(Received for publication, September 15, 1994; and in revised form, December 19, 1994)
From the
Previous work has suggested that a 97-kDa protein (p97) is involved in the signal transduction pathway of granulocyte-macrophage colony stimulating factor (GM-CSF) as well as interleukin 3, erythropoietin, and interleukin 2. We have examined the relationship of p97 to the protein tyrosine kinase Fes in the GM-CSF signal transduction pathway in erythroid and myeloid cell lines. GM-CSF stimulation of three different cell lines induced tyrosine phosphorylation of p97 as well as a number of other phosphotyrosylproteins. Although each cell line expressed the proto-oncogene product Fes, antisera specific for Fes did not recognize p97 in immunoblotting experiments. Furthermore, immunodepletion of Fes did not reduce the amount of p97 in GM-CSF-treated cells. Two-dimensional gel electrophoresis demonstrated that p97 and Fes have similar charge to mass ratios, and limited proteolytic mapping of p97 and Fes suggested that these proteins may be related but are not identical. Our studies demonstrate that p97 is not Fes but is probably a Fes-related protein.
GM-CSF ()stimulates proliferation and differentiation
of myeloid progenitor cells as well as activation of
neutrophils(1, 2) . The receptor for GM-CSF consists
of an
subunit of 85 kDa which binds ligand with low affinity and
a
subunit of approximately 120 kDa which does not bind ligand but
converts the receptor complex to high affinity
binding(3, 4) . Similar to other members of the
hemapoietin receptor superfamily, the components of the GM-CSF receptor
do not contain intrinsic tyrosine or serine/threonine kinase activity.
Recent work has suggested that the rapid increases in protein tyrosine
phosphorylation stimulated by GM-CSF may be mediated through activation
of multiple tyrosine kinases. A number of Src-like tyrosine kinases
(Hck, Lyn, and Yes), as well as JAK2, are stimulated in response to
GM-CSF(5, 6, 7, 8, 9) . In
addition, the work of Hanazono and co-workers (10, 11) has suggested that Fes is activated in
response to GM-CSF as well as IL-3 and erythropoietin. Interestingly, a
number of laboratories have reported a phosphotyrosylprotein similar in
size to Fes (90-100 kDa) in GM-CSF-, IL-3-, or
erythropoietin-stimulated cells (12, 13, 14, 15) . Furthermore, we
previously have described a putative tyrosine kinase of 97 kDa (p97)
phosphorylated on tyrosine residues in response to GM-CSF(12) .
The present study addresses whether p97 is the proto-oncogene product
Fes or potentially a Fes-related protein.
Two factor-dependent human cell lines widely used for study
of the GM-CSF signal transduction pathway are Mo7e and TF-1 cells.
Stimulation of either of these cell lines results in increases in
proliferative response ranging from 20-200 fold over control
(data not shown). Another factor-responsive cell line used for study of
human GM-CSF are HL-60 cells cultured in MeSO for 3 days.
These cells, although approaching a differentiated phenotype, have a
transient capacity to proliferate in response to GM-CSF and have been
used for study of GM-CSF-induced tyrosine phosphorylation(9) .
Shown in Fig. 1A are the results of GM-CSF stimulation
of the Mo7e and HL-60/Me
SO cells. Cells were radiolabeled
for 3 h with [
P]orthophosphate, stimulated with
GM-CSF for 10 min, and lysed, and phosphotyrosylproteins were
immunoprecipitated using PY-20, a monoclonal antibody specific for
phosphotyrosine. Phosphotyrosylproteins were resolved using SDS-PAGE
and visualized using autoradiography. As shown in Fig. 1A, GM-CSF-induced phosphorylation of a 97-kDa
protein as well as phosphotyrosylproteins of 140, 120, 70, 55, and 42
kDa in the Mo7e and HL-60/Me
SO cells. Identical results
were also obtained with the TF-1 cell line (data not shown).
Figure 1:
GM-CSF induces tyrosine
phosphorylation in cell lines expressing Fes. Panel A,
tyrosine phosphorylation in GM-CSF-treated cells. Cells were washed in
phosphate-free RPMI 1640 and radiolabeled with
[P]orthophosphate (1 mCi/ml) for 3 h. One
milliliter of cells (10
/ml) were stimulated with GM-CSF for
10 min, pelleted, lysed, and immunoprecipitated with the
anti-phosphotyrosine monoclonal antibody PY-20. Phosphotyrosylproteins
were eluted, diluted with SDS sample buffer, resolved using
one-dimensional gel electrophoresis, dried, and visualized with
autoradiography. Panel B, expression of Fes. Cells were lysed
and equivalent amounts of protein immunoprecipitated with anti-Fes.
Washed immunoprecipitates were eluted with SDS sample buffer, resolved
using one-dimensional gel electrophoresis, and transferred to
Immobilon. Shown are the results of immunoblotting with monoclonal
antibody against Fes using ECL for visualization. The abbreviation IP designates immunoprecipitation, and the abbreviation IB designates immunoblotting.
Expression of Fes was next examined in the Mo7e, TF-1, and
HL-60/MeSO cell lines. Shown in Fig. 1B are
results of a Fes immunoblot of Fes immunoprecipitates performed on
equivalent amounts of protein isolated from the cells indicated. As
expected, Fes was present in the erythroid and myeloid cells (Mo7e,
HL-60/Me
SO, and TF-1) but not in the lymphoid line, Jurkat.
Previous work has suggested that GM-CSF induces tyrosine
phosphorylation of Fes as well as increases its autophosphorylation (10, 11) . In addition, our prevous work demonstrated
that p97 has a number of features consistent with those of a tyrosine
kinase(12) . To address whether p97 was the proto-oncogene
product Fes, we performed immunoblotting of radiolabeled
phosphotyrosylproteins from untreated or GM-CSF-treated Mo7e cells. As
shown in the autoradiograph in Fig. 2A, GM-CSF
stimulated tyrosine phosphorylation of p97; however, the Fes antisera
did not recognize p97 in the immunoblot shown in Fig. 2B. The identical results were obtained in each of
the three cell lines tested. These experiments were done with
phosphotyrosylproteins immunoprecipitated from 10-fold more cells
(10 cells/point) then the studies shown in Fig. 1to
reduce the probability that the amount of p97 in the immunoprecipitate
was below the threshold of detection of the antibody used to detect
Fes. As another approach to address any potential relationship between
p97 and Fes, we performed immunodepletion studies. Using conditions in
which Fes could clearly be immunodepleted (Fig. 2C), we
found no reduction in the amount of radiolabeled p97 in
anti-phosphotyrosine immunoprecipitates from GM-CSF-stimulated cells (Fig. 2D).
Figure 2:
Fes-specific antibody does not immunoblot
p97. Panel A, GM-CSF-induced tyrosine phosphorylation in Mo7e
cells. Radiolabeled phosphotyrosylproteins were immunoprecipitated
using the PY-20 antibody, resolved using SDS-PAGE, and transferred to
Immobilon. Shown in panel A is the autoradiograph from this
experiment. PanelB, Western blot analysis of Fes in
GM-CSF-induced phosphotyrosylproteins from panelAversus a Fes immunoprecipitate. Enhanced
chemiluminescence (ECL) was used for visualization. The abbreviation IP designates immunoprecipitation, and the abbreviation IB designates immunoblotting. PanelC,
immunodepletion of Fes from Mo7e cells. Lysates from Mo7e cells were
immunoprecipitated with antibody to Fes. The Fes-depleted supernatants
were saved and reimmunoprecipitated with more Fes antibody. Shown are
the Fes immunoblots before and after immunodepletion. PanelD, immunodepletion of Fes does not reduce the levels of
p97. Mo7e cells were radiolabeled with
[P]orthophosphate as described in Fig. 1.
Cells were incubated in the presence or absence of GM-CSF and lysed,
and lysates clarified. The indicated lysate was depleted of Fes through
immunoprecipitation and phosphotyrosylproteins from all the lysates
were immunoprecipitated as described in Fig. 1.
Phosphotyrosylproteins were visualized using
autoradiography.
We next examined whether we could detect
GM-CSFinduced phosphorylation of Fes in vivo (Fig. 3).
Tyrosine phosphorylation of Fes was examined in both Mo7e (Fig. 3A) and MeSO-treated HL-60 cells (Fig. 3B) after stimulation with GM-CSF. Although no
GM-CSF-induced tyrosine phosphorylation of Fes was observed in either
cell line, the Fes immunoblots in Fig. 3(C and D) demonstrate that Fes was present in each of the
immunoprecipitates. We also examined the effects of GM-CSF on
phosphorylation of Fes using [
P]orthophosphate
and found no changes at any time point examined (Fig. 3E). Thus, using anti-phosphotyrosine
immunoblotting as well as radiolabeling procedures, we could not detect
GM-CSFinduced phosphorylation of Fes.
Figure 3:
GM-CSF does not induce phosphorylation of
Fes. Mo7e (panelsA and C) or
MeSO-treated HL-60 cells (panels B and D)
were resuspended at 2
10
/ml in RPMI 1640. Cells
were stimulated for the indicated times, lysed, and immunoprecipitated
with antibody to Fes or a control. Immunoprecipitates were resolved
using SDS-PAGE, transferred to Immobilon, and Western blots performed
using either antibodies specific for phosphotyrosine (panelsA and B) or Fes (panelsC and D). PanelE, Mo7e radiolabeled with
[
P]orthophosphate were stimulated with GM-CSF,
lysed, and immunoprecipitated with antibody specific for Fes.
Immunoprecipitates were resolved using SDS-PAGE and proteins visualized
using autoradiography.
The previous experiments suggested that p97 represented a protein distinct from the Fes tyrosine kinase. To address any potential relationship between Fes and p97, we examined the charge to mass ratio of GM-CSF-induced phosphotyrosylproteins and Fes using two-dimensional gel electrophoresis. Samples were subjected to isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. As shown in Fig. 4, the migration patterns of p97 and of Fes were quite similar. Both Fes and p97 migrated to a relatively basic position in the isoelectric focusing dimension; however, Fes consistently had a slightly broader and more basic position. Somewhat surprisingly, the molecular sizes of p97 and Fes, originally described as a 92-kDa protein, were close to identical when resolved in the second dimension. The basis for this apparent discrepancy relates to the debate over the actual molecular weight of the phosphorylase b marker used for the molecular mass standards. While phosphorylase b was once thought to be a 92-kDa protein, later work demonstrated it to be 97 kDa. Thus it is likely that the apparent molecular mass of Fes may actually be closer to 97 kDa. We also examined migration of Fer, another member of the Fps/Fes family of tyrosine kinases using two-dimensional gel electrophoresis. Interestingly, similar to p97 and Fes, Fer also migrated toward the basic end of the isoelectric focusing gel (data not shown). In contrast, however, in SDS-PAGE gels, Fer migrated at a molecular weight distinctly greater than p97 or Fes. These data suggest that p97 is not Fes or Fer but may represent a related protein. To further test this postulate, we performed limited peptide mapping of p97 and Fes isolated from GM-CSF-stimulated cells. Comparison of the peptide maps of Fes and p97 shown in Fig. 5demonstrates that several, but not all, of the proteolytic fragments from Fes were the same molecular weight as those generated from the digestion of p97. These data, considered in conjunction with the similarity in migration of p97 and Fes using two-dimensional electrophoresis, strongly suggest that these proteins are related but distinct.
Figure 4:
Two-dimensional gel electrophoresis of
GM-CSF-induced phosphotyrosylproteins and Fes. Mo7e cells were
radiolabeled with [P]orthophosphate as described
in Fig. 1. Cells were stimulated with GM-CSF for 10 min and
lysed, and either Fes or phosphotyrosylproteins were isolated using
immunoprecipitation. Proteins were resolved using isoelectric focusing
in the first dimension and SDS-PAGE in the second dimension. The
abbreviation IP designates
immunoprecipitation.
Figure 5: Peptide mapping of p97 and Fes. Proteins were excised from SDS-polyacrylamide gels, rehydrated, equilibrated, digested with 0.05 µg of V8 protease, and subjected to electrophoresis as described under ``Materials and Methods.''
The Fes/Fps family of protein tyrosine kinases
currently includes Fes/Fps, Fer, and a truncated form of Fer designated
FerT(17, 18, 19, 20, 21, 22) .
Of these members, Fer is expressed relatively ubiquitously, FerT is
testes-specific and Fes is expressed predominantly in myeloid cells and
certain erythroid progenitors. Recent work has suggested that Fes may
be activated in response to GM-CSF, IL-3, and
erythropoietin(10, 11) . Fes has also been suggested
to play a role in maintaining viability of granulocytes during
differentiation(23) . Previous work from our laboratory
identified a putative protein tyrosine kinase of approximately 97 kDa
(p97), which was phosphorylated in response to a number of different
hematopoietic growth factors(12) . The objective of this study
was to address the relationship between Fes and the 97-kDa protein
phosphorylated in response to GM-CSF. For this purpose we have used
three cell lines of erythroid and myeloid origin which express Fes and
proliferate in response to GM-CSF. Consistent with previous work, p97
was one of the dominant phosphotyrosylproteins induced by GM-CSF in
each of the cell lines examined (Fig. 1). Several lines of
evidence strongly suggest that p97 is not Fes. First, anti-Fes did not
recognize p97 in the GM-CSF-induced phosphotyrosylproteins (Fig. 2, A and B). Second, immunodepletion of
Fes did not reduce the levels of the radiolabeled p97 in the
anti-phosphotyrosine immunoprecipitates (Fig. 2D).
Third, we did not observe GM-CSF-induced phosphorylation of Fes using
either anti-phosphotyrosine immunoblotting or
[P]orthophosphate radiolabeling (Fig. 3).
Fourth, peptide mapping experiments of Fes and p97 suggested that these
two proteins are distinct but probably related (Fig. 5). In this
regard, the similarity in the migration of p97, Fes, and Fer in
two-dimensional gel electrophoresis further suggests that these
proteins are related (Fig. 4). We have also examined whether p97
is the Fps/Fes family member Fer and found it unlikely based
on the difference in molecular weight of p97 and Fer, as well as the
absence of cytokine-induced increases in Fer autophosphorylation. (
)In summary, these studies demonstrate that p97 is not the Fes proto-oncogene product and suggest that it is a member of
the Fps/Fes family of tyrosine kinases.
Recent work has
suggested a broader role for Fes in mammalian cell physiology than
previously thought. Fes has been observed in embryonic tissues, and Fes
or a Fes-related protein has been reported in murine lymphoid cell
lines(24, 25) . Further more, we have previously noted
a protein of approximately 97-kDa which is phosphorylated on tyrosine
in response to IL-2(12, 26, 27) . Peptide
mapping of the IL-2-induced phosphotyrosylprotein of 97-kDa and a
protein of similar size and pI phosphorylated in response to GM-CSF
from [P]orthophosphate-labeled lymphoid and
myeloid cells has indeed suggested that these proteins are either
closely related or identical(12) . Unfortunately, our efforts
to determine if these proteins are identical through peptide mapping of
p97 from [
S]methionine-labeled
phosphotyrosylproteins from GM-CSF-stimulated myeloid cells and
IL-2-stimulated lymphoid cells have failed. Thus, to date, the
experimental designs necessary to further address the relationship of
the 97-kDa protein found in cytokine-stimulated myeloid and lymphoid
cells have been unsuccessful.
The role of the Fes
proto-oncogene product in cytokine signal transduction has been
somewhat controversial. Hanazano and colleagues (10, 11) have seen increases in Fes
autophosphorylation, as well as increases in its tyrosine
phosphorylation after stimulation of TF-1 cells with GM-CSF, IL-3, or
erythropoietin. In addition, these investigators have reported that
GM-CSF induces Fes to associate with the chain of the GM-CSF
receptor(10) . In contrast, other groups have not found Fes
activated in response to either erythropoietin or GM-CSF(5) .
Thus, more work will be necessary to delineate more conclusively the
role of Fes in signal transduction of hematopoietic cytokines. A
relatively large body of work has also suggested that Fes plays a role
in the differentiated phenotype in myeloid cells. Although
overexpression of Fes partially abrogated CSF-1 dependence in a
macrophage-derived cell line, it appeared to be through a signal
transduction pathway distinct from that utilized by CSF-1(28) .
Glazer and co-workers (29, 30, 31) reported
increases in Fes activity in differentiating myeloid cells and also
found that transfection of the erythroid cell line K562 with fes conferred the capacity for these cells to differentiate toward a
myeloid phenotype More recently, another group has shown that antisense
oligonucleotides specific for fes induced apoptosis in HL-60
cells treated with inducers of granulocytic
differentiation(23) . These investigators concluded that Fes
played a role in preventing apoptosis during granulopoiesis.
Considered in total, these observations strongly suggest that, as in the Src and JAK family of tyrosine kinases, Fes/Fer have members that perform parallel or similar functions in the signal transduction pathways of multiple stimuli. Our studies suggest that p97 is related to Fes and may represent a new member of the Fps/Fes/Fer family of protein tyrosine kinases. Future work will be directed toward cloning new members of this family of tyrosine kinases and better characterizing their role in receptor-effector coupling of hematopoietic cytokines.