-CSFR constructs. Carboxy-terminal to Y704
at the +3 position is Gln; YXXQ represents a consensus Stat3
recruitment and activation motif. Y744 is followed at the +3 position
by Cys (C); YXXC, represents a novel motif implicated in the
recruitment and activation of Stat3. Modeling of the SH2 domain of
Stat3 based on homologous SH2 domains of known structure revealed polar
residues whose side chains contact the +3 position. This substitution
may confer specificity for the Y704- and Y744-based ligands by allowing
H-bond formation between the binding surface and the Gln or Cys found
at the respective +3 position.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
TARGETED DISRUPTION of the mouse
Stat3 gene leads to embryonic lethality at 6.5 to 7.5 days,
indicating that Stat3 is essential for early embryonic
development.1 At the cellular level, Stat3 has been shown
to be involved in the signaling cascade initiated by receptors of
several classes. These include receptors for granulocyte
colony-stimulating factor (G-CSF) and for the interleukin-6 (IL-6)
cytokine family as well as other type I and type II cytokine
receptors2 (IL-2,3 IL-4,4
IL-5,5 IL-10,6 IL-12,7
IL-13,4 interferon-,8 growth
hormone,9 thrombopoietin,10 and
leptin11), receptor tyrosine kinases (epidermal growth
factor receptor [EGFR],12 platelet-derived growth factor
receptor [PDGFR],13 colony-stimulating factor-1 receptor
[CSF-1R],13 and basic fibroblast growth factor receptor [bFGFR]14) and the G-protein-coupled receptors for
angiotensin II15 and the CC chemokines RANTES and
MIP-1.16 G-CSF initiates a signaling cascade in bone
marrow progenitor cells that is critical to their differentiation into
neutrophils.17,18 Activation of Stat3 by the G-CSFR as well
as by the homologous IL-6R chain (gp130) has been shown to be
essential for differentiation of murine myeloid cell lines by their
respective cytokines.19-21
The G-CSF receptor (G-CSFR) is a member of the type I cytokine receptor
family.2 Ligand-induced dimerization of the G-CSFR results
in activation of protein Tyr kinases (PTK) including
Jak1,22Jak2,23-25 and Lyn.26
Activation of receptor-associated PTK results in receptor Tyr
phosphorylation and recruitment of SH2-containing proteins including
additional PTKs such as Syk,26 adapter proteins such as
Shc,27 and members of the STAT protein family including Stat1,23 Stat3,23,28,29 and
Stat5.23 Stat3 activation has been shown to require the
distal cytoplasmic portion of the G-CSFR which contains four Tyr
residues,22,30 suggesting that Stat3, in particular, may be
recruited directly by the receptor through an SH2-phosphotyrosine
interaction involving one or more of these residues.
Two isoforms of Stat3 derived from a single gene have been described in
mice31 and humans28,32 and are designated
Stat3 and Stat3. Unlike Stat3 (p92), Stat3 (p83) appears to
functionally interact with c-Jun.31 Also, Stat3 lacks
Ser727 present in Stat3 that has been shown to be a site of
phosphorylation,33,34 possibly by MAP kinase.33
Serine phosphorylation has been shown to enhance Stat3 DNA
binding33 and to be required for maximal transcriptional
activation.34 In addition, Stat3 inhibits the ability of
Stat3 to activate promoter constructs in transient-transfection assays.32 The distinct transactivating abilities of
Stat3 and Stat3 appear to be biologically important. G-CSF
activated predominantly Stat3 in normal adult human
CD34+ bone marrow cells capable of differentiating in
response to G-CSF.28 In contrast, G-CSF activated
predominantly Stat3 in acute myeloid leukemia cell lines not shown
to differentiate in response to G-CSF.28 Intriguingly, in
studies revealing an essential role for Stat3 in gp130-induced M1 cell
differentiation, the investigators demonstrated that overexpression of
Stat3 behaved in a dominant-negative fashion and inhibited terminal
differentiation of this cell line.20 In contrast,
overexpression of Stat3 blocks v-Src transformation of
fibroblasts.35
Studies examining the ability of full-length and truncated mutant
G-CSFR constructs to induce proliferation and differentiation of
myeloid cells showed that while the N-terminal half of the cytoplasmic
domain was necessary and sufficient for proliferation, differentiation
required the full-length receptor, thereby mapping specific signaling
events required for differentiation to the C-terminal half the
cytoplasmic domain.36,37 We30 and
others25 previously showed that activation of Stat3 did not
correlate with proliferation; rather, it required the C-terminal half
of the cytoplasmic domain of the G-CSFR, which is essential for
differentiation.37 This region contains four Tyr at
positions 704, 729, 744, and 764. STAT proteins have previously been
shown to be recruited to receptor complexes through SH2-phosphotyrosine
interactions.6,38-43 The studies reported here were
undertaken to determine which phosphotyrosine, if any, within the
G-CSFR are responsible for recruitment and activation of Stat3 and
Stat3 and to determine whether any of the phosphotyrosines show
preferential binding activity for either Stat3 isoform.
Our results demonstrate that phosphotyrosine motifs containing Y704 and
Y744 are responsible for Stat3 and Stat3 recruitment and that
each contributes to Stat3/ activation by G-CSF. Neither motif
showed preferential affinity for either Stat3 isoform. Although the
Y704 motif, YVLQ, follows the consensus Stat3 recruitment motif (YXXQ),
the Y744 motif, YENC, represents a novel Stat3 recruitment motif. The
presence of polar amino acid residues, Q and C, at the +3 position
within these phosphotyrosine ligands along with sequence analysis of
the Stat3 SH2 support a model of Stat3 SH2-phosphotyrosine binding in
which the region of the Stat3 SH2 domain that binds the +3 amino acid
forms a polar interacting surface.
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EXPERIMENTAL PROCEDURES |
Cells.
All factor-independent acute myeloid leukemia cell lines were
maintained, as described,28 in RPMI 1640 (Life
Technologies, Gaithersburg, MD) supplemented with heat-inactivated
fetal calf serum (10%), penicillin (100 U/mL), and streptomycin (100 U/mL). The parental pro-B cell line, BAF/BO3, and BAF/BO3 cells
expressing wild-type and truncated mutant G-CSFR constructs were
maintained as described.30 The murine myeloid leukemia cell
line, M1, and clones stably transfected with wild-type human G-CSFR or
mutant constructs containing single tyrosine-to-phenylalanine mutations (Y704F, Y729F, Y744F, and Y764F) and double mutations (Y704/744F) were
kindly provided by Dr Judith Layton (Ludwig Institute for Cancer Research, Victoria, Australia) and were maintained as
described.44
Peptides.
Phosphotyrosine-containing peptides (12 amino acid residues long based
on the sequence surrounding Tyr residues 704, 729, 744, and 764 within
the human G-CSFR) and a Stat3-specific peptide (11 amino acid
residues long starting at the N-terminus with cysteine and containing
the last ten amino acid residues of Stat3) were used in these
studies (Table 1). Peptides were
synthesized at the Department of Molecular Genetics and Biochemistry
Shared Resources Facility of the University of Pittsburgh School of
Medicine on an automated peptide synthesizer (PerSeptive Biosystems,
Inc, Framingham, MA) using standard
fluoren-9-ylmethoxycarbonyl (FMOC) solid-phase synthesis protocols as
described.45 Where indicated, a portion of each peptide was
biotinylated before deprotection at the N-terminus using
sulfo-N-hydroxysuccinimidyl-6-(biotinamido)hexanoate (Sulfo-NHS-LC-Biotin; Pierce Chemicals, Rockford, IL). Peptide composition and purity was confirmed by mass spectroscopy.
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Table 1.
Peptides Synthesized Spanning Either Side of Each of the
Four Tyr Residues Within the G-CSFR Cytoplasmic Domain (1-4) and
Containing Stat3-Specific Sequence (5)
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Cytokines and antibodies.
Recombinant human G-CSF was purchased from Amgen (Thousand Oaks, CA).
Stat3 antibody, C-20 (Santa Cruz Biotechnology, Santa Cruz, CA), was
generated in rabbits immunized with the C-terminal end of murine
Stat3/p92, amino acids 750 to 769. Stat3 monoclonal antibody
(Transduction Laboratories, Lexington, KY) was developed in mice
immunized with a portion of murine Stat3/p92 containing amino acids
1-178.
Chicken IgY specific for Stat3 was generated by an outside vendor
(Charles Rivers PharmServices, Southbridge, MA) by preparation of a
Stat3-specific peptide immunogen corresponding to the unique C-terminal residues that are not found in Stat3 (Table 1).
This peptide was rendered immunogenic by coupling it to the carrier thyroglobulin. This peptide/carrier conjugate (250 µg in complete Freund's adjuvant) was used to immunize a hen subcutaneously followed by booster injections (100 µg in incomplete Freund's adjuvant) every
3 weeks. Upon demonstrating high titer on enzyme-linked immunosorbent
assay (ELISA) to a peptide-bovine serum albumin (BSA) conjugate, the
IgY was fractionated from eggs per the distributor's protocols.
Electrophoretic mobility shift assay (EMSA).
Cells ( 106) in suspension were incubated in 1 mL
phosphate-buffered saline (PBS) with or without cytokine at 37°C.
Whole-cell and nuclear extracts were prepared and EMSAs performed on
4% native polyacrylamide gels using the high-affinity
sis-inducible element (hSIE; m67) as described.30
Phosphorylated and nonphosphorylated peptide inhibition studies were
performed as described.46 Briefly, whole-cell
extracts (20 µg) of cells stimulated with G-CSF (100 ng/mL) for 30 minutes were incubated with tyrosine phosphorylated peptides or nonphosphorylated peptides at 0, 30, 100, 300, and 400 µmol/L for 60 minutes at 37°C before addition
of radiolabeled duplex hSIE and EMSA. The gels were dried and exposed
to Kodak XAR film (Eastman Kodak Co, Rochester, NY) before
developing.
Phosphopeptide and antibody affinity purification and immunoblotting
studies.
Biotinylated peptides (120 pmol) were incubated overnight at 4°C with
streptavidin coated paramagnetic beads from Dynal (Lake Success, NY).
Beads were washed thoroughly with buffer A (20 mmol/L HEPES pH 7.8, 100 mmol/L NaCI, 20 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L Na4P2O7, 1 mmol/L EDTA, 1 mmol/L EGTA, 20% glycerol, 0.05% NP-40, 1 mmol/L dithiothreitol
(DTT), 1 mmol/L phenylmethylsulfonyl fluoride [PMSF], 1 mg/mL
leupeptin, and aprotinin). DER cells were lysed using buffers A (with
1% NP-40) and centrifuged at 14,000 rpm to remove cell debris. The
supernatant was diluted 1:1 with buffer A (without NaC1 and NP-40) and
incubated with Dynal beads prebound to biotinylated peptide for 2 hours
at 4°C and washed three times with buffer A. Phosphopeptide
affinity-purified proteins were separated and immunoblotted as
described.28
Stat3-specific IgY antibody was conjugated to CNBr-activated
Sepharose 4B (Pharmacia, Uppsala, Sweden) following the
protocol supplied by the manufacturer. Whole-cell extracts (1 mg) were incubated with 50 µL of IgY-Sepharose suspension (50% in buffer A)
for 60 minutes at 4°C and washed three times in buffer A. Bound proteins were eluted by boiling in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer, and separated on SDS-PAGE and immunoblotted with Stat3
monoclonal antibody as described.28
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RESULTS |
In vivo activation of Stat3 and
Stat3 in BAF/BO3 cells expressing full-length and
truncated mutants of G-CSFR.
EMSA analysis performed previously on extracts of parental
BAF/BO3 cells and derivatives expressing wild-type and truncated mutant
G-CSFR suggested that maximal activation of Stat3 required full-length G-CSFR.30 To map the region of the G-CSFR
required for activation of Stat3 by EMSA, we developed IgY antibody
specific for Stat3 using as immunogen a peptide containing the
C-terminal seven amino acid residues unique to Stat3 (Table 1). This
antibody immunoprecipitated Stat3 but not Stat3 (Fig
1). EMSA of extracts of G-CSF-stimulated
BAF/BO3 cells transfected with full-length G-CSFR (B-183) or truncated
G-CSFR mutants showed activation of an hSIE-protein complex only in
B-96 and B-183 (Fig 2A), which express G-CSFR constructs that contain one or more Tyr. Supershift analysis of the complexes from these cells demonstrated a low level of
Stat3 activation in B-96; however, maximal activation of Stat3
required the full-length G-CSFR. To confirm this result, we performed
DNA-affinity purification of whole-cell extracts of these cells (Fig
2B). Immunoblotting of hSIE-affinity-purified proteins showed the
presence of Stat3 and Stat3 only in extracts of G-CSF-stimulated
B-96 and B-183 cells. Immunoblotting of whole-cell extracts of each of
these cell lines revealed that preferential activation of Stat3/
in B-96 and B-183 was not due to increased levels of expression of
Stat3 isoforms in these cell lines compared with B-57, B-26, or
parental BAF-BO3 (Fig 2C).
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| Fig 1.
Specificity of Stat3 antibody. Whole-cell protein
extracts (1 mg) of unstimulated cells were incubated with rabbit
polyclonal Stat3-specific antibody (C-20; 1 µg) followed by
protein G-Sepharose (left lane) or with chicken Stat3-specific
IgY conjugated to CNBr-activated Sepharose Beads (right lane). Bound
proteins were eluted by boiling in SDS-PAGE sample buffer and separated
by SDS-PAGE and immunoblotted using Stat3 monoclonal antibody. The
positions of the prestained molecular-weight markers are indicated on
the left and the positions of the Stat3 and Stat3 bands are
indicated on the right. The results shown are representative of two
experiments.
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| Fig 2.
EMSA supershift analysis and DNA-affinity chromatography
of BAF/BO3 cells transfected with full-length and truncated mutants of
G-CSFR. BAF/BO3 cells were stably transfected with vector containing
either wild-type human G-CSFR (HRG-183) or with vector containing
G-CSFR constructs truncated at intracytoplasmic amino acid residue 96 (HGR-96), residue 57 (HGR-57) or residue 26 (HGR-26) as
described.36 The constructs contained one or more of the
homology regions, boxes 1 and 2, and tyrosines (Y) as indicated in
panel A. Cells were extracted before ( ) or after (+) G-CSF or
interferon- stimulation. EMSA was performed without () or with
(+) antibody to Stat3. The position of the SIF-A, B, and C
complexes and the supershifted(SS) Stat3 complex are indicated on
the right. The results shown are each representative of two
experiments. In (B), the stably transfected cell lines (B-26, B-57,
B-96, and B-183) and the parental cell line (BO3) were stimulated with
G-CSF (100 ng/mL for 15 minutes) and extracted. Whole-cell protein
extracts (500 µg each) were bound to and eluted from a hSIE-affinity
purification column. Eluted proteins were separated by SDS-PAGE,
immunoblotted with Stat3 monoclonal antibody, and developed with ECL
chemiluminescence. The position of the prestained molecular-weight
markers are shown on the left and the position of the Stat and
Stat3 bands are shown on the right. In (C), whole-cell protein
extracts of each cell line (50 µg each) were separated by SDS-PAGE
and immunoblotted with Stat3 monoclonal antibody. The positions of
Stat3 and Stat3 are indicated on the right.
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Phosphopeptides based on Y704 and Y744 affinity purify
Stat3 and Stat3.
To determine specifically which, if any, of the Tyr residues within the
C-terminal cytoplasmic domain of the hG-CSFR are involved in the
recruitment of human Stat3 and Stat3, phosphopeptides 12-amino
acid residues each were synthesized spanning either side of each Tyr
within the G-CSFR cytoplasmic domain (Table 1). Each phosphopeptide was
incubated with whole-cell extracts of human myeloid leukemia cell
lines, DER and THP-1, previously shown to express both isoforms of
Stat3 upon immunoblotting of whole-cell extracts.28
Immunoblotting demonstrated bands corresponding to both Stat3 and
Stat3 (Fig 3 and data not shown) in the
peptide-affinity complexes of phosphopeptides based on Y704 and Y744
but not Y729 or Y764 and not within complexes formed with
nonphosphorylated analogues of peptides based on Y704 and Y744. Of
note, neither Stat3 nor Stat3 showed preferential binding to
phosphopeptides based on either Y704 or Y744. Immunoblotting of
proteins affinity purified by phosphopeptides Y704 and Y744 also
revealed a third Stat3 cross-reactive band of
approximately 72 kD, which we recently showed to be a
proteolytic fragment of Stat3.47
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| Fig 3.
Phosphopeptide affinity purification of Stat3 and
Stat3. Biotinylated tyrosine phosphorylated peptide (+P) or
nonphosphorylated peptides (P) were bound to paramagnetic beads and
incubated 2 hours at 4°C with whole cell extracts (1.5 mg) prepared
from unstimulated DER cells. The peptide-protein complexes were
magnetically separated and eluted by boiling in SDS-PAGE sample buffer
followed by SDS-PAGE separation. The proteins were transferred to PVDF
membrane, developed with Stat3 monoclonal antibody, and visualized by
ECL chemiluminescence. The position of prestained molecular-weight
markers are indicated on the left and of Stat3 and Stat3 are
indicated on the right. The results shown are representative of two
experiments.
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Phosphopeptides based on Y704 and Y744 inhibit Stat3
and Stat3 binding to DNA.
Once activated, STAT protein dimers can be specifically destabilized
and inhibited from binding DNA by incubation with phosphopeptides based
on receptor sequences involved in their recruitment and activation.46 We examined whether phosphopeptides based on
Y704 and Y744 could destabilize Stat3 dimers and thereby inhibit their ability to bind DNA. Whole-cell extracts of G-CSF-stimulated cells including DER, THP-1, EM2, and EM3 were preincubated with
phosphopeptides before addition to binding reactions and EMSA.
Phosphopeptides based on Y704 and Y744 nearly completely inhibited hSIE
binding by Stat3/ while phosphopeptides based on Y729 and Y764
and nonphosphorylated analogues of Y704 and Y744 peptides had no effect
(Fig 4 and data not shown). These results
provide additional support for a critical role for Y704 and Y744, but
not Y729 and Y764, in Stat3 recruitment to the G-CSFR.
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| Fig 4.
Phosphopeptide inhibition of Stat3/ binding to
hSIE. Whole-cell extracts (20 µg) of an acute leukemia cell line,
EM2,28 stimulated with G-CSF (100 ng/mL) for 30 minutes
were incubated with the indicated tyrosine phosphorylated peptides
(+P) or nonphosphorylated peptides (P) at 0, 30, 100, 300, and 400 µmol/L for 60 minutes at 37°C before addition of radiolabeled
duplex hSIE and EMSA. The gels were dried and exposed to Kodak XAR film
for before developing. The results shown are representative of three
experiments.
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Y704 and Y744 contribute to optimal activation of
Stat3 and Stat3 by G-CSF in M1 cells.
To determine whether Y704 and Y744 contribute to Stat3/
activation, we examined the effect of mutation of these residues to
phenylalanine on Stat3/ activation in the murine myeloid leukemia
cell line, M1. This cell line was previously shown to differentiate
into macrophages after heterologous expression of the human G-CSFR and
upon exposure to G-CSF.44 M1 cell clones expressing Y704F
and Y744F mutant G-CSFR constructs and especially clones expressing the
Y704/744F double mutant showed dramatically reduced ability to
differentiate in response to G-CSF.44 EMSA supershift
analysis was performed on parental M1 cells and M1 clones expressing
wild-type and mutant G-CSFR (Y704F, Y729F, Y744F, Y764F, and Y704/744F)
at equivalent levels44 using Stat3- or Stat3-specific
antibody (Fig 5). Each antibody supershifts
its respective isoform but also reduces complex formation (Fig 2A and
data not shown). Consequently, we used the nonsupershifted band
following incubation with Stat3-specific antibody for quantitation of Stat3 by PhosphorImager analysis. Clones expressing G-CSFR mutants Y729F and Y764F demonstrated levels of Stat3 activation similar to clones expressing wild-type receptor (Fig 5A and B). In
contrast, levels of Stat3 activation were reduced in M1 clones expressing G-CSFR containing either the Y704F or Y744F mutation. Furthermore, in clones expressing G-CSFR containing the double mutation
(Y704/744F), levels of Stat3 activation were reduced to levels
observed in the parental M1 cells. Similar results were obtained for
Stat3 (Fig 5C and D), although receptors containing Y729F and Y764F
also showed reduced Stat3 activation compared with clones expressing
wild-type receptor.
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| Fig 5.
Stat3 and Stat3 supershift analysis of M1 cells
clones transfected with wild-type and mutant human G-CSFR constructs.
M1 parental cells (P) or clones (two each) expressing either wild-type
(WT) human G-CSFR or constructs containing tyrosine-to-phenylalanine
mutations at the indicated tyrosine residues were stimulated with G-CSF
(1 ng/mL for 15 minutes) and extracted. EMSA was performed in the
presence of antibody specific for either Stat3 (A and B) or Stat3
(C and D). (A and C) Autoradiographs of EMSA gels of M1 parental cells
and a representative clone containing each construct. The position of
the supershifted and nonsupershifted Stat3 and Stat3 are
indicated on the right. The signal remaining within the nonsupershifted
Stat3 band (B) and the nonsupershifted Stat3 band (D) obtained
from M1 parental cells and both clones containing each construct were
quantitated by PhosphoImager analysis and the mean ± SEM shown.
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DISCUSSION |
Evaluation of Stat3-deficient mice showed that Stat3 plays an essential
and nonredundant role in early embryonic development. In addition,
Stat3 activation is critical for myeloid differentiation in murine cell
line models in response to G-CSF19 and
IL-6.20,21 G-CSF-dependent myeloid differentiation maps to
the distal cytoplasmic region of the human G-CSFR,37,48
which contains four tyrosines (Y704, Y729, Y744, and Y764), and
especially to Y704 and Y744.44 To map the tyrosine(s)
required for Stat3 recruitment and activation, we performed DNA
affinity purification of whole-cell extracts of G-CSF-activated
BAF/BO3 cells containing full-length and truncated G-CSFR constructs
and supershift analysis, which suggested that Y704 and possibly one or
more of the distal tyrosines participated in the recruitment of
Stat3 and Stat3. We showed that both Stat3 isoforms were affinity
purified using phosphopeptides based on Y704 and Y744 but not by
nonphosphorylated peptide analogues or by peptides bases on Y729 and
Y764. In addition, phosphopeptides based on Y704 and Y744 completely
inhibited the DNA-binding activity of both Stat3 and Stat3 while
nonphosphorylated peptide analogues of each and phosphopeptides bases
on Y729 and Y764 had no effect. EMSA analysis of M1 parental cells and
M1 cell clones expressing wild-type or Y-to-F mutant human G-CSFR
constructs confirmed the contribution of Y704 and Y744 to activation of
both Stat3 and Stat3.
The regions of the G-CSFR surrounding Y704 and Y744 have previously
been implicated as important in the function of G-CSFR based either on
homology to regions within related receptors or on gene activation
studies. Y744 is contained within the box 3 domain determined by
sequence homology between the G-CSFR, gp130, and the
LIFR.49 Y704 is contained within a region of the G-CSF essential for activation of acute phase response genes when wild-type or truncated receptors were transfected into human hepatoma cell lines.36 More recently, these two tyrosine sites especially Y744 have been shown by Nicholson et al44 to be critical
for G-CSF-induced differentiation of the murine myeloid leukemic cell line, M1, transfected with wild-type and Y-to-F mutant G-CSFR constructs. This group was unable to consistently demonstrate a
contribution of any G-CSFR tyrosine to Stat3 phosphorylation and
activation. However, they examined extracts of cells stimulated with a
G-CSF at 10 ng/mL which is 100-fold higher than the concentration of
G-CSF that resulted in macrophage differentiation in 50% of cells
containing wild-type G-CSFR (EC50WT), 100-fold greater than the EC50Y764F, 14- to 17-folder greater than the
EC50Y704F and EC50Y724F and 7-fold greater than
the EC50Y744F. We also were unable to detect clear
differences in levels of Stat3 activation between M1 cells containing
the different Y-to-F mutant constructs at G-CSF concentrations 10
ng/mL (data not shown). However, in the results shown (Fig 5), Stat3
activation was examined in extracts from cells stimulated with a
concentration of G-CSF nearer the EC50 of most of the
Y-to-F mutant ie, 1 ng/mL. The membrane proximal cytoplasmic region of
the G-CSFR is responsible for association with and activation of
Jak2,23,25,50 which in turn may directly recruit and
activate Stat3.51-54 Consequently, it is possible that Stat3 activation by direct Jak2 interaction may obscure differences in
Stat3 activation mediated through receptor phosphotyrosine interactions
at higher G-CSF concentration (10 ng/mL).
Stat3 has been shown to be essential for differentiation induced by
ligand-activated G-CSFR19 and gp130,20,21 a
receptor whose intracellular domain is homologous to that of the
G-CSFR. These findings together with the results of Nicholson et al and our laboratory strongly support the following hypothesis:
phosphotyrosine residues at position 704 and 744 within the G-CSFR are
responsible for the recruitment and activation of Stat3 which, in turn,
is critical for G-CSF-induced myeloid differentiation.
Our results indicate that phosphorylated Y704 and Y744 each recruit
Stat3 directly, resulting in Stat3 activation. In contrast, the
findings of reduced Stat3 activity, especially Stat3, in M1 cells
containing Y729F and Y764F constructs suggest that Y729 and Y764 each
may affect Stat3 activation indirectly, possibly by decreasing
phosphorylation of Y704 and Y744 within the G-CSFR, as suggested by the
findings of Yoshikawa et al,55 or by altering the secondary
structure of the cytoplasmic region around Y704 and Y744, thereby
interfering with their ability to recruit Stat3. Alternatively, Y729
and/or Y764 may activate a signaling pathway that regulates
Stat3 DNA binding activity downstream of tyrosine phosphorylation in a
positive manner, through activating serine phosphorylation of Stat3 or,
in a negative manner, by activating one of the proteolytic pathways
demonstrated to be involved in STAT protein inactivation, ie,
proteosomal degradation, caspases or serine proteases.47 Of
note is the finding that Y764 may be involved in the activation of Ras
and MAP kinase through its recruitment of Shc/GRB2,27
suggesting a linkage between Y764 and Stat3 serine phosphorylation.
Also notable are the findings of two groups44,55 that
implicate Y729 (or Y728 in the murine G-CSFR) in G-CSF-induced
differentiation of M1 and LGM-1 cells, respectively. Their findings,
together with the results reported here, suggest that this tyrosine
activates a differentiation-promoting signaling pathway distinct from
Stat3.
We have previously shown that Stat3 is preferentially activated by
G-CSF in myeloid precursor cells capable of differentiating into
neutrophils in response to this ligand while Stat3 activation predominated in myeloid precursor cells incapable of G-CSF-induced neutrophilic differentiation. The isoform of Stat3 preferentially activated by G-CSF in a given cell line corresponded to the predominant isoform that was expressed within extracts of that cell
line.28 The studies reported here were undertaken, in part,
to examine whether selective recruitment of either Stat3 isoform to the
G-CSFR might also contribute to preferential activation. Our results, however, do not support this hypothesis.
Each STAT protein contains a single SH2 domain.8 Critical
ligands for STAT protein SH2 domains include receptor
phosphotyrosine-containing motifs and phosphotyrosine-containing motifs
located near the C-terminus of the STAT proteins
themselves.6,8,38-43 Interaction with the receptor
phosphotyrosines causes juxtapositioning of STAT proteins with
activated receptor-associated PTK, including members of the JAK and Src
family resulting in phosphorylation of STAT proteins on their
C-terminal tyrosine. Interaction between the SH2 domain of one STAT
protein with the C-terminal phosphotyrosine of another STAT protein
results in homodimerization or heterodimerization, the requisite
configuration for STAT proteins to bind DNA. The amino acid residues
surrounding the phosphotyrosine greatly influence the affinity of the
SH2-phosphotyrosine interaction.56 Of the surrounding
residues, the one at the C-terminal +3 position has been shown to have
the greatest impact on SH2 binding. The phosphotyrosine-containing motif at position Y704 is YVLQ. This motif contains the polar amino
acid residue, Gln (Q), at the position identical to all previously
described motifs shown to recruit Stat3 including those within human
gp130 (YRHQ, YFKQ, YLPQ, and YMPQ) and the receptors for hLIF (YQPQ and
YKPQ), hEGFR (YINQ and YYHQ), and mIL-10 (YQKQ and
YLKQ).6,39,40 The phosphotyrosine-containing motif at position Y744 is YLRC. This is the first motif demonstrated to recruit
Stat3 that contains the polar amino acid residue, Cys, at the +3
position. In contrast, both Y729 and Y764 are followed at the +3
position by the nonpolar amino acid residue Leu.
To better understand the specificity of the Stat3 SH2 domain for the
Y704- and Y744-based ligands, this domain was modeled using SH2 domains
of known structure and high sequence homology as templates (Fig
6). CLUSTAL
analysis57 of the SH2 domain within Stat3 to other proteins
in the Brookhaven data base identified the following proteins as having
greatest homology, in order of decreasing homology: the SH2 domains of
v-Src, p56-Lck, murine Syp, and human Grb-2. Also included in the
CLUSTAL analysis for comparison were the sequences of identified
homologues of Stat3: the SH2 domains of Stat1, Stat2, Stat4, Stat5A,
Stat5B, and Stat6. Very recently, the crystal structure of a truncated
Stat1 homodimer complexed to DNA has been determined.58
Although the STAT protein SH2 domains differ in sequence from other SH2
domains, there appears to be remarkable conservation with respect to
SH2 structure (2.6 Å rms deviation of Stat1 with respect to v-Src).
The alignments between STAT and non-STAT SH2 domains were based on
structural alignment of the SH2 domains of v-Src and
Stat1.58 Gaps in the alignment were generally limited to
loops between -helices and/or sheet elements (the
location of these elements are shown schematically in Fig 6). SH2
domain structures were examined using
O.59
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[in a new window]
| Fig 6.
Alignment of partial SH2 domains of Stat proteins with
homologous SH2 domains of known structure. All sequences were
downloaded from GenBank, except for SH2 domain of Stat3, which was used
in the search query. The numbering of the peptide fragments and
accession codes are as follows: Grb2 67-140, P29354; Syp 120-202, P35235; p56 Lck 134-213, P06239; v-Src 154-234, P15054; Stat3 590-661;
Stat1, 572-646, P42224; Stat2, 387-459, P52630; Stat4, 579-651, P42228;
Stat5A 599-665, P42229; Stat5B 599-665, P51692; and Stat6 543-611, P42226. STAT sequences (in black) were aligned using CLUSTAL, as were
nonSTAT sequences (in gray). The relative alignments between STAT and
non-STAT proteins were based on alignments of Chen et al,58
which used direct comparison of the crystal structures of the SH2
domains of v-Src and Stat1. Secondary structural elements observed in
structural studies of v-Src and Stat1 SH2 domains are indicated below
the sequences (in gray and black, respectively); -sheets are
indicated as arrows, and -helices as boxes with diagonal stripes.
Residue sidechains, which have been shown in high resolution studies to
interact directly with the phosphotyrosine of the ligand, are boxed and
highlighted. The v-src sidechains of which form the binding pocket for
the +3 residue of the ligand are highlighted (Y202 and I214).
Residues of Stat3, which are hypothesized to interact with bound
ligand, are also highlighted (E638, Y640, and Y657).
|
|
In all of these examined SH2 domains of known structure, the side
chains that directly interacted with the phosphotyrosine of the bound
ligand were conserved (see residues corresponding to v-Src R155, R175,
S177, E178, and T179). Similarly conserved residues were also present
in the STAT proteins. Of particular note was the observed variability
that occurred in the binding pocket that makes direct contact with the
+3 residue of the phosphotyrosine ligand in the crystal structures. In
v-Src, p56-Lck, Syp, and Grb-2, this residue is strictly hydrophobic
(Ile, Ile, Val, or Phe, respectively), and in each case the
corresponding +3 ligand residue which it contacts is also hydrophobic.
In our model of human Stat3, which is based on the highly conserved
Stat1 structure (56% sequence identity), the corresponding SH2
residues modeled in this binding site are polar and hydrophilic. The
schematic representation of the interaction of v-Src with its ligands,
and the proposed interactions involved in Stat3 recognition of the G-CSF receptor are shown in Fig 7. In
v-Src, the hydrophobic pocket is comprised of Ile214, as well as some
contributions from the gamma and delta carbons of Tyr202 (ie, the edge
of the aromatic ring, distal relative to the hydroxyl, also lies at the
surface of the binding pocket). In human Stat3, the corresponding
residues that map to the surface of the binding site are Glu638,
Tyr640, and Tyr 657, dramatically changing the chemical nature of this site. Of note, the other STAT proteins also have polar hydrophilic residues at these positions, suggesting they also may bind
phosphotyrosine-containing motifs with hydrophilic residues at the +3
position. Very recently, the three-dimensional structure of Stat3
homodimer bound to DNA was reported.60 However, the
electron density obtained for the SH2 domain and the phosphopeptide
region were not well defined. Consequently, additional useful
information relevant to our model was not obtained from these studies.
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[in a new window]
| Fig 7.
v-Src residues directly interacting with ligand and
corresponding putative residues involved in Stat3 binding of the G-CSF
receptor. v-Src residues which directly interact with the
phosphorylated ligand were determined by observing the crystal
structures of v-Src (Brookhaven accession codes and , with
ligands pTyr-Val-Pro-Met and pTyr-Leu-Arg-Val, respectively) using
O59 with a 3.5 Å cut-off. The residues interacting
with the central ligand are in white typeface in gray boxes, and the
+3 residue of the ligand is shown in gray. The corresponding residues
in the Stat3 model are shown in black typeface in white boxes, and the
+3 residue is shown in black. Of note, the Stat3 residues proposed to
interact with the phosphotyrosine are conserved or homologous to those
in v-Src, but those defining the binding pocket for the +3 residue in
Stat3 have hydrophilic substitutions that may hydrogen bond with the
more hydrophilic G-CSF ligand.
|
|
| |
ACKNOWLEDGMENT |
We thank Judith E. Layton and members of her laboratory, especially
Sandra E. Nicholson, for providing the M1 cell clones.
| |
FOOTNOTES |
Submitted July 30, 1998;
accepted October 13, 1998.
Supported in part by National Institutes of Health Grant No. CA 72261.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to David J. Tweardy, MD, Division of
Infectious Diseases, University of Pittsburgh Medical Center, Suite
501, Kaufmann Bldg, 200 Lothrop St, Pittsburgh, PA 15213; e-mail:
tweardy+{at}pitt.edu.
| |
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Top
Abstract
Introduction