(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 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.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) ()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.
Although the 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.
Previous studies done using a series of truncated mutants
from the C terminus revealed that the cytoplasmic domain of 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.
To fully understand the
mechanism of signal transduction of hGM-CSFR, we analyzed the signaling
potential of various 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.
The 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.
Figure 1:
Schematic structure of the 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.
Figure 2:
Short term proliferation of BA/F3
transfectants expressing 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.
Figure 3:
Potential of 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.
Figure 4:
Tyrosine phosphorylation of the 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).
The subregion covering aa 544 and 589 contains one
tyrosine residue at position 577 (Tyr). 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.
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 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.
Figure 5:
Tyrosine phosphorylation of SH2-containing
proteins in BA/F3 transfectants. The factor-deprived BA/F3
transfectants (5 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
p52
and p46
(A)
and PTP1D (B) are shown on the right by arrows.
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
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.
We analyzed functional residues of the cytoplasmic domain of
the 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.
Figure 6: A putative model for signal transduction of GM-CSFR.
The 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.
In contrast to box 1, the box 2
motif of the 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.
We also showed that Tyr 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.
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 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. (
)
Our observations imply that tyrosine phosphorylation of
the 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.