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(Received for publication, October 24, 1995; and in revised form, January 17,
1996) From the
Granulocyte-macrophage colony-stimulating factor (GM-CSF)
provokes a proliferative response and induction of early-response genes
such as c-fos in target cells. It also induces rapid tyrosine
phosphorylation of cellular proteins, including the
Granulocyte-macrophage colony-stimulating factor (GM-CSF) ( Although the Previous studies done using a series of truncated mutants
from the C terminus revealed that the cytoplasmic domain of To fully understand the
mechanism of signal transduction of hGM-CSFR, we analyzed the signaling
potential of various
The
Figure 1:
Schematic structure of the
Figure 2:
Short term proliferation of BA/F3
transfectants expressing
Figure 3:
Potential of
Figure 4:
Tyrosine phosphorylation of the
The subregion covering aa 544 and 589 contains one
tyrosine residue at position 577 (Tyr
Cell lysates were prepared from either unstimulated or
hGM-CSF-stimulated BA/F3 transfectants. Immunoprecipitations were
performed using the anti-Shc antibody, and the proteins precipitated
were subjected to Western blot analysis. This antibody
immunoprecipitated 46- and 52-kDa species of Shc proteins (p46
Figure 5:
Tyrosine phosphorylation of SH2-containing
proteins in BA/F3 transfectants. The factor-deprived BA/F3
transfectants (5
Immunoprecipitates obtained using an anti-PTP1D antibody from BA/F3
transfectants were analyzed by Western blotting. As shown in Fig. 5B, exposure of hGM-CSF to cells expressing
We analyzed functional residues of the cytoplasmic domain of
the
Figure 6:
A putative model for signal transduction
of GM-CSFR.
The In contrast to box 1, the box 2
motif of the We also showed that Tyr Previous studies have shown that both
induction of the c-fos/c-jun mRNAs and activation of
the Ras/Raf/mitogen-activated protein kinase cascade by hGM-CSF depend
on the membrane-distal region, thereby implying that these events are
related(8, 13) . Ras is indeed required for induction
of the c-fos mRNA, as the dominant-negative Ras mutant
completely inhibited the hGM-CSF-dependent activation of the c-fos promoter via the wild-type Our observations imply that tyrosine phosphorylation of
the
Volume 271,
Number 13,
Issue of March 29, 1996 pp. 7587-7592
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit
(c) of its functional receptor. However, locations and functions
of phosphorylated tyrosine residues within the c are unclear. To
elucidate the mechanism of the human GM-CSF receptor signal
transduction, mutational analyses were made of the cytoplasmic domain
of the c, using murine BA/F3 cells. Deletion of the conserved box
1 motif resulted in loss of tyrosine phosphorylation of the c,
thereby indicating an essential role for this motif in activating the
tyrosine kinase which phosphorylates c. A C-terminal truncated
mutant at position 589 activated the c-fos promoter, and this
activation was diminished by a substitution at tyrosine 577
(Tyr). However, the same substitution in the full-length
c did not completely abrogate the c-fos promoter
activation, hence, redundant signaling pathways probably exist. When we
analyzed signaling molecules functioning downstream of the c we
found that Tyr is essential for Shc phosphorylation,
while tyrosine phosphorylation of PTP1D was mediated through
Tyr
as well as through other site(s). We suggest that
GM-CSF stimulates at least two modes of signals leading to Ras
activation, an event which ultimately gives rise to promoter activation
of c-fos.
)is a multifunctional cytokine which supports survival and
proliferation of hematopoietic stem cells or progenitor cells, and also
enhances multiple functions of mature neutrophils, macrophages, and
eosinophils(1, 2) . A functional high-affinity GM-CSF
receptor (GM-CSFR) complex is composed of the ligand-specific 
subunit(s) and the shared subunits (c), both belonging to
the type I cytokine receptor superfamily (also known as hematopoietin
receptor family)(3, 4, 5) . The cytoplasmic
domains of both the and the c are essential for
GM-CSF-induced signaling, however, the c has a relatively large
cytoplasmic domain composed of about 430 amino acid residues (aa) (4, 6) and is likely to play pivotal roles in signal
transduction. and c subunits do not contain
characteristic motifs of kinase or phosphatase, GM-CSF does induce a
rapid and reversible tyrosine phosphorylation of various cellular
proteins which seem to be critical for biological functions, as
revealed by experiments using tyrosine kinase
inhibitors(6, 7, 8) . JAK2, a member of the
Janus kinase family, is associated with the membrane-proximal region of
the human (h) c and is activated by hGM-CSF
stimulation(9) . Lyn and Fes/Fps are also
activated(10, 11) , but their roles in signaling
remain unknown. One substrate for GM-CSF-activated tyrosine kinases is
c (4, 12) . The locations of phosphorylated
tyrosine residues and their functional significance require further
study.c
contains two functional regions required for
signaling(6, 8, 13) . The membrane-proximal
region, which contains the conserved box 1 motif, is involved in a
proliferation signal and induction of c-myc mRNA. The distal
region is essential for activation of the cascade of events involving
Shc, Ras, Raf, mitogen-activated protein kinase, and for induction of
the c-fos and c-jun mRNAs. Shc is an adaptor protein
which contains the SH2 (Src homology 2) domain and the phosphotyrosine
interaction domain (or phosphotyrosine-binding domain) (14, 15, 16) and is associated with
phosphotyrosine residues of several growth factor receptors being
phosphorylated, binding to Grb2, which in turn activates Ras by
recruiting the Ras-guanine nucleotide exchange factor
Sos(17, 18) . Tyrosine phosphorylation of PTP1D (also
known as SH-PTP2 or Syp), a cellular protein tyrosine phosphatase which
contains two SH2 domains (19, 20, 21) , also
results in its association with Grb2 and is therefore thought to induce
the activation of Ras(22, 23) . It has been reported
that GM-CSF induces tyrosine phosphorylation of PTP1D and its
association with Grb2 and the p85 subunit of phosphatidylinositol
3`-kinase(24) . The functional region of the GM-CSFR
responsible for this event is unknown.c mutants by reconstituting high-affinity
receptors in combination with the wild-type subunit in murine
interleukin-3 (mIL-3)-dependent BA/F3 cells. We found that tyrosine at
position 577 (Tyr) of
c is important for activation
of the c-fos promoter by hGM-CSF and is one of the sites of
phosphorylation, while activation of the kinase which phosphorylates
the c depends on the membrane-proximal box 1 motif. We also show
that hGM-CSF induces in a different manner tyrosine phosphorylation of
two signaling molecules Shc and PTP1D, functioning in Ras activation.
Cells and Culture
A mIL-3-dependent pro-B cell
line, BA/F3, was maintained in RPMI 1640 medium supplemented with 10%
fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin,
and 0.25 ng/ml mIL-3. Purified recombinant hGM-CSF produced in Escherichia coli was provided by Schering-Plough Corp. The
mIL-3 expressed in silkworm (Bombyx mori) was purified as
described previously(25) .Antibodies
Rat monoclonal antibodies against the
human common c, 5A5, were prepared as described
previously(26) . Rabbit polyclonal antibodies against the
extracellular domain of the human c were obtained from Medical
& Biological Laboratory Co. Ltd. (Nagoya, Japan).
Anti-phosphotyrosine monoclonal antibodies (4G10) and antisera against
Shc were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY),
and antisera against PTP1D from Santa Cruz Biotechnologies (Santa Cruz,
CA).Plasmids
The hGM-CSFR and c (KH97)
cDNAs were originally cloned into pCEV4 and pME18S vectors,
respectively, as described previously(4) . The c-fos promoter-luciferase gene fusion plasmid contains the human
c-fos promoter fragment (-404 to +41) fused to the
luciferase fragment and was constructed as described
elsewhere(27) .763, 626, and 544 mutants
were generated as described(6) . The c mutants newly
prepared in this study were constructed by polymerase chain
reaction-mediated site-directed mutagenesis (28) using
appropriate oligonucleotide primers. The accuracy of all the nucleotide
sequences of the fragments derived from polymerase chain reaction was
confirmed by dideoxy sequencing using an automated sequencer (Applied
Biosystems Inc.). To establish stable transfectants of the c
mutants, mutant cDNAs were inserted between the XhoI site and
the XbaI site of the pME18S vector containing a neomycin
resistance gene.Establishment of Stable Transfectants
The
expression plasmid for the c containing a neomycin resistance gene
was transfected by electroporation into BA/F3 cells which stably
express the wild-type hGM-CSFR subunits. Cells suspended in 200
µl of ice-cold phosphate-buffered saline (137 mM NaCl, 2.7
mM KCl, 4.3 mM Na
HPO
, 1.4
mM KHPO, pH 7.5) (2 
10
cells) were mixed with 15 µg of plasmid DNA. Electroporation
was then carried out using a Gene Pulser (Bio-Rad) set at 250
microfarads and 300 V. Transfectants were recovered and maintained in
complete medium for 3 h. Viable cells were counted and aliquots were
put into 96-well plates (3 10 cells in 100
µl/well) in complete medium. Following 2 days of culture, G418 was
added at a final concentration of 1 mg/ml and the selection was carried
out for about 10 days. Surface expression of the transfected c
gene products of the G418-resistant clones was confirmed by flow
cytometry with FACScan (Becton Dickinson), using anti-c antibody
5A5.Cell Proliferation Assays
Cell proliferation was
measured by the colorimetric assay using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
originally developed by Mosmann(29) . Cells were washed twice
with factor-free medium containing 4% fetal calf serum and aliquots
were put into 96-well plates (5 10
cells in 100
µl/well) with the same medium in the presence of various
concentrations of hGM-CSF or mIL-3, or without cytokine. Following 24 h
incubation, 10 µl of MTT solution (5 mg/ml) was added to each well
and incubation was continued for a further 5 h at 37 °C. Finally
100 µl of isopropyl alcohol containing 0.04 N HCl was
added and the cells were thoroughly suspended. The absorbance value
(OD) was measured using an Emax microplate
reader (Wako Pure Chemical Industries, Ltd.).
Transient Transfection and Luciferase
Assays
Plasmid DNAs were transiently transfected into BA/F3
cells using the DEAE-dextran method(27) . For each
transfection, 3 µg of c-fos-luciferase fusion plasmid and
5 µg of the wild-type hGM-CSFR cDNA plasmid, in combination
with 5 µg of various c mutants or control vector, were used.
Three million cells were washed twice with TBS(++) (25 mM Tris-HCl, pH 7.5, 137 mM NaCl, 5 mM KCl, 0.5
mM NaHPO, 0.49 mM MgCl, 0.68 mM CaCl) and then
mixed with DNA and DEAE-dextran (0.5 mg/ml) in 4 ml of
TBS(++). Following a 30-min incubation at room temperature,
cells were washed and cultured in complete media for 12 h, and then
were separated into three aliquots. After factor depletion for 6 h in a
mIL-3-free medium containing 10% fetal calf serum, the cells were
stimulated with hGM-CSF (5 ng/ml), mIL-3 (1 ng/ml), or no cytokine for
6 h, then harvested and used for luciferase assay. Proteins were
extracted from cells by three cycles of freezing and thawing.
Luciferase activity was measured using a luminometer (model LB9501;
Berthold Lumat Co. Ltd., Japan). Protein concentration was determined
using the BCA protein assay reagent (Pierce) according to the
manufacturer's instruction.Immunoprecipitation
Cells were resuspended in
factor-free medium at 4 10 cells/ml and incubated
at 37 °C for 60 min, then either stimulated with 10 ng/ml hGM-CSF
for 10 min or left unstimulated. The reaction was stopped by adding
ice-cold phosphate-buffered saline. Cells were harvested, washed once
with ice-cold phosphate-buffered saline, and then lysed in 200 µl
of lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1 mM EDTA, 1% Nonidet P-40, 100 µM NaVO, 50 mM sodium fluoride, 1
mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1
µg/ml pepstatin A) for 1 h at 4 °C. Cell lysates were
centrifuged to remove the insoluble material and the supernatants were
then incubated for 1 h at 4 °C with the indicated antibody. Protein
G-Sepharose was added to the reaction mixture and the incubation was
continued for an additional 1 h. The beads were pelleted by
centrifugation, washed five times with cold lysis buffer, then were
resuspended in 1 Laemmli's sample buffer (30) without 2-mercaptoethanol, boiled for 5 min, and then
2-mercaptoethanol was added in all cases except in case of
immunoprecipitation of Shc.Western Blot Analysis
The protein samples were
electrophoresed on an SDS-polyacrylamide gel, and electrophoretically
transferred onto Immobilon polyvinylidene difluoride membrane
(Millipore) in transfer buffer (25 mM Tris base, 192 mM glycine, 20% methanol). The membrane was blocked with TBS (25
mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 5%
bovine serum albumin (Fraction V, Sigma) for 1 h at room temperature
and washed three times with TBS containing 0.05% Tween 20 (TBS-T). The
membrane was incubated with the indicated primary antibody for 1 h at
room temperature, washed again three times with TBS-T, and then
incubated with the appropriate secondary antibody conjugated with
horseradish peroxidase for 1 h at room temperature. Following five
washes with TBS-T, the blot was developed with the ECL system
(Amersham) and exposed to x-ray film (Kodak), according to the
manufacturer's instructions.
The Membrane-proximal Box 1 Motif Is Essential to
Transduce Proliferation Signals
The membrane-proximal region (aa
456-517), which is required for the proliferation signal and
activation of the c-myc gene, contains box 1 (from Trp to Pro
) and is adjacent to box 2 (from Val
to Cys
). These motifs are conserved in the
membrane-proximal regions of several type I cytokine receptors and have
been shown to play critical roles in signaling events(31) . We
asked whether boxes 1 and 2 of the GM-CSFR are also important for
signaling and for this, internal deletion mutants of these motifs,
box1 and box2, were used, respectively (Fig. 1). To
determine their functions in supporting short-term proliferation, BA/F3
transfectants expressing the wild-type c (wild), box1,
or box2, in combination with the wild-type subunit were
cultured for 24 h in the presence of various concentrations of hGM-CSF
and MTT assays were performed. Cells expressing either wild or
box2 proliferated in medium containing 1 ng/ml hGM-CSF, although
the latter did show a slightly reduced sensitivity to hGM-CSF (Fig. 2, A and C). In contrast, box1 at
the same concentration of hGM-CSF did not transmit short-term
proliferation signals (Fig. 2B). The response observed
at a high concentration (100 ng/ml) was due to endogenous mouse c
(AIC2B), which stimulates cell proliferation in combination with the
human subunit(5) . These results suggest that box 1, but
not box 2, is essential to support hGM-CSF-induced short-term
proliferation.
c
mutants used in this study. The extracellular portions are abbreviated.
Positions of wild-type tyrosine residues are indicated as solid
lines, while white lines are for substitutions for
phenylalanine. Dotted lines mean the portions internally
deleted in the mutants.
c mutants. The BA/F3 transfectants
expressing the wild-type hGM-CSFR subunit together with the
wild-type c (A), box1 (B), or box2 (C) were incubated for 24 h in the presence of 0-100
ng/ml hGM-CSF, and cell growth was examined by the MTT colorimetric
assay. As a control, the cells were cultured with 1 ng/ml mIL-3. Vertical axis indicates the relative MTT reduction value
normalized to the value for cells incubated with 1 ng/ml mIL-3. All
values are the average of duplicated samples and standard deviations
are shown as error bars. Numbers show independent clones of
transfected BA/F3 cells.
Box 1 Is Also Essential for c-fos Promoter Activation
Signals
Previous studies demonstrated that the membrane-distal
region of the c is required for the hGM-CSF-dependent induction of
the c-fos mRNA(8, 13) . However, these
studies were carried out by using the c mutants truncated from the
C terminus, and it was unclear if the distal region alone was
sufficient to transduce signals. We examined effects of the deletion of
either box 1 or box 2 on the potential to activate the c-fos promoter by means of the transient transfection assay, using a
reporter plasmid. The reporter plasmid carrying the c-fos promoter fused to the luciferase gene was co-transfected with
expression constructs encoding for both the wild-type subunit and
a series of c mutants. After stimulation with cytokine, cell
lysates were prepared and the luciferase activities were determined.
The wild activated the c-fos promoter in response to
hGM-CSF (Fig. 3). However, box1 did not activate the
c-fos promoter even though it retained an intact distal
region. Deletion of the box 2 motif resulted in a partial loss of the
potential to activate the c-fos promoter, which means that box
2 is involved in, but is not essential for this signaling. Therefore,
the distal region alone is insufficient for and the membrane-proximal
region, especially box 1, is also required for c-fos promoter
activation signals.
c mutants to activate
the c-fos promoter. Activation of c-fos promoter by
each mutant c was measured by the transient transfection assay
using the c-fos promoter-luciferase fusion construct as a
reporter gene. Continuously growing BA/F3 cells were transfected with
plasmids containing the wild-type hGM-CSFR subunit cDNA and those
containing each of the c mutants together with the c-fos promoter-luciferase reporter plasmid, as described under
``Materials and Methods.'' After 6 h factor depletion, cells
were either left unstimulated or stimulated with 5 ng/ml hGM-CSF or 1
ng/ml mIL-3 for 6 h. Cell lysates were prepared and subjected to the
luciferase assay. The c-fos promoter activity was calculated
by dividing the luminescence intensity (relative light units per
min/µg of total protein) of no stimulation or hGM-CSF stimulation
by that of mIL-3 stimulation, and are presented as a percentage of that
of the wild. All values are the average of at least two
experiments and standard deviations are shown as error
bars.
Box 1 Is Required for Activation of the Tyrosine Kinase
Which Phosphorylates the
We next examined whether or not
box 1 is involved in tyrosine phosphorylation of the cc. Stable
transfectants expressing hGM-CSFR composed of the wild-type and
the c mutant, were either left unstimulated or stimulated with
hGM-CSF, and the immunoprecipitation was performed followed by Western
blot analysis. As shown in Fig. 4A, the c appeared
in duplicated bands due to differences in glycosylation, since only a
single band with a molecular mass (in agreement with the value
predicted from the amino acid sequence of the c) was detected
after treatment with tunicamycin, ()the specific inhibitor
of N-linked glycosylation. Blotting with anti-phosphotyrosine
antibody (4G10) revealed that the box1 mutant was not
phosphorylated following hGM-CSF stimulation. In contrast, box2
induced hGM-CSF-dependent tyrosine phosphorylation of the c. These
results indicate that box 1, but not box 2, is essential for tyrosine
phosphorylation of the c, and that this motif is important for
activating tyrosine kinase phosphorylating the c.
c.
The factor-deprived BA/F3 transfectants (1 10
cells
each) stably expressing either the wild-type c (A and B), box1, box2 (A), 589, 544,
wild;Y577F, or 589;Y577F (B) were either left
unstimulated or stimulated with 10 ng/ml hGM-CSF for 10 min at 37
°C. Cells were lysed and immune complexes with anti-c
monoclonal antibody 5A5 were precipitated. Protein samples were
separated by SDS-7.5% polyacrylamide gel electrophoresis and Western
blot analyses were performed using anti-phosphotyrosine monoclonal
antibody 4G10 (left column) or anti-c polyclonal
antibodies (right column).
Tyr
To delineate the essential residue(s) or motif(s)
for signaling leading to the c-fos promoter activation, we
examined the potential of previous Within the Cytoplasmic Domain of
the
c Is Important for Signaling to Activate the c-fos
Promoterc mutants as well as newly
constructed ones (Fig. 1) to activate the c-fos promoter by the transient transfection assay. As shown in Fig. 3, 589, possessing only about 140 aa of the
cytoplasmic domain, was still capable of activating the promoter at a
level comparable to that seen with the wild. However, the
potential of the c to activate the c-fos promoter was
markedly reduced by truncation at aa 544. These data demonstrate
requirement of the subregion covering aa 544 and 589 for this signaling
event. These results, however, are inconsistent with other data that
626 is incapable of inducing the c-fos mRNA(8, 13) . The cDNA construct for 626 was
confirmed to be correct. However, we observed that the
BaF/626 clone used in previous studies expresses a protein
which does not match the expected size, only a smaller one. ()). In the case of
growth factor receptors, tyrosine residues are involved in signaling
through phosphorylation. Since the hGM-CSF also induces tyrosine
phosphorylation of the
c(6, 12) , we examined the
possible requirement of Tyr for signaling by using
mutants in which Tyr
was substituted for by
phenylalanine. The level of the c-fos promoter activation by
589, which is comparable to that of wild, was remarkably
reduced by this substitution (589;Y577F). However, the same
substitution of phenylalanine for Tyr in full-length
c (wild;Y577F) did not significantly impair the function of
the receptor. These data demonstrated that: 1) Tyr is an
essential residue for activation of the c-fos promoter, at
least in
589, and 2) other sites located C-terminal to aa 589 in
the full-length c play a similar, if not identical, role.Tyr
As it seemed clear that
Tyrof the
c Is Phosphorylated
following Ligand Stimulation is involved in signaling from the
c, we next
examined whether or not this tyrosine residue is actually the target
site for the hGM-CSF-dependent phosphorylation and here we used
immunoprecipitation and Western blot analysis. As shown in Fig. 4B, hGM-CSF induces tyrosine phosphorylation of
either 589 or wild, but not 544 or
Tyr-mutated
589. Since Tyr is the only
tyrosine residue within the region between aa 545 and 589, these
results show that Tyr
is a target site of
phosphorylation. However,
wild;Y577F, the full-length c with
a single substitution of phenylalanine for Tyr was still
phosphorylated following ligand stimulation. Therefore, hGM-CSF induces
phosphorylation of multiple tyrosine residues in the
c, including
Tyr. This result is consistent with findings that
wild;Y577F is capable of activating the c-fos promoter.Tyr
Next, we investigated the
nature of the signaling molecules functioning downstream of the
phosphorylated of the
c Is Required for
Tyrosine Phosphorylation of Shcc. Our previous study demonstrated that the hGM-CSF
induces tyrosine phosphorylation of Shc through the membrane-distal
region of the c (13) , therefore, we searched for the
possible involvement of Tyr for activation of Shc.
and p52
, respectively) from BA/F3 transfectants (Fig. 5A, lower panel). Blotting with an
anti-phosphotyrosine antibody revealed that p46
was
phosphorylated in a constitutive manner.
wild and 589
apparently induced tyrosine phosphorylation of p52 in a
ligand-dependent manner (Fig. 5A, upper panel).
However, phosphorylation of this protein did not occur in cells
expressing
544 or wild;Y577F. Such being the case,
Tyr is likely to be essential for tyrosine
phosphorylation of Shc and is probably the critical site for
Shc-mediated Ras activation.
10 cells each) were either left
unstimulated or stimulated with 10 ng/ml hGM-CSF for 10 min at 37
°C. Cells were lysed and immune complexes with anti-Shc antibody (A) or anti-PTP1D antibody (B) were precipitated.
Protein samples were separated by SDS-7.5% polyacrylamide gel
electrophoresis and tyrosine-phosphorylated proteins were identified by
Western blot using anti-phosphotyrosine antibodies (upper
panels). Immunoprecipitated Shc or PTP1D proteins were identified
by blotting with anti-Shc antibodies or anti-PTP1D antibodies (lower panels), respectively. Molecular size standards are
shown in kDa on the left. The positions of
p52and p46
(A)
and PTP1D (B) are shown on the right by arrows.
Tyrosine Phosphorylation of PTP1D Is Mediated by
Tyr
As indicated above, the full-length as Well as by Other Functional Sites of the
cc
containing a single substitution at Tyr is unable to
induce tyrosine phosphorylation of Shc yet activates the c-fos promoter. This implies the existence of Shc-independent signaling
pathways leading to activation of the c-fos promoter. Several
lines of evidence strongly suggest that PTP1D functions as a positive
regulator in growth factor receptor signaling, via
Ras(22, 23, 32, 33) . We next
examined
c mutant-induced tyrosine phosphorylation of PTP1D. wild led to the induction of tyrosine phosphorylation of PTP1D,
and there was a slight shift in mobility. Stimulation of cells
expressing 589 also resulted in the same pattern of
tyrosine-phosphorylated proteins including PTP1D, which was
significantly diminished by a single substitution at Tyr.
However,
wild;Y577F still induced tyrosine phosphorylation of
PTP1D and co-immunoprecipitated proteins. Thus, while Tyr is involved in tyrosine phosphorylation of PTP1D, unlike Shc, it
is not essential for this event. The activation of PTP1D is mediated by
Tyr
as well as by other functional sites located at
position C-terminal to aa 589.
c, and found that: 1) the box 1 motif is essential for
activation of the tyrosine kinase which phosphorylates c and 2)
Tyr is one phosphorylation site within the
c and is
critical for the activation of Shc (Fig. 6). Our results also
suggest that GM-CSF stimulates at least two signaling pathways, one is
mediated by Shc and the other by PTP1D, both leading to activation of
Ras and ultimately to transcriptional activation of the c-fos gene.
c subunit of the hGM-CSFR contains the conserved
box 1 and box 2 motifs, and we found that box 1 has a critical role in
hGM-CSF-induced signaling. The box1 mutant failed to induce
short-term proliferation in response to hGM-CSF. Box 1 also proved to
be essential for activation of the c-myc promoter in the
transient transfection assay using the c-myc-CAT reporter
plasmid. ()In addition to these signaling events, box 1 is
essential for c-fos promoter activation signals for which only
the membrane-distal region of the c has heretofore thought to be
required. The box1 mutant did not induce tyrosine phosphorylation
of the c following hGM-CSF stimulation. This indicates that box 1
is required for activation of some tyrosine kinase which phosphorylates
c. The requirement of box 1 for c-fos promoter activation
seems reasonable since tyrosine phosphorylation of the c plays a
role in signaling from the distal region. However, the molecular nature
of the tyrosine kinase which phosphorylates the c will need to be
clarified. Recently, it was reported that box 1 of the c directly
interacts with the N-terminal portion of JAK2(34) . We also
found that box 1 is necessary for activation of JAK2 and that
overexpression of both JAK2 and the c in COS7 cells results in
tyrosine phosphorylation of the c. Thus, it is
possible, although not yet proven, that JAK2 phosphorylates the
tyrosine residues of the c.c is dispensable for signaling, the box2 mutant
was capable of stimulating short-term proliferation. However, cells
expressing box2 showed a slightly reduced sensitivity to hGM-CSF,
and the level of the c-fos promoter activation by box2
was about half that by the wild. Therefore, box 2 of the c
appears to enhance activity of the receptor. It should be noted that in
other type I cytokine receptors, such as the interleukin-2 receptor
subunit, gp130, and the erythropoietin receptor, box 2 is
essential for cell proliferation and other signaling
events(31, 35, 36) . The molecular basis for
this discrepancy and functions of box 2 will need further attention. within the cytoplasmic domain
of the
c is phosphorylated following ligand stimulation, and is
crucial for hGM-CSF-dependent signal transduction. Substitution at
Tyr abrogated the signals leading to activation of the
c-fos promoter by
589, and reduced those by the
full-length c. However, as wild;Y577F partially activated the
c-fos promoter means that other active site(s) located at
position(s) C-terminal to aa 589 perform a similar, if not identical,
function and that either Tyr or the other site(s) alone
is sufficient to transduce signals. Since tyrosine phosphorylation of
the
c occurs at multiple sites, including Tyr,
phosphorylated tyrosine(s) other than Tyr
are likely to
have a role in signaling.
c in the transient transfection
assay. (
)Thus, it appears that the signaling pathways
originating from both Tyr and other functional sites
independent of Tyr
merge upstream of Ras. Both Shc and
PTP1D, when tyrosine phosphorylated, are known to associate with Grb2
and subsequently activate Ras. Our data concerning activation of these
molecules strongly suggest that hGM-CSF stimulates at least two
independent molecular events leading to Ras activation, and that
residues of the
c responsible for these events are different
although overlapping. As tyrosine phosphorylation of Shc was abrogated
by the single substitution at Tyr of the full-length
c, Tyr has an essential role in Shc activation. It
has been reported that Shc protein associates with the phosphorylated
c and that this association is mediated through its SH2
domain(37) . Although co-immunoprecipitating Shc with the
c or vice versa has yet to be done, it is of considerable interest
as to whether or not Shc directly binds to phosphorylated
Tyr. In contrast to Shc, both
589 and
wild;Y577F are capable of inducing tyrosine phosphorylation of
PTP1D, indicating that activation of PTP1D is mediated by multiple
sites, including Tyr. Since PTP1D contains two SH2
domains, it may be that this molecule associates with the receptor in
such a manner that each SH2 domain interacts, either directly or
indirectly, with distinct phosphotyrosine residue of the
c, one of
which is sufficient for signaling. The N-terminal SH2 domain of PTP1D
was predicted to bind to a consensus sequence of
Tyr-Ile/Val-Xaa-Val/Ile/Leu/Pro with low selectivity(38) . The
sequence surrounding Tyr (Tyr
-Leu-Gly-Pro)
partially matches this sequence. There is no available evidence that
PTP1D directly interacts with
c. It is noteworthy that
589;Y577F slightly stimulates phosphorylation of PTP1D and this
may partly account for the observation that 544 or 589;Y577F
induces slightly higher levels of the c-fos promoter
activation than seen with box1 or the vector control. One possible
explanation for this weak phosphorylation is that activation of the
tyrosine kinase which phosphorylates PTP1D depends on the region
located N-terminal to aa 544, while efficient phosphorylation is
mediated by Tyr and other sites located more C-terminal
by recruiting the molecule onto the receptor. This notion is given some
support by the finding that the overexpression of both JAK2 and PTP1D
in COS7 cells results in tyrosine phosphorylation of PTP1D. (
)c is crucial for signal transduction of hGM-CSFR. Based on
this, we are now analyzing functional tyrosine residues of the c
other than Tyr as well as their downstream signaling
molecules. All these observations will help unravel mechanisms which
regulate proliferation and differentiation of hematopoietic cells by
GM-CSF.
))))))
We thank Dr. T. Kinoshita for helpful suggestions, and
M. Ohara and M. Dahl for comments on the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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