From the Arthritis and Inflammation Research Centre,
University of Melbourne, Department of Medicine, Royal Melbourne
Hospital, Parkville, Victoria, Australia 3050, the
§ Co-operative Research Centre for Chronic Inflammatory
Diseases, Royal Melbourne Hospital, Parkville, Victoria, Australia
3050, the ** Ludwig Institute for Cancer Research, Parkville,
Victoria, Australia 3050, and the
Cell Cycle Proteolysis
Laboratory, Trescowthick Research Laboratories, Peter MacCallum Cancer
Institute, A'Beckett Street, Melbourne, Australia 3006
Received for publication, January 10, 2001, and in revised form, March 26, 2001
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ABSTRACT |
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Macrophage colony stimulating factor
(M-CSF or CSF-1) acts to regulate the development and function of cells
of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with
the CSF-1 receptor (FD/WT) adopt a macrophage-like morphology when
cultured in CSF-1. This process is abrogated in FDC-P1 cells
transfected with the CSF-1 receptor with a tyrosine to phenyalanine
substitution at position 807 (FD/807), suggesting that a molecular
interaction critical to differentiation signaling is lost (Bourette,
R. P., Myles, G. M., Carlberg, K., Chen, A. R., and
Rohrschneider, L. R. (1995) Cell Growth Differ. 6, 631-645). A detailed examination of lysates of CSF-1-treated FD/807
cells by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE)
revealed a number of proteins whose degree of tyrosine phosphorylation
was modulated by the Y807F mutation. Included in this category were
three phosphorylated proteins that co-migrated with
p46/52Shc. Immunoprecipitation, Western blotting, and
in vitro binding studies suggest that they are indeed
p46/52Shc. A key regulator of differentiation in a number
of cell systems, ERK was observed to exhibit an activity that
correlated with the relative degree of differentiation induced by CSF-1
in the two cell types. Transfection of cells with a
non-tyrosine-phosphorylatable form of p46/52Shc prevented
the normally observed CSF-1-mediated macrophage differentiation as
determined by adoption of macrophage-like morphology and expression of
the monocyte/macrophage lineage cell surface marker, Mac-1. These
results are the first to suggest that p46/52Shc may play a
role in CSF-1-induced macrophage differentiation. Additionally, a
number of proteins were identified by two-dimensional SDS-PAGE whose
degree of tyrosine phosphorylation is also modulated by the Y807F
substitution. This group of molecules may contain novel signaling
molecules important in macrophage differentiation.
CSF-11 (or M-CSF) acts
to regulate the proliferation, differentiation, activation, and
survival of cells of the macrophage lineage (reviewed in Ref. 1). CSF-1
binds to the class IV cytokine receptor family member, the CSF-1R,
which is the product of the c-fms proto-oncogene (2). Upon
ligand binding the CSF-1R undergoes dimerization and subsequent auto-
and trans-phosphorylation (3-5). The phosphorylation of the CSF-1R is
thought to create docking sites for downstream signal transduction
molecules containing SH2 domains (6-8). Such interactions are thought
to mediate both the formation and also the binding, directly or
indirectly, to the phosphorylated CSF-1R of signaling molecule
complexes containing Grb2, Sos, p150, PI 3-kinase and Shc (9-11),
p125FAK (12), SHP-1, and c-Cbl (13) and Stats 1,3, 5a, and
5b (14, 15).
The signaling events that mediate either proliferation or
differentiation in cells of the macrophage lineage are poorly
understood. Previously, the function of the CSF-1R in growth and
differentiation has been examined by transfecting the CSF-1R into the
murine hemopoietic FDC-P1 cells (FD) (16). These cells became
responsive to CSF-1 and underwent changes in morphology suggestive of
macrophage differentiation (17). Single and multiple substitutions of
tyrosines for phenylalanines in the CSF-1R at positions 697 (FD/697),
706 (FD/706), and 721 (FD/721) were not observed to affect
proliferation or differentiation (16). This lack of an effect on
proliferation or differentiation occurred despite the inhibitory
effects of the Y697F mutation on Grb-2 binding to the CSF-1R (18), the
Y721F mutation on PI 3-kinase activation (9, 19), and the Y706F
mutation on Stat-1 activation (14). The Y807F substitution was observed
to impair phospholipase C In contrast to FD/807 cells where there is a defect in differentiation,
fibroblasts transfected with the human CSF-1RY809F, the human
equivalent of the Y807F mutation, were found to be incapable of
proliferation (3). No differentiation phenotype exists for the
CSF-1R-transfected fibroblasts, and the Y809F substitution did not
modulate the accumulation of mRNA of two transcription factors,
c-Fos and JunB (21). In other cell systems, modulation of
c-fos mRNA levels has been associated with macrophage
lineage differentiation (22, 23) as well as with osteoclast
differentiation (24). Taken together, these reports suggest that the
murine Tyr807 (or human Tyr809) may be
involved in signaling pathway(s) that, depending on the cell system,
are involved in either differentiation and/or proliferation.
The p46/52Shc adaptor protein is widely expressed and is
composed of a C-terminal SH2 domain and an N-terminal protein tyrosine binding domain (25). Treatment of human monocytes with CSF-1 results in
the tyrosine phosphorylation of p46/52Shc and its
association with Grb2 (12). A complex of Grb2 and p46/52Shc
was found to co-immunoprecipitate CSF-1R following treatment of FD/WT
cells with CSF-1 (9). In other cell systems, p46/52Shc also
forms complexes with p125FAK and PI 3-kinase (11, 12).
Given the recurring observation of Grb-2 and p46/52Shc
association in a range of cell types, it is interesting that the
CSF-1R-mediated tyrosine phosphorylation of p46/52Shc is
unaffected by the Y697F substitution at the Grb2 binding site (9). This
implies that there may be a CSF-1R site(s) involved in
p46/52Shc phosphorylation that has yet to be identified.
While the Tyr to Phe substitution at position 807 of the CSF-1R in FD
cells leads to a loss of the ability to differentiate into
macrophage-like cells in response to CSF-1, the molecular basis for
this is yet to be determined. Examination of the tyrosine phosphorylation profile of Nonidet P-40 lysate proteins in
CSF-1-treated FD/WT and FD/807 cells by one-dimensional SDS-PAGE has
failed to reveal any differences (16). We used the higher resolving power of two-dimensional SDS-PAGE to examine the phosphotyrosine patterns of CSF-1-treated FD/WT and FD/807 cells to identify
tyrosine-phosphorylated proteins that may be involved in macrophage differentiation.
Chemicals and Reagents--
DMEM, streptomycin, and penicillin
were purchased from ICN-Flow Laboratories, Sydney, New South Wales,
Australia), and fetal bovine serum and heat-inactivated new born calf
serum from Commonwealth Serum Laboratories (Parkville, Victoria,
Australia). Other reagents were obtained as follows: recombinant human
CSF-1 from Chiron Corp., recombinant murine IL-3 from Biotechnology
Australia Pty. Ltd. (Roseville, Sydney, Australia). The following
primary antibodies were used: mouse monoclonal 4G10-conjugated to HRPO
(4G10-HRPO), and p85 subunit of PI 3-kinase, biotin-conjugated 4G10
(Upstate Biotechnology, Inc.), phospho-ERK rabbit polyclonal
(New England Biolabs Inc.) anti-Erk-2 goat polyclonal and mouse
monoclonal anti-p52Shc antibodies (Santa Cruz
Biotechnology). The following secondary antibodies were used:
HRPO-conjugated, affinity-purified, swine immunoglobulins to rabbit
immunoglobulins (Dako), HRPO-conjugated rabbit immunoglobulins
to mouse immunoglobulins (Dako), and HRPO-conjugated streptavidin
(Zymed Laboratories Inc.).
Cells--
The murine hemopoietic FD/WT and FD/807 cell lines
were obtained from Drs. K Carlberg and L. Rohrschneider (Department of Pathology, University of Washington, Seattle) and have been described previously (16).
M1 murine myeloid cells were transfected with wild type
c-fms (M1/WT) or c-fms with the tyrosine mutated
to phenylalanine (M1/807). Briefly, wild type c-fms
(obtained from D. Hume, University of Queensland, Brisbane, Australia)
and a Y807F c-fms mutant were cloned into the pEF-BOS
expression vector (26). Mutation of tyrosine 807 to phenylalanine was
generated by PCR using mutagenic primers that substituted the codon of
Y807F from TTC to TAC. All PCR fragments were generated using the
Expand High Fidelity PCR system (Roche Molecular Biochemicals),
and mutant clones were fully sequenced prior to experimental use. The
PCR fragments containing the mutations were then cloned into the wild
type c-fms gene. The expression vectors were introduced into
M1 cells as described previously (27). The capacity of M1/WT cells to
adopt a macrophage morphology in response to treatment with CSF-1 was
examined as before (27) and confirmed to be similar to that of FD/WT
cells (16). Similarly, the inability of M1/807 cells to adopt a
macrophage morphology in response to treatment with CSF-1 was examined
and found to be similar to that of FD/807 cells (16).
Murine DNA constructs encoding Myc-tagged forms of either wild type
p52Shc or a non-tyrosine-phosphorylatable form of
p52Shc with Tyr to Phe substitutions at positions 239, 240, and 317 were a kind gift from Dr. Benjamin Margolis (28). They were transfected using the FuGENETM 6 transfection
reagent (Roche Molecular Biochemicals) as per the
manufacturer's instructions. Briefly, 3 µl of FuGENETM 6 transfection reagent was diluted in 100 µl of serum-free DMEM for 10 min. To this, 1 µg of cDNA was added to achieve a final concentration of 0.1 µg/ml. After incubation at room temperature for
15 min, this mixture was added dropwise to 2 ml of M1/WT cells at
1 × 105/ml. Cells were then cultured for 14 days in
DMEM with 10% (v/v) heat-inactivated new born calf serum containing
puromycin (2 µg/ml). Selected cells expressing either the wild type
p52Shc (M1/WTWTShc) or a
non-tyrosine-phosphorylatable form of p52Shc with Tyr to
Phe substitutions at positions 239, 240, and 317 (M1/WTF3Shc) were isolated. Pooled transformants were used
in all experiments.
Cell Culture--
FD and M1 cell lines were grown,
growth-arrested, and treated with either CSF-1 or IL-3 as described
previously (16, 27).
Morphologic Analysis of M1 Cells Macrophage
Differentiation--
5 × 104 cells were cytospun and
subjected to May Grunwald Giemsa staining as described
previously (27).
Nonidet P-40 Lysate Preparation--
Pelleted cells were lysed
in cell lysis buffer (50 mM Hepes, pH 7.4, containing 0.5%
(v/v) Nonidet P-40, 100 mM NaF, 10 mM sodium
pyrophosphate, 5 mM EDTA, 1 mM EGTA, 10% (v/v)
glycerol, 200 µM sodium orthovanadate, 10 µg/ml
leupeptin, pepstatin, and Pefabloc). The samples were
centrifuged at 1300 rpm, 5 min in a Heraeus Biofuge 13 (Radiometer
Pacific) and the pellets discarded.
Two-dimensional SDS-PAGE--
Immobiline dry strips (pH 3-10;
linear; Amersham Pharmacia Biotech) were rehydrated overnight (8 M urea, 0.5% (v/v) Triton X-100, 0.5% (v/v) Pharmalytes
3-10, 0.01 M dithiothrietol). 100 µg of cell lysate,
diluted 1:4 in sample solution (9 M urea, 0.06 M dithiothreitol, 2% (v/v) Pharmalytes 3-10, 0.5% (v/v)
Triton X-100), was applied to the rehydrated strips and run on a
horizontal Multiphor II Electrophoresis unit (Amersham Pharmacia
Biotech) at 20 °C for a total of 100,000 Vh. The second
dimension separation was performed as described previously (29).
Western Blot Analysis of SDS-PAGE Gels--
Gels were analyzed
by Western blotting as before (29). In some cases the blots were
stripped of antibodies by extensive washing in stripping buffer
containing 62.5 mM Tris/HCl, 0.1 M Immunoprecipitation--
Lysates were precleared by incubating
500 µg of Nonidet P-40 lysates, from appropriately treated cells,
with 50 µl of a 25% (v/v) slurry of protein A-Sepharose 4B (Amersham
Pharmacia Biotech) with rotation for 30 min at 4 °C. The samples
were then centrifuged at 13,000 rpm for 5 min and the pellets
discarded. They were then incubated with 1 µg of 4G10 antibodies
(Upstate Biotechnology, Inc.) overnight with rotation at 4 °C. Fifty
microliters of a 25% (v/v) slurry of protein A-Sepharose 4B was added
and the mixture incubated for 1 h at 4 °C. The beads were
pelleted and washed three times with cell lysis buffer. Samples were
boiled for 5 min in SDS-PAGE sample buffer, subjected to
one-dimensional SDS-PAGE on 10% SDS-PAGE gels, and transferred to
nitrocellulose for Western blot analysis.
Protein Staining--
Membranes were stained with Colloidal Gold
Total Protein Stain (Bio-Rad). SDS-PAGE gels were stained
overnight with 0.1% (w/v) Coomassie Blue (PhastGel Blue R350
Stain; Amersham Pharmacia Biotech) in 25% (v/v) methanol with
10% (v/v) acetic acid, destained in 25% (v/v) methanol with 10%
(v/v) acetic acid, and dried.
Quantitative Determination of Phosphorylation--
ECL
autoradiographs were scanned using a computerized laser
densitometer (Molecular Dynamics, CA) and the images
analyzed using Image Quant software (Molecular Dynamics).
Proteomic Analysis of Tyrosine Phosphorylation in CSF-1-stimulated
FD/WT and FD/807 Cells--
Nonidet P-40 lysates of unstimulated and
CSF-1-stimulated (5 min) FD/WT and FD/807 cells were resolved by
two-dimensional SDS-PAGE, transferred to nitrocellulose, probed with
biotinylated 4G10 antibodies, and developed with
streptavidin-conjugated HRPO. Cursory examination of the
two-dimensional SDS-PAGE Western blots revealed dramatic up-regulation
of tyrosine phosphorylation of many proteins following CSF-1 addition
(Fig. 1, panels A-C).
Coomassie Blue staining was used as an indication of the consistency of the loading and resolution (Fig. 1, panels D and
E).
Closer examination of the two-dimensional SDS-PAGE phosphoprotein
profiles of FD/WT and FD/807 cells revealed both common and distinct
tyrosine phosphorylation of proteins by CSF-1 (Fig. 1, panels
A-C). Major phosphoproteins were classified on the basis of their
molecular weights and pI. For example, a protein with a molecular
mass of 74 kDa and a pI of 6.0 was designated 74/6.0 (Fig. 1,
panels A-C). Each of the phosphoproteins or "spots"
indicated in this fashion in Fig. 1 (panels A-C) was
subjected to densitometric analysis and quantification (Fig.
2).
The CSF-1-induced tyrosine phosphorylated proteins were classified as
belonging to one of two groups: those whose tyrosine phosphorylation
was consistently "unaffected" by the Y807F substitution and those
whose tyrosine phosphorylation was consistently "affected." These
phosphorylation patterns are depicted qualitatively by a representative
series of 4G10 antibody Western blots in Fig. 1, panels
A-C. In each case, we have attempted to show as complete a
two-dimensional SDS-PAGE gel as possible, focusing on the region of
greatest interest, namely that containing the highest number of clearly
identifiable affected and unaffected phosphoproteins. The
average intensities, from five different experiments, are represented
quantitatively in Fig. 2. As an example, spots 74/6.0, 67/7.8, 66/6.5,
64/5.8, 62/6.7, 62/5.9, 60/7.0, 27/7.4, and 27/6.7 belong to the
unaffected group (Fig. 1, panels A-C; Fig. 2, panels A-I, respectively). The affected group was divided into those spots whose CSF-1-dependent phosphorylation state was
"diminished" by the Y807F substitution or, alternatively, those
whose CSF-1-dependent tyrosine phosphorylation was
"elevated" by the Y807F substitution. The diminished group included
tyrosine-phosphorylated proteins, 74/5.7, 66/6.1, 55/7.7, 52/6.9,
52/6.7, 46/6.3, 42/6.7, 37/6.7, 27/6.1, and 24/6.1 (Fig. 1,
panels A-C; Fig. 2, panels J-S, respectively); the elevated group of molecules included the 100/8.5 and 100/8.0 (Fig.
1, panels A-C; Fig. 2, panels T and
U, respectively). Since the tyrosine phosphorylation status
of the affected group of proteins correlates with a change in the
capacity of the FD cells to differentiate in the presence of CSF-1, it
is possible that proteins in this group may play a role in the
differentiation process.
Differential Tyrosine Phosphorylation of
p46/52Shc--
The phosphorylation of spots 52/6.9,
52/6.7, and 46/6.3 was dramatically abrogated by the Y807F substitution
(Figs. 1, panels A-C; Fig. 2, panels M-O,
respectively). Their molecular weights suggested that these proteins
might be p46/52Shc; however no such
Y807F-dependent modulation in the tyrosine phosphorylation status of p46/52Shc was reported before in CSF-1-treated FD
cells (9). We, therefore, addressed this question further. The
two-dimensional SDS-PAGE blots were stripped and re-probed with
anti-p46/52Shc antibodies. The identification of the
52/6.9, 52/6.7, and 46/6.3 proteins by both anti-p46/52Shc
and 4G10 antibodies suggested that they were p46/52Shc and
that the CSF-1-dependent tyrosine phosphorylation evident in FD/WT cells was abrogated in FD/807 cells (Fig.
3). In addition to phosphoproteins
52/6.9, 52/6.7, and 46/6.3, which were all identified by
p46/52Shc-reactive antibodies, a number of
p46/52Shc proteins (Fig. 3, panels
D--F) did not appear to co-migrate with CSF-1-sensitive phosphoproteins (Fig. 3, panel B),
suggesting that not all the p46/52Shc isoforms are
phosphorylated. Similarly, the phosphorylation of the 52/6.9, 52/6.7,
and 46/6.3 phosphoprotein p46/52Shc isoforms would appear
to alter their pI mobility, probably by the addition of charged
phosphate species. It is interesting to note that the pI distribution
of p46/52Shc isoforms in unstimulated FD/WT and
CSF-1-treated FD/807 cells are nearly identical, possibly corresponding
to an "unphosphorylated" pattern and are different to that of
CSF-1-treated FD/WT cells, which may represent a "phosphorylated"
pattern. The identity of these phosphoproteins was further examined by
amino acid sequence determination as described previously (31).
Consistent with data presented above, phosphoproteins 52/6.9, 52/6.7,
and 46/6.3 were found to be the p46/52Shc adapter protein
(data not shown).
Given this result, we sought to determine directly whether
p52Shc was tyrosine-phosphorylated to different degrees in
FD/WT and FD/807 cells treated with CSF-1. Nonidet P-40 lysates from
untreated and CSF-1-stimulated FD/WT and FD/807 cells were either
immunoprecipitated with 4G10 or anti-p52Shc antibodies,
resolved by one-dimensional SDS-PAGE, transferred to nitrocellulose,
and probed with the converse antibody, that is, either
anti-p52Shc or 4G10 (Fig. 4).
Each approach revealed that the maximal CSF-1-stimulated tyrosine
phosphorylation of p52Shc protein was dependent on the
tyrosine at position 807 (Fig. 4). To determine whether this
observation was simply cell type-specific we repeated the above
examination with another murine myeloid cell system, namely the M1/WT
and differentiation-deficient M1/807 cells (see "Experimental
Procedures"). Again, the Y807F mutation in the CSF-1R resulted in a
dramatically reduced tyrosine phosphorylation of p52Shc in
response to CSF-1 treatment (Fig. 4). The combination of these approaches suggests that the Tyr at position 807 plays a part in
p52Shc tyrosine phosphorylation rather than simply in the
association, direct or otherwise, with tyrosine-phosphorylated
proteins.
As a check on the specificity of our observations for
p52Shc, we examined, for each cell type, the tyrosine
phosphorylation of the p85 subunit of PI 3-kinase using the same
approach and in this case found it to be CSF-1-dependent
and Y807F mutation-independent (Fig. 4). These findings suggest that
p52Shc may have a role in CSF-1-mediated macrophage
differentiation in both of these cell systems.
The Y807F Substitution Prevents Maximal CSF-1-mediated ERK
Activation--
ERK family members can play a role in cellular
differentiation (32-37). The loss of p46/52Shc binding to
the epidermal growth factor receptor family member ErbB3 has been shown
to result in the loss of ERK activation (38). Accordingly, given the
loss of p52Shc tyrosine phosphorylation following the Y807F
substitution, we sought to examine the consequent effect of this
mutation on ERK activity. The phospho-ERK immunoreactivity of M1/WT and
M1/807 cells treated with CSF-1 for 4, 10, and 30 min was examined. The antibody binds specifically to the active forms of ERK and revealed that the Y807F mutation dramatically abrogated ERK activation (Fig.
5). Interestingly, the specific MEK
inhibitor, PD98059, inhibits both CSF-1-induced ERK activation and
differentiation of M1 cells.2
Therefore, it could be that, in a fashion analogous to the ErbB3 system
(35), ERK activation and differentiation are compromised through the
loss of p52Shc activation following the Y807F mutation.
Expression of a Non-tyrosine-phosphorylatable Form of
p46/52Shc Prevents CSF-1-mediated Macrophage
Differentiation--
As the tyrosine phosphorylation of
p46/52Shc was abrogated by the Tyr to Phe substitution at
position 807 of the CSF-1R, which also dramatically affects macrophage
differentiation, we sought to determine whether the prevention of
p46/52Shc tyrosine phosphorylation could similarly abrogate
macrophage differentiation. Myc-tagged forms of either wild type
p52Shc or a non-tyrosine-phosphorylatable form of
p52Shc with Tyr to Phe substitutions at positions 239, 240, and 317 were transfected into M1/WT cells to produce
M1/WTWTShc or M1/WTF3Shc cells, respectively.
Quiescent M1/WTWTShc and M1/WTF3Shc cells were
either left untreated or stimulated with CSF-1 and Nonidet P-40 lysates
prepared. These were subjected to Western blot analysis using
p46/52Shc-, Myc-, or phosphotyrosine-reactive antibodies
following immunoprecipitation with either p46/52Shc or
Myc-reactive antibodies (Fig. 6).
p46/52Shc underwent CSF-1-induced tyrosine phosphorylation
in M1/WTWTShc, but not M1/WTF3Shc, cells
consistent with previous reports (Fig. 6; Ref. 28). Interestingly, the
endogenous p46Shc and p52 Shc forms failed to
be tyrosine-phosphorylated in M1/WTF3Shc cells in response
to CSF-1 treatment, suggesting a dominant negative effect.
The functional significance of the loss of p46/52Shc
tyrosine phosphorylation in M1/807 and M1/WTF3Shc cells was
next investigated. M1/WT, M1/807, M1/WTWTShc, and
M1/WTF3Shc cells were cultured in DMEM containing either
HINBCS alone or with CSF-1 for 4 days. Consistent with previous reports
in analogous FDC-P1 cells transfected with the CSF-1R (16), the wild
type CSF-1R permitted CSF-1-dependent macrophage
differentiation in M1 cells as determined by cell spreading, adhesion,
blebbing, and loss of blast-like morphology (Fig.
7A). Similarly, these cell
lines were assessed by flow cytometry for their capacity to express the
monocyte/macrophage lineage marker, Mac-1, in response to CSF-1
treatment (Fig. 7B). The observation that both M1/807 and
M1/WTF3Shc cells were incapable of CSF-1-mediated
differentiation (Fig. 7, A and B) suggests that
the Tyr807 of the CSF-1R-dependent macrophage
differentiation process in this system is mediated through the tyrosine
phosphorylation of p46/52Shc.
The association of the CSF-1R with p46/52Shc following
specific ligand stimulation is well established (9, 10). We, therefore, sought to determine the effect of the F3 mutation of Shc, given the
impairment of CSF-1-mediated macrophage differentiation, on the
association of p46/52Shc and the CSF-1R.
p46/52Shc was immunoprecipitated from CSF-1-treated M1/WT,
M1/807, M1/WTWTShc, and M1/WTF3Shc cell
lysates, resolved by one-dimensional SDS-PAGE, transferred to
nitrocellulose, and probed with HRPO-conjugated 4G10 antibodies (Fig.
8, panel A).
p46/52Shc was observed to be phosphorylated maximally at 1 and 5 min post-stimulation in M1/WT and M1/WTWTShc cells
and this diminished progressively at 15 and 30 min, respectively, in a
manner consistent with previous reports (39). This phosphorylation was
dramatically reduced in M1/807 cells as described earlier (see Fig. 4).
Interestingly, M1/WTF3Shc cells contained significantly
higher levels of phosphorylated p46/52Shc relative to
M1/807 cells, presumably corresponding to endogenous p46/52Shc, but this was dramatically reduced relative to
that seen in M1/WT and M1/WTWTShc cells (Fig. 8,
panel A).
Stripping and re-probing of the membrane depicted in Fig. 8,
panel A, with anti-c-Fms antibodies was performed (Fig. 8,
panel B). The expected CSF-1-dependent
association of p46/52Shc and c-Fms was observed in M1/WT
and M1/WTWTShc cells. Of significant interest was the
reduced association of p46/52Shc and c-Fms in both M1/807
and M1/WTF3Shc cells. This is, presumably, due to the
inability of a nonphosphorylated p46/52Shc to bind to
either the CSF-1R or other proteins that are themselves associated with
the CSF-1R. As M1/WTF3Shc cells contain an otherwise normal
CSF-1R, unlike that expressed in M1/807 cells, this lack of association
is unrelated to the activation of the CSF-1R itself.
The molecular basis for Y807F-dependent loss of
macrophage differentiation in FD/807 cells was examined.
One-dimensional SDS-PAGE and Western blot analysis of tyrosine
phosphorylated proteins in Nonidet P-40 lysates revealed an increased
and largely conserved pattern of tyrosine-phosphorylated proteins in
FD/WT and FD/807 cells following CSF-1 treatment (16). In contrast, a
more detailed examination by two-dimensional SDS-PAGE revealed dramatic
differences in the tyrosine phosphorylation patterns of FD/WT and
FD/807 cells treated with CSF-1. A number of tyrosine-phosphorylated
proteins were observed to be modulated by treatment with CSF-1,
including the 52/6.9, 52/6.7, and 46/6.3 proteins that were also
identified by p46/52Shc-reactive antibodies and whose
tyrosine phosphorylation was down-regulated by the Y807F mutation.
Subsequent immunoprecipitation, Western blotting, and in
vitro binding studies supported the view that the 52/6.9, 52/6.7,
and 46/6.3 proteins were p46/52Shc. The transfection of a
non-tyrosine-phosphorylatable form of p52Shc with Tyr to
Phe substitutions at positions 239, 240, and 317 conferred a
differentiation-incapable phenotype to M1/WT cells that closely
reflected the differentiation-deficient behavior of M1/807 cells. These
results are the first to suggest that p46/52Shc, and more
specifically its tyrosine phosphorylation, may play a role in
macrophage differentiation.
These findings are in contrast to a previous report showing the
tyrosine phosphorylation of p52Shc was unaffected by the
Y807F in FD cells (9). This prior study reported that
immunoprecipitates of p52Shc from FD/WT and FD/807 cells
exhibited similar 4G10 antibody immunoreactivities (9). These
discordant findings are difficult to reconcile. One possibility is that
in the earlier study (9), the comparative analysis of
p52Shc phosphorylation in FD/WT and FD/807 cells did not
include an unstimulated control group for either of these cells.
Rather, parental FD cells were used, which cannot respond to CSF-1. As in our studies above, the inclusion of unstimulated FD/WT and FD/807
cells as controls may have revealed the effects of the Y807F mutation
on the phosphorylation state of p52Shc. In addition, we
have used complementary and reverse combinations of Western blot and
immunoprecipitation analysis with anti-p52Shc and 4G10
antibodies to observe that p52Shc tyrosine phosphorylation
is diminished in FD/807 cells compared with FD/WT cells in response to
CSF-1. As an added check on our findings, the analysis was repeated in
another myeloid cell line, namely M1 cells, to exclude the possibility
of a cell line-specific artifact. More importantly, the ability of a
non-tyrosine-phosphorylatable form of p52Shc to abrogate
CSF-1-dependent monocyte/macrophage differentiation in
otherwise competent M1/WT cells provides the strongest evidence that
the tyrosine phosphorylation of p52Shc is important in
these processes.
There are other reports in growth factor/receptor systems that suggest
a role for p46/52Shc in cellular differentiation (37, 38,
40-46). The c-Mpl ligand, MGDF (or TPO), induces basophil-like
differentiation in 32D cells transfected with murine c-Mpl (37, 38).
This differentiation has been shown to involve p46/52Shc
phosphorylation (38). The Tyr to Phe substitution at position 559 on
the c-Mpl receptor in WEHI3D-B+ cells abrogates both
differentiation and p46/52Shc phosphorylation (40), further
suggesting a role for p46/52Shc in hemopoietic lineage
differentiation. Indeed, a mutant form of p46/52Shc lacking
a phosphotyrosine interaction domain blocks c-Mpl-mediated granulocytic
differentiation (41). It has been shown in neuronal cells that
differentiation signals are controlled by the NGF Receptor/Trk binding
sites for p46/52Shc and phospholipase C- The inability of FD/807 cells to differentiate in response to CSF-1
suggests that a molecular interaction critical to differentiation signaling is lost. Our work has revealed that p46/52Shc may
be part of the CSF-1-mediated differentiation signaling pathway. The
mechanism by which the loss of p46/52Shc activation may
lead to an abrogation of differentiation remains unclear. A mutation of
the p46/52Shc binding site on epidermal growth factor
receptor family member ErbB3 leads to the loss of heregulin activation
of MAP kinases (35). The activation of ERKs is considered important in
cellular differentiation signaling in a number of cell systems
(32-34). For example, retinoic acid-induced myeloid differentiation of HL-60 cells involves the actions of MEK and ERKs (36). Of greater direct relevance is the report that ERK activation is impaired in
Tyr809 to Phe mutant forms of the CSF-1R-transfected
NIH-3T3 cells (46). Consistent with these findings in other cell
systems is our observation that differentiation-deficient FD/807 cells
exhibit impaired CSF-1-dependent Erk1/2 activation, and
this may be related to or caused by the loss of p52Shc phosphorylation.
Another possible role for p46/52Shc in macrophage
differentiation may relate to its reported association with
cytoskeletal proteins (15, 47). p46/52Shc has been shown to
co-immunoprecipitate with actin following growth factor stimulation
(47). A number of workers have demonstrated the importance of actin
polymerization in cellular differentiation (47-54), an example of
which is that the specific actin polymerization inhibitor,
cytochalasin, inhibits cellular differentiation (48, 50, 52, 53). Of
more direct interest is the report that cytochalasins B and D are
potent inhibitors of key events associated with phorbol ester-mediated
HL-60 macrophage differentiation (54). Yeung et al. (15)
have reported that a CSF-1R-containing complex is formed following
CSF-1 treatment of BAC1.2F5 cells and that this complex contains a
number of structural cytoskeletal proteins, including actin as well as
p46/52Shc (15). These reports provide evidence for a
possible role for p46/52Shc in macrophage actin
cytoskeleton re-organization.
Of additional importance from our findings is the identification of at
least 10 proteins by two-dimensional SDS-PAGE whose tyrosine
phosphorylation is modulated by the Y807F substitution. Among these
proteins are 55/7.7 and 37/6.7 whose CSF-1-dependent phosphorylation is down-regulated by the Y807F substitution. These molecular weights are suggestive of the pp57 and pp37 that have been
reported to be bound to the CSF-1R signaling complex (15). In
particular, the pp37 is reported to correspond to a novel phosphatase substrate that may play a role in cytoskeletal regulation (15). This
report would be consistent with the view that the pp37 may be the
37/6.7 and that these molecules may in turn be involved in
CSF-1-mediated differentiation signaling in myeloid cell lines. The
remaining group of Y807F substitution-dependent molecules may contain novel and as yet uncharacterized signaling molecules that
may be involved in macrophage differentiation. The purification, sequence determination, and characterization of these molecules may
provide important insights into the currently poorly understood field
of hemopoietic differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-2 activation, which appears to act
with PI 3-kinase during CSF-1-induced FD/WT differentiation to
macrophages (20). Nevertheless, the exact signaling mechanism(s) that
are abrogated by the Y807F mutation and critical to differentiation are
poorly understood (16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 2% SDS, blocked with a 3% (w/v) bovine serum albumin, 1% (w/v) ovalbumin solution for 1 h at room temperature, then re-probed with different antibodies as described elsewhere (30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (63K):
[in a new window]
Fig. 1.
Two-dimensional SDS-PAGE analysis of
tyrosine-phosphorylated proteins in Nonidet P-40 lysates of FD/WT and
FD/807 cells treated with CSF-1. Cells were growth-arrested in
medium containing 1% serum for 18 h (unstimulated) and
then treated with CSF-1 (5000 units/ml) for 5 min (+CSF-1).
Nonidet P-40 lysates were standardized for protein and resolved by
isoelectric focusing pH 3-10 and SDS-PAGE (see "Experimental
Procedures"). Gels were either transferred to nitrocellulose and
probed with 4G10 antibodies (panels A-C) or stained for
protein loading with Coomassie blue (panels
D-F). The location of phosphoproteins of interest
(see "Results"), their pI and molecular masses are indicated with
arrows.
View larger version (35K):
[in a new window]
Fig. 2.
Densitometric analysis of protein tyrosine
phosphorylation. The relative intensity of the tyrosine
phosphorylation is summarized for each of the selected phosphoproteins
(Fig. 2) in FD/WT cells (WT) and FD/807 cells
(807), which were either untreated (U/S) or
treated with CSF-1 (5000 units/ml) for 5 min (+CSF-1). The
relative intensities are the averages of between three and five
different experiments and the S.D. values are indicated.
Phosphoproteins are grouped on the basis of consistent and significant
sensitivity to the Y807F substitution: when phosphorylation is
relatively unaffected by the Y807F substitution (panels
A-I), when phosphorylation is diminished by the Y807F
substitution (panels J-S), and when phosphorylation is
enhanced by the Y807F substitution (panels T and
U).
View larger version (38K):
[in a new window]
Fig. 3.
Two-dimensional SDS-PAGE examination of
tyrosine phosphorylation of p46/52Shc in Nonidet P-40
lysates of FD/WT and FD/807 cells treated with CSF-1. Cells were
growth-arrested in medium containing 1% serum for 18 h
(U/S) and then treated with CSF-1 (5000 units/ml) for 5 min
(+CSF-1). Nonidet P-40 lysates were standardized for protein
and resolved by isoelectric focusing pH 3-10 and 10%
SDS-PAGE. Gels were transferred to nitrocellulose and either probed
with 4G10 antibodies (panels A-C) or with
anti-p46/52Shc antibodies (panels
D-F). The locations of phosphoproteins 52/6.9,
52/6.7, and 46/6.3 are indicated with arrows.
View larger version (26K):
[in a new window]
Fig. 4.
Immunoprecipitation analysis of the tyrosine
phosphorylation state of p52Shcc in
Nonidet P-40 lysates of FD/WT, FD/807, M1/WT, and M1/807 cells treated
with CSF-1. Cells were growth-arrested in medium containing 1%
serum for 18 h ( ) and then treated with CSF-1 (5000 units/ml) for 5 min (+). Nonidet P-40 lysates were
standardized for protein and immunoprecipitated with 4G10 antibodies,
resolved by one-dimensional SDS-PAGE, transferred to nitrocellulose,
and probed with either anti-p52Shc or anti-PI 3-kinase p85
subunit antibodies. p52Shc, immunoglobulin heavy chain, and
the p85 subunit of PI 3-kinase are indicated with
arrows.
View larger version (19K):
[in a new window]
Fig. 5.
Analysis of the activation of ERK in Nonidet
P-40 lysates of M1/WT and M1/807 cells treated with CSF-1. Cells
were growth-arrested in medium containing 1% serum for 18 h
( ) and then treated with CSF-1 (5000 units/ml) for 4, 10, or 30 min; Nonidet P-40 lysates were standardized for protein, resolved
by 10% SDS-PAGE, transferred to nitrocellulose, and probed with
anti-phospho-ERK (pERK) antibodies. Subsequently, ERK-2
loading was determined by reprobing with anti-ERK-2 antibodies.
View larger version (45K):
[in a new window]
Fig. 6.
Analysis of the tyrosine phosphorylation
state of p52Shc in Nonidet P-40 lysates of
M1/WTWTShc and M1/WTF3Shc cells treated with
CSF-1. Cells were growth-arrested in medium containing 1% serum
for 18 h ( ) and then treated with CSF-1 (5000 units/ml) for 5 min (+). Nonidet P-40 lysates were
standardized for protein and immunoprecipitated with either Shc- or
Myc-reactive antibodies, resolved by one-dimensional SDS-PAGE,
transferred to nitrocellulose and probed with either Shc,
phosphotyrosine, or Myc-reactive antibodies.
View larger version (61K):
[in a new window]
Fig. 7.
Examination of the criticality of
p52Shc tyrosine phosphorylation on
the capacity of M1/WT cells to differentiate along the
monocyte/macrophage lineage in response to CSF-1. M1/WT,
M1/807, M1/WTWTShc, and M1/WTF3Shc cells were
cultures in DMEM with either heat-inactivated new born calf serum alone
or with CSF-1 (5000 IU/ml) 4 days. A, cells were cytospun
and stained with May Grunwald Giemsa (see "Experimental
Procedures"). B, cells were stained with either
Mac-1-reactive or isotype control antibodies and assessed for Mac-1
expression by flow cytometry (27). These experiments were repeated
three times.
View larger version (43K):
[in a new window]
Fig. 8.
Analysis of the tyrosine phosphorylation
state and association of p52Shc and CSF-1R in Nonidet P-40
lysates of M1/WT, M1/807, M1/WTWTShc, and
M1/WTF3Shc cells treated with CSF-1. Cells were
growth-arrested in medium containing 1% serum for 18 h and then
treated with CSF-1 (5000 units/ml) for 0, 1, 5, 15, and 30 min and
Nonidet P-40 lysates prepared as described under "Experimental
Procedures." Panel A, p46/52Shc
immunoprecipitates were resolved by 10% one-dimensional SDS-PAGE gel,
transferred to nitrocellulose, and probed with 4G10 antibodies directly
conjugated with HRPO. p46/52Shc is indicated with
arrows. Panel B, the membrane described above was
stripped and reprobed with anti-c-Fms antibodies. c-Fms and IgG are
indicated with arrows.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(41, 43,
44). It is interesting to note that the tyrosine phosphorylation of
phospholipase C-
2 in CSF-1-treated FD/807 cells is diminished
(20) in a fashion that parallels the PC12 neuronal cell differentiation
model (43). The Y577F substitution in the GM-CSF receptor in
murine pro-B BA/F3 cells resulted in the loss of p52Shc
phosphorylation without affecting the activation of the
p21ras/Raf-1/MEK/MAPK cassette, the JAK/STAT pathway, and
proliferation, suggesting that p52Shc phosphorylation in
not involved in proliferation (45).
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ACKNOWLEDGEMENT |
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We thank Dr. Benjamin Margolis for the generous gift of the cDNAs encoding the WT and F3 forms of p52Shc.
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FOOTNOTES |
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* This work was supported by a Merk Medical School Grant and by grants from the National Health and Medical Research Council of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Arthritis and Inflammation Research Centre, University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville 3050, Victoria, Australia. Tel.: 613-8344-5478; Fax: 613-9347-1863; E-mail: xfc@unimelb.edu. au.
Recipient of the Viertel Senior Medical Research Fellowship.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M100213200
2 N. J. Wilson, V. Kanangasundarum, S. T. Moss, G. Whittey, J. A. Hamilton, and X. F. Csar, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: CSF-1, colony-stimulating factor-1; M-CSF, macrophage-CSF; CSF-1R, colony-stimulating factor-1 receptor; SH2, Src homology 2; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; HRPO, horseradish peroxidase; PI, phosphatidylinositol; PCR, polymerase chain reaction; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MAPK, mitogen-activated protein kinase; JAK, Janus kinase; STAT, signal transducers and activators of transcription.
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