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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1933-1942
Identification of a 14-3-3 Binding Sequence in the Common  Chain of
the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF),
Interleukin-3 (IL-3), and IL-5 Receptors That Is Serine-Phosphorylated
by GM-CSF
By
F.C. Stomski,
M. Dottore,
W. Winnall,
M.A. Guthridge,
J. Woodcock,
C.J. Bagley,
D.T. Thomas,
R.K. Andrews,
M.C. Berndt, and
A.F. Lopez
From The Cytokine Receptor Laboratory, The Hanson Centre for Cancer
Research and Institute of Medical and Veterinary Science, Adelaide,
Australia; and the Baker Research Institute, Melbourne, Australia.
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ABSTRACT |
The common  chain (c) of the
granulocyte-macrophage colony-stimulating factor (GM-CSF),
interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit
of these receptors coupling ligand binding to multiple biological
activities. It is thought that these multiple functions arise as a
consequence of the recruitment of specific signaling molecules to
tyrosine-phosphorylated residues in the cytoplasmic domain of
c. However, the contribution of serine phosphorylation
in c to the recruitment of signaling molecules is not
known. We show here the identification of a phosphoserine motif in the
cytoplasmic domain of c that interacts with the adaptor
protein 14-3-3 . Coimmunoprecipitation and pull-down experiments with
a glutathione S-transferase (GST):14-3-3 fusion protein showed that
14-3-3 directly associates with c but not the GM-CSF receptor  chain. C-terminal truncation mutants of c
further showed that a region between amino acids 544 and 626 in
c was required for its association with 14-3-3. This
region contains the sequence 582HSRSLP587,
which closely resembles the RSXSXP (where S is
phosphorylated) consensus 14-3-3 binding site identified in a number of
signaling molecules, including Raf-1. Significantly, substitution of
582HSRSLP587 for EFAAAA completely abolished
interaction of c with GST-14-3-3. Furthermore, the
interaction of c with GST-14-3-3 was greatly reduced in
the presence of a peptide containing the 14-3-3 binding site, but only
when 585Ser was phosphorylated. Direct binding experiments
showed that the peptide containing phosphorylated 585Ser
bound 14-3-3 with an affinity of 150 nmol/L. To study the regulation
of 585S phosphorylation in vivo, we raised antibodies that
specifically recognized 585Ser-phosphorylated
c. Using these antibodies, we showed that GM-CSF
stimulation strongly upregulated 585Ser phosphorylation in
M1 myeloid leukemic cells. The proximity of the SHC-binding site
(577Tyr) to the 14-3-3-binding site
(582HSRSLP587) and their conservation between
mouse, rat, and human c but not in other cytokine
receptors suggest that they form a distinct motif that may subserve
specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN granulocyte-macrophage
colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are
pleiotropic cytokines capable of stimulating normal and transformed
hematopoietic cells.1-4 With each, the initiating event for
signal transduction is the binding of GM-CSF, IL-3, and IL-5 to their
surface receptors that are composed of a cytokine-specific  chain
and a common chain (c).5-7 Engagement of
c by the binding of GM-CSF, IL-3, and IL-5 to surface
receptors results in the stimulation of cell survival, proliferation,
and differentiation and mature cell effector function in the
appropriate lineage, a fact that emphasises the major signaling role
played by c in mediating GM-CSF, IL-3, and IL-5
biological activities.8
One of the first events in GM-CSF, IL-3, and IL-5 activation of their
receptors and in the initiation of the signaling cascade is tyrosine
phosphorylation of c.9 This is a common
theme among cytokine receptor signaling subunits and can be seen in homodimeric receptors such as the erythropoietin (EPO) receptor, thrombopoietin (TPO) receptor, and granulocyte colony-stimulating factor (G-CSF) receptor as well as in heterodimeric receptors such as
in the IL-6 and IL-2 receptors.10-13 In the latter case, as
in the GM-CSF, IL-3, and IL-5 receptor system, tyrosine phosphorylation is also restricted to its signaling subunit.14
Tyrosine phosphorylation of cytokine receptor signaling subunits
appears to be a critical step in the creation of docking sites for the
association of signaling molecules. For example, ligand-induced
tyrosine phosphorylation of c is considered critical for
binding of SH2-containing signaling molecules such as STATs, SHP1,
SHP2, and SHC. In the case of STAT5, this is recruited to phosphotyrosines in c and is then phosphorylated by
JAK2, resulting in STAT activation, dimerization, and translocation to
the nucleus, where it directly regulates gene
transcription.15,16 Previously, it has been shown that 6 of
the 8 tyrosine residues of c allow the docking of STAT5
and its subsequent activation, indicating a high degree of redundancy
as to which tyrosine residues are critical for STAT5
activation.17 The specific phospho-tyrosine residue
responsible for recruitment of SHC to c is
577Tyr. The bound SHC recruits grb2 into the complex and
grb2 binds sos, which then activates p21ras nucleotide
exchange. This leads to the activation of ras and downstream partners
of the mitogen-activated protein (MAP) Kinase pathway,
including Raf.17-20 SHP2, a prominent tyrosine
phospho-protein, is able to bind 3 tyrosine residues of the activated
c (577Tyr, 612Tyr, and
695Tyr), although only 577Tyr and
612Tyr allow SHP2 to interact with grb2.17,21
Despite the well-documented importance of tyrosine phosphorylation of
cytokine receptors, it is becoming apparent that signaling can proceed
in its absence, suggesting that other mechanisms may be at work. This
is demonstrated in the EPO and TPO receptors, in which the substitution
of all tyrosines failed to abolish their activities.22,23
In the case of c, mutation of all 8 cytoplasmic tyrosine
residues does not abolish activation of JAK2 and induction of tyrosine
phosphorylation of STATs and many other cellular proteins. The only
significant defect in this mutation is the reduction in phosphorylation
of SHP2 and SHC.17 In the case of mutant mice that have
STAT5a and STAT5b genes deleted, there is no hematopoietic phenotype,
suggesting that other pathways may substitute for STAT5 activation.24 We and others have also shown that FDCP-1
cells transduced with a c mutant in which all
intracellular tyrosines were substituted for phenylalanines were able
to proliferate in response to GM-CSF.16,17,25
We show here the identification in the common chain of the GM-CSF,
IL-3, and IL-5 receptors of a sequence responsible for its association
with the adaptor protein 14-3-3. Furthermore, we show that GM-CSF
regulates the phosphorylation of 585Ser within the
14-3-3 binding sequence. Given the conservation of this region in
c of different species but not in other cytokine receptors, we suggest that it may be involved in GM-CSF, IL-3, and IL-5
receptor-specific functions.
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MATERIALS AND METHODS |
Mutagenesis of human c and expression plasmid
constructs.
Substitution mutations of 2 sequences within the cytoplasmic domain of
the human c cDNA were constructed using
oligonucleotide-directed mutagenesis (Altered-sites; Promega, Sydney,
New South Wales, Australia), as described previously.26
Both mutants were essentially poly-alanine substitutions. Mutagenesis
oligonucleotides encoding nonalanine residues were included to
facilitate restriction enzyme screening of mutant clones. The first
motif was 582HSRSLP587 mutated to
582EFAAAA587, and the second was
820RSKPSSP826 mutated to
820EFAAAAA826. The point mutant S585A was also
constructed; however, this mutant created a cryptic proteolytic site in
c and was not able to be used (see Results). The
mutations were confirmed by nucleotide sequencing and the mutant
c cDNAs subcloned into the eukaryotic expression
vector pcDNA1 (Invitrogen, San Diego, CA). The c
deletion mutant cDNAs were a kind gift of Dr A. Miyajima (University of Tokyo, Tokyo, Japan). The 14-3-3 zeta clone was isolated from a human
monocyte lambda gt11 cDNA library (Clontech Laboratories, Palo Alto,
CA) and subcloned into a pGEX-2T vector.
GM-CSF and IL-3.
Recombinant human IL-3 and GM-CSF were produced in Escherichia
coli essentially as described before.27,28 Cytokine
purity and quantitation was determined by high-performance liquid
chromatography (HPLC) analysis and Coomasie staining of
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)-separated proteins. The activity of the cytokines based on
the ED50 values in a TF-1 proliferation assay29
was 0.03 ng/mL for GM-CSF and 0.1 ng/mL for IL-3.
Antibodies.
The monoclonal antibodies (MoAbs) 8E4 and 1C1 directed against the
c were generated as previously described.30
The anti-phospho-585Serc antibody was
raised by immunizing New Zealand White rabbits with the phosphorylated
CLGPPHSRSLPDILG peptide conjugated to keyhole limpet
hemocyanin (Sigma, St Louis, MO). The antipeptide antibody
was firstly affinity purified with the immunizing peptide conjugated to
sepharose and then absorbed with the nonphosphorylated CLGPPHSRSLPDILG
peptide conjugated to sepharose. The specificity of the
anti-phospho-585Serc antibody was verified
by dot immunoblots against the CLGPPHSRSLPDILG peptide in either
the nonphosphorylated or serine-phosphorylated form and a
scrambled peptide LPLSGPDSHIRGPL. The corresponding phosphorylated
serine in each peptide is underlined. Peptides were synthesized by
Chiron Mimotopes (Melbourne, Australia). The anti-14-3-3 antibody
was kindly provided by Dr A. Aitken (Edinburgh University, Edinburgh, UK).
Cell culture and DNA transfection.
The HEK293T cell line was maintained in RPMI-1640 supplemented with
10% vol/vol fetal calf serum (FCS). On the day before transfection,
1.4 × 106 cells were plated into 6-cm tissue culture
dishes to adhere overnight. Four hours after a medium change, 6 µg of
wild-type or mutated c cDNA was added to cells in the
form of a calcium phosphate precipitate,31 and the cells
were placed in an incubator for 4 to 6 hours to permit the uptake of
the DNA-calcium phosphate precipitate. The cells were then washed,
replated, and placed in the incubator for 48 hours before cytokine
treatment. M1 cell line expressing GM-CSF receptor chain and
c wild-type was maintained in RPMI-1640 supplemented
with 10% vol/vol FCS. The M1 cell line was kindly provided by Dr N. Nicola (Walter and Eliza Hall Institute of Medical Research, Australia).
Surface marker analysis by flow cytometry.
Expression of receptors on transfected cells was verified by flow
cytometry. Briefly, cells were incubated with the anti-c MoAb (1C1)32 or anti-GM-CSFR MoAb (4H1)30
for 20 minutes on ice, washed, and subsequently incubated with
fluorescein isothiocyanate (FITC)-conjugated antimouse IgG antibody
(Silenus Laboratories, Hawthorn, Victoria, Australia) for 20 minutes on
ice. Cells were then washed and resuspended in FACS FIX and analyzed
using a Profile II (Coulter Electronics, Hileah, FL).
Immunoprecipitations.
Cells were lysed in lysis buffer (150 mmol/L NaCl, 10 mmol/L Tris-HCl
[pH 7.4], 1% Digitonin with protease inhibitors [10 µg/mL
leupeptin, 2 mmol/L phenylmethlysulfonyl fluoride, and 10 µg/mL
aprotinin], and 2 mmol/L sodium vanadate) for 30 minutes at 4°C
followed by centrifugation of the lysate for 15 minutes at
12,000g at 4°C. After 1 hour of preclearance with
Protein-A-sepharose (Pierce, Rockford, IL) at 4°C, the supernatant
was incubated for 2 hours with 5 µg/mL antibody. Protein-Ig complexes
were captured by incubation for 1 hour with Protein-A-sepharose
followed by 6 subsequent washes in lysis buffer. Samples were boiled
for 5 minutes in SDS sample load buffer in the presence or absence of 2-mercaptoethanol before separating immunoprecipitated proteins by
SDS-PAGE.
Precipitations.
Cells were lysed in lysis buffer for 30 minutes at 4°C followed by
centrifugation of the lysate for 15 minutes at 12,000g at
4°C. After 1 hour of preclearance with glutathione S-transferase (GST)-sepharose at 4°C, the supernatant was incubated for 2 hours with GST-14-3-3-sepharose followed by 3 subsequent washes in lysis buffer. Samples were boiled for 5 minutes in SDS sample load buffer in
the presence of 2-mercaptoethanol before separating precipitated proteins by SDS-PAGE.
Competition of precipitations and immunoprecipitations by peptides.
Cell lysates were precipitated or immunoprecipitated in the presence of
various concentrations of the following peptides: c
peptide sequence CLGPPHSRSLPDILG in either the
nonphosphorylated or serine-phosphorylated form and a scrambled peptide
CLPLSGPDSHIRGPL. Raf1 peptides corresponding to the sequence
CLSQRQRSTSTPNVHM were also used and were either
nonphosphorylated or serine-phosphorylated. The corresponding
phosphorylated serine in each peptide is underlined. Peptides were
synthesized by Chiron Mimotopes. The presence of c in
either the immunoprecipitation or precipitation experiment was
determined by Western blotting with anti-c antibody
(MoAb 1C1).
SDS-PAGE, immunoblot, and enhanced chemiluminescence
(ECL).
Immunoprecipitated proteins separated by SDS-PAGE were transferred to
nitrocellulose membrane by electroblotting. Dot blots of peptides were
performed by spotting either 10 or 100 ng of each peptide onto
nitrocellulose membranes. Routinely, nitrocellulose membranes were
blocked in a solution of phosphate-buffered saline (PBS)/0.05%
(vol/vol) Tween 20 (PBT) containing 1% (wt/vol) blocking reagent 1096 176 (Boehringer Mannheim, Mannheim, Germany) and probed
with anti-c (1C1), anti-14-3-3,33 or
anti-phospho-585Serc, followed by either
anti-mouse or anti-rabbit peroxidase-conjugated antibodies. Immunoreactive proteins were detected by chemiluminescence using the ECL kit (Amersham, Little Chalfont, UK) following the manufacturer's instructions.
Binding of the 125I-labeled 14-3-3 to synthetic
peptides.
The recombinant 14-3-3 protein was 125I-labeled using
IODOBEADS (Pierce). The synthetic peptides were solubilized in
distilled water and then diluted to 50 µg/mL in 0.1 mol/L
NaHCO3, pH 9.2. The peptides were coated onto microtiter
wells (Immunolon II Removawells; Dynatech Laboratories, Chantilly,
VA) by incubation at 22°C for 6 hours and then at 4°C
overnight. The peptide-coated microtiter wells were blocked at 22°C
with 5% bovine serum albumin for 2 hours and then with
125I-labeled 14-3-3 protein for 2 hours. After 3 washes,
microtiter well-bound radioactivity were estimated in a  -counter.
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RESULTS |
Association of c with 14-3-3.
In examining the cytoplasmic region of c, we identified
a small region encompassing amino acids 566 to 589 that exhibits unusually high sequence identity (72%) between mouse and human c when compared with the overall identity of the
cytoplasmic domain of c (55% identity;
Fig 1). Previous studies have implicated this region as being important for cell survival and
differentiation.34 Closer examination of this region
showed, in addition to Tyr577 (binding site for SHC), a
possible 14-3-3 binding motif, 582HSRSLP587
that is conserved in c from different species. This
sequence closely resembles the prototypic 14-3-3 binding consensus
(RSXSXP) identified in Raf-1 (where S is
phosphorylated) and in other proteins involved in signal transduction
(Fig 2).
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| Fig 1.
Schematic representation of the cytoplasmic domain of
c showing the overall sequence identity between human
and mouse c and the seventh region of highest identity.
The positions of box-1 and box-2 are shown as well as the functional
role ascribed to different parts of human
c.34 The position of the conserved tyrosine
residues and of 585Ser is also shown.
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| Fig 2.
Comparison of sequences in signaling molecules known to
bind 14-3-3 and the derived consensus 14-3-3 binding motif. The
sequences in human, mouse, and rat chains similar to the consensus
are also shown.
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To test the possibility that human c was able to
associate with 14-3-3 and to determine the region of interaction, we
transfected HEK 293T cells with wild-type c and a series
of C-terminal truncation mutants and examined the ability of
c to associate with 14-3-3 by coimmunoprecipitation.
Whereas wild-type c and C-terminal truncation mutants up
to amino acid 626 coimmunoprecipitated with 14-3-3, further truncations
resulted in no detectable association of c and 14-3-3 (Fig 3A). Similar results were obtained in
GST-14-3-3 pull-down experiments. When compared with GST controls,
GST-14-3-3 interacted with wild-type c and C-terminal
truncation mutants up to amino acid 626. However, additional
truncations resulted in a loss of detectable association of
c and GST-14-3-3 (Fig 3). These experiments indicate
that (1) c interacts with 14-3-3 and (2) that the region
of interaction lies between amino acids 544 and 626.
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| Fig 3.
Human c associates with 14-3-3, and
this association is mediated by the 544-626 region of c.
HEK 293T cells were either mock-transfected (mock) or transfected with
wild-type c (wt) or c containing
C-terminal deletions. Lysates were prepared from transfected cells and
either immunoprecipitated with anti-14-3-3 antibody (A) or
precipitated with either 14-3-3-GST sepharose (B) or GST-sepharose
(C). All proteins were separated on 7.5% SDS-PAGE under reducing
conditions before Western blotting with anti-c antibody
(MoAb 1C1).
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We then examined whether 14-3-3 interacted with c via
the 582HSRSLP587 motif that lies within the
544-626 region identified in Fig 3. A substitution mutant
(c-582HSRSLP587 EFAAAA)
and a point mutant (c-585SA) in the putative
14-3-3 binding site were constructed as well as a control mutant
(c 820RSKPSSP826EFAAAAA). These mutants
were expressed in HEK 293T cells and examined for their ability to
interact with GST-14-3-3 in pull-down experiments. Whereas wild-type
c and the control mutant c interacted with GST-14-3-3, no detectable interaction was observed for the c 582HSRSLP587EFAAAA
mutant (Fig 4). These results indicate that
14-3-3 associates with c via the
582HSRSLP585 sequence. Experiments examining
the association of c-585SA point mutant with
GST-14-3-3 were not possible, because it is likely that this mutation
introduced a cryptic proteolysis cleavage site. Flow cytometry and
Western blot analysis indicated that this mutant was proteolysed and
failed to be expressed on the cell surface (data not shown).
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| Fig 4.
14-3-3 specifically binds the HSRSLP motif of the
c cytoplasmic domain. (A) HEK 293 cells were used either
untransfected (UT) or transfected with wild-type c (wt),
with c containing the sequence
581PHSRSLP587 mutated to
581GEFAAAA587, or with c
containing the sequence 820RSKPSSP826 mutated
to 820EFAAAAA826. Lysates were made and
immunoprecipitations were performed using GST-14-3-3-sepharose. The
presence of c was determined by Western blotting with an
anti-c antibody (MoAb 1C1). (B) The level of expression
c in the lysates was determined by Western blotting with
an anti-c antibody (MoAb 1C1).
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Role of 585Ser in c interaction with 14-3-3.
14-3-3 is known to be a phospho-serine binding protein that interacts
with the RSXSXP motif, where S is phosphorylated.
It would then be expected in the case of c that
585Ser phosphorylation within the
582HSRSLP587 motif would be required for 14-3-3 association. The inability to express full-length
c-585SA precluded using this mutant in either
coimmunoprecipitation or pull-down experiments to examine the
requirement of c 585Ser phosphorylation for
14-3-3 interaction. As an alternative, we synthesized a
c peptide containing a nonphosphorylated
585Ser (C578LGPPHSRSLPDILG591) and
a c peptide containing a phosphorylated
585Ser and examined their ability to inhibit
c interaction with GST-14-3-3 in a pull-down
experiment. Whereas the peptide containing phosphorylated
585Ser inhibited c association with
GST-14-3-3, no inhibition of association was observed for the peptide
containing the nonphosphorylated 585Ser
(Fig 5). As a comparison, nonphosphorylated
and phosphorylated peptides corresponding to the 14-3-3 binding site in
Raf-1 were also tested. We found that the serine-phosphorylated Raf-1
peptide was also able to inhibit c association with
14-3-3, whereas the nonphosphorylated peptide did not (Fig 5).
Furthermore, the ability of the c-phosphorylated peptide
to inhibit the association of c with 14-3-3 was
dose-dependent and specific as another phosphorylated peptide with
sequence corresponding to a different region of c failed
to inhibit this association (Fig 6). Direct
binding experiments and Scatchard analysis demonstrated that the
phosphorylated peptide (C578LGPPHSRSLPDILG591) bound to
14-3-3 with an affinity of approximately 150 nmol/L (Fig 7). This affinity is comparable to the
affinities reported by other investigators (55 to 190 nmol/L)35,36 for 14-3-3 binding. Together, these
experiments show that the 582HSRSLP587 sequence
in c directly binds to 14-3-3 and that this binding is
dependent on 585Ser being phosphorylated.
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| Fig 5.
Inhibition of c association with 14-3-3
by phosphorylated but not by unphosphorylated c and
raf-1 peptides. Lysates of HEK293T cells transfected with
c were immunoprecipitated with GST-14-3-3-sepharose in
the absence or in the presence of chemically synthesized peptides (100 µmol/L) containing a c sequence (CLGPPHSRSLPDILG) or a
Raf-1 sequence (CLSQRQRSTSTPNVHM). In the phosphorylated peptides, the
relevant phosphorylated serine is underlined. The experiment was
performed on 7.5% SDS-PAGE under reducing conditions. The presence of
c in the precipitation experiment was determined by
Western blotting with anti-c antibody (MoAb 1C1).
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| Fig 6.
Specific inhibition of c association with
14-3-3 by a phosphorylated peptide encompassing the 579-592 region
of c. Lysates of HEK293T cells transfected with
c were precipitated with GST-14-3-3-sepharose in the
absence or in the presence of various concentrations of chemically
synthesized peptides. Two c peptide sequences were used,
CLGPPHSRSLPDILG in either the nonphosphorylated (A) or
serine585-phosphorylated (B) form and
CPLSLRSKPSPGPGP in either the nonphosphorylated (C) or
serine-phosphorylated (D) form. The appropriate phosphorylated serine
in each peptide is underlined. The experiment was performed on 7.5%
SDS-PAGE under reducing conditions. The presence of c in
the immunoprecipitates was determined by Western blotting with
anti-c antibody (MoAb 1C1).
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| Fig 7.
Binding of 125I-labeled 14-3-3 to
synthetic peptides corresponding to the 14-3-3 binding region of
c. Microtiter wells were coated with synthetic peptides
CLGPPHSRSLPDILG either nonphosphorylated or phosphorylated on the
second serine (underlined). Various concentrations of
125I-labeled recombinant 14-3-3 protein were added to
microtiter wells and incubated at 22°C for 2 hours. (Insert)
Scatchard analyses of 14-3-3 interaction with the serine-phosphorylated
peptide.
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In vivo regulation of 585Ser phosphorylation.
Having identified the requirement for c
585Ser to be phosphorylated to allow 14-3-3 binding, we
then examined whether 585Ser was phosphorylated in vivo and
whether its phosphorylation was regulated by GM-CSF. These
possibilities were initially addressed using
32P-orthophosphate-labeled HEK 293T cells transfected with
the GM-CSF receptor ( and c subunits). These cells
were stimulated with GM-CSF, and total c was
immunoprecipitated and examined for 32P-labeling. No
detectable change in c serine/threonine phosphorylation in response to GM-CSF stimulation was observed (data not shown). This
is most likely due to the presence of 60 serine and threonine residues
in the intracellular region of c, some of which may be
constitutively phosphorylated. To directly address the phosphorylation status of c 585Ser in vivo, we raised a
rabbit antiserum against a peptide containing the 14-3-3 binding site
identified in c:
C578LGPPHSRSLPDILG591. This antibody
preparation, termed anti-phospho-585Ser, specifically
recognized the CLGPPHSRSLPDILG peptide containing a
phosphorylated 585Ser but not a peptide containing a
nonphosphorylated 585Ser or a peptide containing a
scrambled 14-3-3 binding site (Fig 8). The
specificity of the anti-phospho-585Ser antibody was
further confirmed by Western blotting of immunoprecipitated c from GM-CSF receptor-transfected HEK 293T cells. In
these experiments, the phosphorylated CLPPHSRSLPDILG peptide
was able to inhibit anti-phospho-585Ser recognition of
c, whereas the nonphosphorylated and scrambled peptides
did not (Fig 8B). In addition, pretreatment of c
immunoprecipitates with calf intestinal phosphatase before Western blot
analysis completely abolished the anti-phospho-585Ser
signal, and immunoprecipitation of either the wild-type
c or the
582HSRSLP587EFAAAA mutant from
HEK293T transfected cells followed by Western blot analysis with the
anti-phospho-585Ser antibody demonstrated that this
antibody specifically recognized the wild-type but not the mutant
receptor (data not shown).
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| Fig 8.
585Ser in c is phosphorylated
in vivo by GM-CSF. (A) The
anti-phospho-585Serc antibody specifically
recognizes the phosphorylated CLGPPHSRSLDILG peptide. Dot
blots were prepared on nitrocellulose filters of either the
nonphosphorylated or the serine-phosphorylated
CLGPPHSRSLPDILG peptide and the scrambled peptide
CLPLSGPDSHIRGPL before probing with
anti-phospho-585Serc antibody. (B) The
serine-phosphorylated CLGPPHSRSLPDILG peptide specifically
inhibits the binding of
anti-phospho-585Serc antibody to
c. Lysates of HEK293T cells transfected with wild-type
c were immunoprecipitated with anti-c
antibody (MoAb 8E4). Immunoprecipitates were run on 7.5% SDS-PAGE
under reducing conditions.
Anti-phospho-585Serc antibody was then
preincubated with either medium (none), 100-fold molar excess of the
serine-phosphorylated (1) or nonphosphorylated (2) CLGPPHSRSLPDILG
peptide, or the scrambled peptide CLPLSGPDSHIRGPL (3). The filters were
then Western blotted and probed with the pretreated
anti-phospho-585Serc antibody. (C)
Upregulation of 585Ser phosphorylation by GM-CSF. M1 cells
expressing GM-CSFR and c were either untreated ( )
or stimulated with GM-CSF (2 ng/mL) for 30 seconds (+). Lysates of
the M1 cells were immunoprecipitated with anti-c
antibody (MoAb 8E4) and the immunoprecipitates were run on 10%
SDS-PAGE under reducing conditions. The filters were then Western
blotted with either anti-phospho-585Serc
antibody or the anti-c antibody (MoAb 1C1).
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Using these anti-phospho-585Ser specific antibodies, we
then examined the regulation of c 585Ser
phosphorylation after GM-CSF stimulation of MI myeloid leukemic cells.
M1 myeloid leukemic cells were stimulated with 2 ng/mL GM-CSF,
c was immunoprecipitated, and immunoprecipitates were probed with the anti-phospho-585Ser antibody. As shown in
Fig 8, GM-CSF stimulation strongly upregulated 585Ser
phosphorylation of c.
| |
DISCUSSION |
We show here the identification of a 14-3-3-binding motif in the
common chain of the GM-CSF/IL-3/IL-5 receptors. It is shown that
this motif mediates the association of c with 14-3-3 in vitro and in vivo and requires the phosphorylation of
585Ser, but it diverges from the canonical 14-3-3 binding
sequence, having an His at position 1 instead of the usual Arg.
Furthermore, we obtained evidence that phosphorylation of
585Ser is regulated by GM-CSF, demonstrating for the first
time that not only tyrosine, but also serine phosphorylation of
c is subjected to ligand-mediated regulation. Given the
proximity of the 14-3-3 binding sequence
(582HSRSLP587) to the SHC-binding sequence
(577Tyr), we suggest that these form a novel motif perhaps
involved in certain specialized functions associated with the GM-CSF,
IL-3, and IL-5 receptors.
We have demonstrated c:14-3-3 interaction using several
approaches. Firstly, we were able to coimmunoprecipitate
c with 14-3-3, indicating that these proteins interact
in vivo. Secondly, we have shown in pull-down experiments that
GST-14-3-3 interacts with c. Using a series of
C-terminally truncated mutants, we have localized this interaction to a
region between amino acids 544 and 626. Closer examination of this
region showed a possible 14-3-3-binding motif,
582HSRSLP587. This sequence closely resembles
the 14-3-3-binding consensus identified in Raf-1 and several other
signaling molecules (582RSXSXP587,
where S is phosphorylated). Thus, phosphorylation of
Ser585 within 582HSRSLP587 was an
expected prerequisite for 14-3-3 binding to c. A
substitution mutant of this motif
(582EFAAAA587) abolished the interaction of
c with 14-3-3. A S585A mutant was not able to be tested
for its ability to associate with 14-3-3 due to its poor expression
(see Results); however, phosphopeptide competition experiments suggest
that the association is dependent on Ser585 phosphorylation.
Although the phosphorylation of the second serine in the HSRSLP
sequence and the presence of a proline at the end are consistently found in 14-3-3 binding sequences, from the 14-3-3 crystal structure it
is predicted that the proline in the docking protein produces a
critical 90° bend in the chain, allowing the remainder of the protein to exit the binding cleft.36 The
presence of an His at the beginning of the sequence is unusual, because
the most frequent residue found at this position is Arg (Fig 2),
although other amino acids can be found and, in fact, the sequence seen in IRS-1 contains an His. It is becoming clear that there is no fixed
14-3-3 binding sequence but instead a defined hierarchy in which
certain amino acids are preferred over others.36 In terms
of the sequence found in c, the affinity of GST-14-3-3 for a peptide encompassing the phosphorylated 14-3-3 binding motif was
152 nmol/L, which is in line with the affinity of other previously reported 14-3-3 binding motifs.35,36
The notion that the 14-3-3 binding motif in c may be
functionally important is supported by the ability of GM-CSF to
regulate the phosphorylation of 585Ser in the M1 myeloid
cell line (Fig 8). This was shown through the development of antibodies
specific for the CLGPPHSRSLPDILG sequence in which the second
serine was phosphorylated. In previous experiments, we have found that
the overall serine/threonine phosphorylation of the cytoplasmic region
of c (which contains 60 serines and threonines) was
similar before and after stimulation by GM-CSF (data not shown),
indicating a substantial basal level of phosphorylation that obscured
any specific serine and threonine phosphorylation event regulated by
ligand. These results make 585Ser more akin to tyrosine
residues in c, because the phosphorylation of these is
ligand-dependent. Analogous to tyrosine-phosphorylated sites in
c that serve as docking sites for STAT-517,
SHC,16,17,19 and PTP137 with clear functional
consequences, the ligand-dependent phosphorylation of
585Ser may also initiate distinct signaling events. In
other experiments using a CTL-EN cell line expressing the human IL-3
receptor, we found that stimulation with IL-3 increased the association
of c to 14-3-3 by approximately 5-fold (data not shown).
The importance of serine/threonine phosphorylation in mediating the
biological activity of cytokines and growth factors is underscored by
the recent findings of several groups that several receptor (GHR, EPOR,
TPOR, and G-CSF) mutants in which all cytoplasmic tyrosines have been
substituted for phenylalanine are able to mediate most, if not all, of
the biological activity of the wild-type receptors. We25
and others16,17 have also shown that a mutant
c in which all 8 cytoplasmic tyrosines are substituted
for phenylalanine is able to support both proliferation and survival of
FDCP1 and BaF3 cells in response to GM-CSF. Thus, it is tempting to
speculate that this may be achieved, at least in part, by
ligand-dependent 585Ser phosphorylation, which activates
either the same or alternative and redundant pathways.
Although our results show that 14-3-3 binds 585Ser of
c, little is known about the possible role of 14-3-3 in
receptor signaling. The 14-3-3 family of proteins consists of 7 different isoforms and is expressed ubiquitously from yeast to
humans.38,39 The ability of 14-3-3 to bind phosphoserine
motifs in a wide range of signaling molecules40-50 suggests
that 14-3-3 proteins participate in a number of cell signaling pathways
that may include mitogenesis, transformation, and survival.
Although 14-3-3 has been shown to bind a number of signaling molecules,
it has been more difficult to determine how 14-3-3 can regulate
signaling events. However, the recent x-ray crystallographic structure
of 14-3-3 has provided several hints.51,52 The 14-3-3 proteins dimerize through an N-terminal domain to form 2 large acidic
grooves. It is proposed that this groove can bind 2 separate 14-3-3-binding motifs, raising the possibility that 14-3-3 could function as an adaptor, bringing together 2 different cellular proteins.41,53 Thus, one possible function of 14-3-3 could be to act as an adaptor linking c to other
14-3-3-binding molecules involved in GM-CSF signaling. Another
possibility is that 14-3-3 could be involved in the regulation of
c cytoplasmic domain contact with cellular membranes in
a manner similar to that described for BAD and Raf-1.
Although the exact functions and signaling pathways involved remain to
be elucidated, it is noteworthy that the
582HSRSLP587 14-3-3 binding sequence in
c lies proximal to the 577Tyr binding site
for SHC.16,17 Because these sequences form a distinct motif
conserved in c of different species and not found in
other cytokine receptors, we propose that they subserve specific
functions associated to the GM-CSF, IL-3, and IL-5 receptors. In
support of this concept, we note that this new motif lies within a
region of c involved in myeloid cell survival and
differentiation34; however, functional studies will be
necessary to test this hypothesis.
| |
ACKNOWLEDGMENT |
The authors thank Anna Nitschke for excellent secretarial assistance
and Dr A. Aitken for a gift of antibodies to 14-3-3 and useful discussions.
| |
FOOTNOTES |
Submitted March 10, 1999; accepted May 12, 1999.
Supported by grants from the NH&MRC of Australia.
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 A.F. Lopez, MD, PhD, The Hanson Centre for
Cancer Research, IMVS, Frome Road, Adelaide, SA 5000, Australia;
e-mail: angel.lopez{at}imvs.sa.gov.au.
| |
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INTRODUCTION