From the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Stimulation of macrophages with
colony-stimulating factor-1 (CSF-1) results in the protein tyrosine
phosphorylation of the CSF-1 receptor (CSF-1R) and many other,
primarily cytosolic, proteins. Stimulation by CSF-1 at 4 °C was used
to facilitate the purification and identification of the proteins of
the cytosolic anti-phosphotyrosine (PY)-reactive fraction (PY-RF)
involved in downstream signaling pathways. Confocal microscopy revealed
that the PY proteins are in close proximity to the CSF-1R at the plasma
membrane. The
PY-RF contained pre-existing complexes of PY proteins
and non-PY proteins which generally increased in size and PY protein
content following CSF-1 stimulation. PY proteins identified by
microsequencing and Western blotting include Cbl, STAT3, STAT5a,
STAT5b, SHP-1, Shc, and two novel proteins pp57 and pp37. Other
proteins included cytoskeletal/contractile proteins (paxillin,
vimentin, elongation factor-1
, F-actin, tropomyosin, and myosin
regulatory light chain), Ras family signaling proteins (p85
(phosphoinositide 3-kinase), Vav, Ras-GTPase-activating protein SH3
domain-binding protein, and Grb2), DnaJ-like protein, and
glyceraldehyde-3-phosphate dehydrogenase. CSF-1 induced the de
novo recruitment of Cbl, STAT3, STAT5a, STAT5b, p85, SHP-1, Shc,
vimentin, and Grb2 to complexes and caused pre-existing complexes
involving Vav, elongation factor-1
, and F-actin to increase in size.
These studies indicate that CSF-1-induced protein tyrosine
phosphorylation is associated with the reorganization of complexes of
cytoskeletal, signaling, and other proteins that mediate
CSF-1-regulated motility and growth.
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INTRODUCTION |
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The survival, proliferation, and differentiation of mononuclear phagocytes are primarily regulated by colony-stimulating factor-1 (CSF-1)1 (reviewed in Refs. 1 and 2). CSF-1 also regulates macrophage morphology in vitro (3, 4) and in vivo (5-7). In vitro studies of regulation by CSF-1 have utilized the CSF-1-dependent mouse macrophage cell line BAC1.2F5 (8). BAC1.2F5 cells morphologically resemble primary macrophages and, like primary macrophages, require CSF-1 for both survival and proliferation (8). They also exhibit similar chemotactic and morphological responses to CSF-1 (4, 8, 9). When cultured overnight in the absence of CSF-1, BAC1.2F5 cells round up and become less mobile. In response to re-addition of CSF-1, these cells rapidly (5 min) spread and produce membrane ruffles and lamellipodia and regain their motility (4, 8, 9). The early responses to CSF-1 are associated with the reorganization of the actin cytoskeleton and the appearance of new actin cables (4, 10).
The effects of CSF-1 are mediated by a specific receptor tyrosine
kinase (11-13) encoded by the c-fms protooncogene (14). Incubation of BAC1.2F5 macrophages with CSF-1 causes dimerization, activation, and tyrosine phosphorylation of the CSF-1 receptor (CSF-1R), followed by the tyrosine phosphorylation of several primarily
cytoplasmic proteins (15-19). The identities of the majority of these
proteins are still unknown. At 37 °C, their appearance is maximally
stimulated by 60 s (18). They may represent direct substrates of
the activated CSF-1R or substrates of non-receptor tyrosine kinases
activated in the CSF-1 response, or alternatively, the increased
tyrosine phosphorylation may be due to CSF-1-induced inhibition of
protein tyrosine phosphatases. At 4 °C, these proteins are
tyrosine-phosphorylated with slower kinetics than at 37 °C (16, 18),
but the rates of phosphotyrosine dephosphorylation are differentially
lowered. Thus, CSF-1-stimulated tyrosine phosphorylation reaches a
relatively stable maximum between 90 and 180 min (16, 18), and a higher
level of tyrosine phosphorylation is obtained than the level achieved
by incubation for optimum periods at 37 °C. Analysis of the
stimulation of cells with CSF-1 at 4 °C has enabled differences in
the kinetics of the appearance of the tyrosine-phosphorylated proteins
to be resolved (16). Furthermore, by stimulating BAC1.2F5 cells with
CSF-1 at 4 °C for 2 h, it is possible to carry out large scale
stimulation and subcellular fractionation in order to isolate
sufficient amounts of anti-phosphotyrosine (PY)-reactive fraction
(PY-RF) proteins from the subcellular fractions for their
identification (20). Essentially the same pattern of protein tyrosine
phosphorylation is observed following CSF-1 stimulation at either
4 °C for 2 h or 37 °C for 1 min. However, at 4 °C,
subsequent events, e.g. Raf activation and phosphorylation
(21), are blocked. Thus, analysis of the stimulation of cells with
CSF-1 at 4 °C has been very useful in analyzing the very early
events in CSF-1 signal transduction.
As indicated above, with the exception of the CSF-1R, a 260-kDa protein
which we now know to be a multi-ubiquitinated form of the CSF-1R (22,
23) and a few additional proteins, the proteins tyrosine-phosphorylated
in response to CSF-1 are predominantly cytoplasmic (18). Hence, we have
focused on the identification of the PY proteins in the cytosolic
fraction. In our analysis of the cytosolic PY-RF from
CSF-1-stimulated and unstimulated BAC1.2F5 macrophages, the protein
tyrosine phosphatase SHP-1 was identified by microsequencing of the
purified protein (20) and the proto-oncogene product Cbl, by Western
blotting analysis (22). Tyrosine phosphorylation of both SHP-1 and Cbl
was stimulated by CSF-1. We and others have shown that Shc is
tyrosine-phosphorylated in macrophages (22) and myeloid cells (24) in
response to CSF-1, and others have shown that phospholipase C-
2
(25), p150Ship (26), Tyk2, STAT3, STAT5a and STAT5b (27,
28) are tyrosine-phosphorylated in myeloid cells in response to CSF-1.
In this report, we describe the use of anti-PY affinity chromatography
for the isolation of PY proteins and the non-PY proteins associated
with them from the cytosolic fraction of CSF-1-stimulated macrophages.
By using Western blotting with antibodies to known proteins and direct
purification and microsequencing approaches, we have identified a
number of PY and non-PY proteins in the cytosolic PY-RF, and we
demonstrate that these proteins are present in various complexes. We
also demonstrate that there is significant reorganization of proteins
in these complexes in response to CSF-1 stimulation.
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MATERIALS AND METHODS |
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Cell Culture, Protein Purification, and Sequencing--
BAC1.2F5
macrophages (8) were cultured in 100-mm tissue culture dishes and
stimulated with 13.2 nM CSF-1 (human recombinant macrophage
colony-stimulating factor, a gift from Chiron Corp.) at 4 °C in the
presence of 8 mM iodoacetic acid (IAA, Fluka) to increase
the yield of PY proteins, as described previously (15). Subcellular
fractionation of the cells after CSF-1 stimulation, purification of
PY-RF from the cytosol, trypsin digestion, and separation of tryptic
peptides for micro-sequencing were performed exactly as described
previously (20) with the exception that the C4 reverse
phase-high performance liquid chromatography (RP-HPLC) was performed at
55 °C and the C8 RP-HPLC at 40 °C. Briefly, cells were disrupted by Dounce homogenization, and subcellular fractions were
separated by differential centrifugation. The
PY-RF from the
100,000 × g post-microsomal cytosolic fraction was
prepared by affinity column chromatography using anti-PY antibody (Ab1) coupled to Sepharose 4B (Calbiochem). Proteins reactive to the anti-PY
antibody were eluted with 5 mM phenyl phosphate (Sigma). Proteins in the eluted cytosolic
PY-RF were denatured and reduced in
6 M guanidine HCl (GdnHCl, Pierce) and 0.1 M
-mercaptoethanol, and separated by denaturing size exclusion
chromatography on a Superose 6 (S6) column (1 × 30 cm, Amersham
Pharmacia Biotech) in the presence of 6 M GdnHCl and 0.1 M
-mercaptoethanol. Fractions containing proteins of
interest were pooled and separated by C4 RP-HPLC at
55 °C. Each protein purified from the C4 RP-HPLC was digested with trypsin (sequencing grade, Boehringer Mannheim), and the
peptides generated were resolved by C8 RP-HPLC at 40 °C. Sequences of the peptides were determined using an Applied Biosystem 477A protein sequenator.
Antibodies--
Anti-CSF-1R antibodies against two cytoplasmic
domain peptides (an interkinase domain peptide,
EGDSSYKNIHLEKKYVRRDSGFC, and a C-terminal domain peptide,
NNDGDYANLPSSGGSGSDSC) were raised in goat by immunizing with a mixture
of the two keyhole limpet hemocyanin-coupled peptides (29). The
antibodies specific to each peptide were purified from the serum by
peptide affinity chromatography and a 1:1 mixture of the two affinity
purified antibodies was used. FITC-coupled donkey anti-mouse and
Cy3-coupled donkey anti-goat antibodies were purchased from Jackson
ImmunoResearch. Anti-actin (clone C4) was obtained from Boehringer
Mannheim. Anti-vimentin and anti-tropomyosin were obtained from Sigma.
Anti-Cbl, anti-STAT5a, anti-STAT5b, and anti-Vav were purchased from
Santa Cruz. Anti-paxillin, anti-Grb2, anti-Shc, anti-STAT1, anti-STAT3,
anti-PY (PY20, mouse monoclonal), and horseradish peroxidase-conjugated
PY antibody (horseradish peroxidase-RC20) were from Transduction
Laboratory. Anti-SHP-1 was raised in rabbit against the C-terminal
peptide, KREEKVKKQRSADKEKS (20). Anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was raised in rabbit against the peptide
DNEYGYSNRVVDLMAYMA. Chicken anti-elongation factor-1
(EF-1
)
antiserum was raised against the C-terminal peptide AGAGKVTKSAQKAQKAK
and purified by peptide affinity chromatography (30). Anti-human p85
(regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase))
antibody was a gift from Dr. Jonathan Backer of the Albert Einstein
College of Medicine. An affinity purified antibody to the C-terminal
half of the chicken gizzard myosin regulatory light chain (MRLC) was kindly provided by Dr. Kathleen Trybus of Brandeis University.
Immunofluorescence Microscopy-- The distribution of PY proteins and the CSF-1R in CSF-1-stimulated and unstimulated cells was assessed by confocal immunofluorescence microscopy. Cells were plated onto chamber slides (21 × 20 mm well, Lab-Tek, Nalge Nunc International) and cultured for at least 24 h. Cells were incubated without CSF-1 for 18 h and then stimulated with 13.2 nM CSF-1 at 4 °C for 10 min and 2 h as described previously (15). Stimulation medium was removed at the end of incubation, and cells were fixed with 3.7% formaldehyde in buffer F (5 mM PIPES, pH 7.2, 1.1 mM Na2HPO4, 0.4 mM KH2PO4, 137 mM KCl, 4.0 mM NaHCO3, 2 mM MgCl2, 2 mM EGTA, and 5.5 mM glucose) for 5 min at 37 °C. Subsequent procedures were then carried out at room temperature. The fixed cells were extracted with 0.5% Triton X-100 in buffer F for 10 min and incubated in 0.1 M glycine in buffer F for 10 min to quench aldehyde autofluorescence. After washing 5 times (5 min each time) with the blocking solution (1% bovine serum albumin, 5% normal donkey serum, 20 mM Tris-HCl, pH 8.0, 154 mM NaCl, 0.1 mM sodium orthovanadate, and 0.02% NaN3), cells were incubated with the same blocking solution for a further 30 min to block nonspecific immunoreactive sites. Anti-PY (4 µg/ml) and anti-CSF-1R (20 µg/ml) antibodies dissolved in blocking solution were incubated with the cells for 1 h. Unbound antibodies were removed by washing the cells 5 times (5 min each time) with the washing solution (1% bovine serum albumin, 20 mM Tris-HCl, pH 8.0, and 154 mM NaCl). The cells were then incubated for 1 h with donkey anti-goat antibody (3.75 µg/ml, Cy3-labeled) and donkey anti-mouse antibody (7.5 µg/ml, FITC-labeled) dissolved in blocking solution. After thorough washing (5 times for 5 min each time) with washing solution, the cells were mounted in a medium containing 50% glycerol, 20 mM Tris-HCl, 154 mM NaCl, and 100 mg/ml 1,4-diazabicyclo-(2.2.2)octane (Sigma) and examined under a Bio-Rad MRC 600 Laser Scanning Confocal Microscope.
Immunoprecipitation and Immunoblotting-- Immunoprecipitation and immunoblotting were performed as described previously (15, 20). All immunoblots were developed with horseradish peroxidase-coupled secondary antibody and ECL reagent (Amersham Pharmacia Biotech).
Two-dimensional Gradient Gel Electrophoresis--
The first
dimension non-denaturing electrophoresis utilized a 1-mm thick vertical
slab gradient (5% acrylamide (10% bis-acrylamide), 15% acrylamide
(5% bis-acrylamide)) gel containing 0.01% Triton X-100 polymerized on
Gel Bond film (FMC bioproduct) backing and using the buffer system of
Davis (31). 32P labeling of the cells was performed as
described previously (16). The 32P-labeled PY-RFs from
CSF-1-stimulated and unstimulated cells were diluted in stacking gel
buffer containing 4% sucrose and 1% Triton X-100 at pH 6.8 for
loading, and the gel was run at 12 V/cm for 24 h at 4 °C.
Following the first dimension electrophoresis, the gel was removed from
the glass plates, blotted to remove excess buffer, wrapped in a layer
of plastic food wrap, and exposed to x-ray film for 5 h. The gel
lanes comprising the first dimension were cut out with reference to the
autoradiogram, immersed in sodium dodecyl sulfate-polyacrylamide
(SDS-PAGE) sample buffer (1% SDS, 50 mM Tris-HCl pH 6.8, 0.1 M
-mercaptoethanol, 6% glycerol, and 0.01%
bromophenol blue) without shaking for 2 h at 37 °C and then
secured on top of the stacking gel of a 1.5-mm thick,
SDS-polyacrylamide gradient slab gel (7.5-17.5% acrylamide, 2.7%
bis-acrylamide, Laemmli (32) system) with 0.5% agarose in SDS sample
buffer without glycerol. The gel was electrophoresed at 8 V/cm for
18 h at 16 °C, stained with Coomassie Blue, dried, and
autoradiographed.
Non-denaturing Size Exclusion Chromatography--
An S6
column equilibrated in a buffer containing 20 mM HEPES, pH
7.0, 200 mM NaCl, 0.1 mM sodium orthovanadate,
0.8% octyl glucoside, and 1 mM benzamidine was run at a
flow rate of 0.2 ml/min at 4 °C. The PY-RF (0.2 ml, ~1 mg/ml)
was clarified by centrifugation at 13,000 × g for 15 min prior to injection. Protein concentration was monitored at 280 nm,
and 0.3-ml fractions were collected. Starting from the fraction
containing the protein eluted at the void volume of the column,
fractions were pooled in groups of four to yield 10 large
fractions of 1.2 ml each, for further analysis.
Denaturing Size Exclusion Chromatography--
To 100 µl of
PY-RF (containing ~0.8 mg of protein), 110 mg of GdnHCl, 9.5 µl
of 2 M Tris-HCl, pH 8.5, and 1.4 µl of
-mercaptoethanol were added to yield 190 µl of
PY-RF in 6 M GdnHCl, 0.1 M Tris-HCl, pH 8.5, and 0.1 M mercaptoethanol. The resulting solution was incubated at
room temperature for 2 h and centrifuged at 13,000 × g for 15 min. The reduced, denatured, and clarified
PY-RF
was then injected into an S6 column equilibrated at room temperature in
6 M GdnHCl, 0.5% dodecyl trimethyl ammonium bromide
(Sigma), 0.1 M
-mercaptoethanol, and 20 mM
Tris-HCl, pH 6.5, at a flow rate of 0.2 ml/min. Protein concentration
was monitored at 280 nm, and 0.3-ml fractions were collected.
DNase I Precipitation of G-actin--
DNase I (Sigma) was
coupled to Sepharose 4B (Amersham Pharmacia Biotech) at a concentration
of 1 mg of protein per ml of gel by cyanogen bromide activation, and
the DNase I binding reaction was performed as described previously
(33). Cytosolic extract (60 µg) or the PY-RF (approximately 6 µg
from stimulated and 4 µg from unstimulated cells) prepared from 6 mg
of cytosolic protein was incubated with 50 µl (packed volume) of the
DNase I-Sepharose beads in buffer A (2 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM dithiothreitol, and 0.8% octyl glucoside) at 4 °C for 3 h. The beads were then washed 4 times each with 0.5 ml of buffer A containing 0.2 M
NH4Cl (salt wash) and then two times with buffer A. The
bound proteins were eluted by incubating the beads with equal volume of
2× SDS-PAGE sample buffer at 60 °C for 30 min.
Biotinylated Phalloidin Precipitation of F-actin from the
Cytosolic PY-RF--
The cytosolic
PY-RF from both
CSF-1-stimulated and unstimulated cells prepared from 10 confluent
100-mm culture dishes (~80 × 106 cells) was
incubated with 2 nmol of either phalloidin-XX-biotin (Molecular Probes)
or 2 nmol of biotin (Sigma) in buffer B (20 mM HEPES, pH
7.3, 150 mM NaCl, 0.1 M sucrose, 0.8% octyl
glucoside, 0.1 mM sodium orthovanadate, and 0.1 mM phenylmethylsulfonyl fluoride) in a final volume of 20 µl for 18 h at 4 °C. The reaction mixtures were then
incubated for 2 h with 25 µl (packed volume) avidin-agarose (Sigma) equilibrated in buffer B at 4 °C, with continuous agitation. The agarose beads were pelleted by centrifugation and washed 6 times
each with 0.5 ml of buffer B at 4 °C. The initial supernatant and
the first 2 washes were pooled and concentrated by microcon 30 (Amicon)
to 20 µl. The thoroughly washed agarose beads were incubated with
equal volume of 2% SDS and heated at 60 °C for 30 min to elute the
bound proteins.
Cytochalasin D Treatment-- Cytochalasin D (Sigma) was dissolved in dimethyl sulfoxide (Me2SO, Sigma) at 5 mg/ml. Cells were incubated without CSF-1 for 18 h. Cytochalasin D solution (2.8 µl) was added to each dish (final concentration, 5 µM), and the cells were incubated for a further 3 h without CSF-1 at 37 °C. Control cells received the same volume of Me2SO. CSF-1 was then added (final concentration, 13.2 nM), and the cells were lysed with Nonidet P-40 buffer at various times as described previously (16).
Other Techniques-- Proteins were resolved by gradient SDS-PAGE (7.5-17.5% acrylamide) (20). Silver staining of protein in SDS-PAGE was carried out by the method of Morrissey (34). Protein determination was carried out as described previously (20).
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RESULTS |
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Distribution of the CSF-1R and PY Proteins in BAC1.2F5
Cells--
For all of the following experiments with the exception of
experiment shown in Fig. 9, BAC1.2F5 macrophages were preincubated without CSF-1 for 18 h at 37 °C and then incubated for 10 min or 2 h at 4 °C in the presence or absence of CSF-1. The
distribution of PY proteins in relation to distribution of the CSF-1R
in cells under these conditions was determined by confocal microscopy
of cells incubated with mouse anti-PY and goat anti-CSF-1R antibodies and visualized with FITC-coupled anti-mouse and Cy3-coupled anti-goat secondary antibodies, respectively. Under the conditions used, at 10 min after addition of CSF-1, the CSF-1R represents 50% of the
tyrosine-phosphorylated proteins. By 2 h after CSF-1 addition, it
represents
25% (15, 16, 18). At 10 min after addition of CSF-1,
strong overlapping and punctate expression of CSF-1R and protein
tyrosine phosphorylation were observed at the plasma membrane (Fig.
1). Some CSF-1R was localized in an
internal perinuclear structure, probably the Golgi complex, which
contains the CSF-1R precursor, representing ~25% of the total
cellular CSF-1R as determined by its lower molecular mass and by its
content of N-linked oligosaccharides of the high mannose
type (35). In cells stimulated with CSF-1 for 2 h, the intensity
of anti-PY staining was significantly increased, and the staining was
more evenly distributed in the vicinity of the plasma membrane, whereas
the CSF-1R staining maintained the punctate appearance seen at 10 min
of stimulation (Fig. 1). Thus the proteins exhibiting an increase in
phosphotyrosine in response to CSF-1 at 4 °C, despite their
predominance in the cytosolic fraction (18), are found close to the
plasma membrane.
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PY Proteins in the Cytosol Are in Multimeric Complexes That Change
following Stimulation with CSF-1--
The existence of multimeric
complexes in the cytosolic PY-RF of the BAC1.2F5 macrophages was
demonstrated by two-dimensional gradient gel electrophoresis. In the
first dimension, proteins in 32P-labeled cytosolic
PY-RF were separated according to size by non-denaturing gradient
gel electrophoresis. The gel lanes from the first dimension were cut
out, denatured, and reduced in SDS sample buffer containing
mercaptoethanol and placed horizontally on an SDS gradient gel to
separate the proteins present in each complex. The resulting
autoradiogram (Fig. 2) showed that CSF-1 not only stimulated an increase in phosphorylation of existing proteins
but also the assembly of larger complexes involving phosphorylated proteins of 37, 57, 75, 120, and 160 kDa.
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Purification and Identification of Proteins in the Cytosolic
PY-RF--
To understand better the relationship between
CSF-1-stimulated protein tyrosine phosphorylation and formation of
these complexes, proteins were identified by a combination of Western
blotting with antibodies to known proteins and direct microsequencing
of purified proteins in the
PY-RF. The first step in the
purification of these proteins was non-denaturing anti-PY affinity
chromatography. To be sure of the identity of proteins exhibiting
CSF-1-stimulated tyrosine phosphorylation, purification was carried out
in parallel from cytosolic fractions of both CSF-1-stimulated and
unstimulated cells. To increase the yield of PY and associated
proteins, cells used for purification of
PY-RF were incubated with
CSF-1 at 4 °C for 2 h in the presence of 8 mM IAA.
As shown in Fig. 3A,
incubation with IAA increased the overall intensity of cytosolic
protein tyrosine phosphorylation in both stimulated and unstimulated
cells, without changing the number of tyrosine-phosphorylated
bands. In fact, tyrosine phosphorylation of some proteins, for example at 57 and 37 kDa, was augmented so profoundly by IAA that the effect of
CSF-1 stimulation was no longer apparent. Comparison of the
silver-stained protein profiles of the
PY-RF (Fig. 3B) with the anti-PY Western blotting profiles (Fig. 3A) of the
same fractions reveals that the
PY-RF prepared from the cytosol of BAC1.2F5 cells contained many proteins that were not
tyrosine-phosphorylated. IAA treatment not only increased the amount of
PY protein in this fraction but also increased the amount of associated
non-PY protein to a similar degree, suggesting that these non-PY
proteins are specifically associated with the PY proteins. Furthermore,
despite the fact that some of the non-PY proteins were abundant
cytoplasmic proteins (see below), the
PY-RF obtained by repeated
anti-PY affinity chromatography (Fig. 4)
comprised only 0.1% of the total cytosolic protein (Fig. 4 and Table
I), indicating that the non-PY proteins
that co-purify with the PY proteins are specifically associated with
the latter and their presence is not due to contamination.
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Analysis of the Multimeric Complexes in the Cytosolic
PY-RF--
When the S6 size exclusion chromatography elution
profiles of the non-denatured proteins in the cytosolic
PY-RF (Fig.
6A, upper) are compared with those of denatured proteins
(Fig. 5), it was apparent that many of the proteins were present in
complexes of different sizes. One of these was actin. Western blotting
analysis of fractions from the non-denaturing S6 size exclusion
chromatography (Fig. 6B) showed that actin (44 kDa) is
present in complexes varying in apparent Mr from
>6 million (Fig. 6B, fraction 1, at void volume of the column) down to ~100,000 (Fig. 6B, fraction
8). In fractions 1-4, which contained protein complexes of 669 kDa or larger, more actin was detected in the CSF-1-stimulated
preparation. However, more actin was detected in the unstimulated
preparation for fractions containing protein complexes of 250 kDa or
less. In the original cytosolic
PY-RF, there was little change (Fig.
6B) or a decrease (Fig. 7,
lanes 1 and 2) in actin content with stimulation.
Vav, EF-1
, GAPDH, and pp37 showed a very similar distribution to
actin in that their presence in the high molecular size fractions
(>334 kDa) was increased by CSF-1, whereas their levels in the lower molecular size fractions (<334 kDa) were decreased by CSF-1
stimulation. Although it was not obvious that Vav was
tyrosine-phosphorylated (Fig. 6B), immunoprecipitation of
Vav and Western blotting with
PY antibody indicated that it was
tyrosine-phosphorylated in the response of BAC1.2F5 cells to
CSF-1.2 Grb2, p85, SHP-1,
Cbl, STAT3, STAT5a, STAT5b, Shc, vimentin, and pp57 showed a
CSF-1-stimulated increase in all fractions in which they were present
(Fig. 6B). The three isoforms of Shc showed significant
differences in their distribution as follows: p68 Shc was eluted in
fractions of 150-400 kDa, p58 Shc in all fractions from 44 kDa to 6 million Da, and p54 Shc in fractions of 150 kDa to 6 million Da. p54
Shc and p68 Shc were dramatically increased in the CSF-1-stimulated
fractions. p58 Shc exhibited a similar dramatic CSF-1-stimulated
response in the very high molecular weight fractions (Fig.
6B, 1 and 2). Whereas p58 Shc was
increased in the lower molecular weight fractions, CSF-1 stimulated a
smaller fold increase in p58 Shc because a significant amount of p58
Shc was present in the unstimulated fractions. Vimentin, a 56-kDa intermediate filament protein, was present only in fractions of proteins >669 kDa (Fig. 6B). SHP-1 was present in
CSF-1-stimulated fractions of both high and low molecular weight (Fig.
6B). There was no effect of CSF-1 on the amount of complexed
tropomyosin, paxillin, or MRLC. Tropomyosin was eluted at 350 kDa,
paxillin at 200 kDa, and the MRLC at 80-44 kDa (Fig. 6B).
The F-actin severing and capping protein, gelsolin, could not be
detected in the cytosolic
PY-RF. The analysis of fractions from two
additional non-denaturing S6 columns yielded similar results.
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Analysis of Actin and Identification of Actin-associated Proteins
in the Cytosolic PY-RF--
Actin is one of the major
silver-stained proteins present in all non-denaturing S6 chromatography
fractions (Fig. 6A, upper). As we considered it likely that
actin forms multiple complexes with several proteins in the cytosolic
PY-RF, it was studied further. Actin can exist as G-actin or short
F-actin fragments in the cytosol. DNase I has been shown to bind
G-actin but not F-actin (33). To characterize the form of actin present
in the cytosolic
PY-RF, DNase I affinity precipitation was performed (Fig. 7). DNase I could not bind the actin present in the
PY-RF from
either stimulated or unstimulated cells (Fig. 7, lanes
1-6), despite the fact that it bound about 70% of the actin in
the cytosol (Fig. 7, lanes 10-13). Actin of the cytosolic
PY-RF was quantitatively recovered in the DNase I flow-through (Fig.
7, lanes 7 and 8). These results suggest that the
actin present in the
PY-RF is F-actin and that G-actin, an abundant
cytosolic protein, is absent.
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Effect of Cytochalasin D on CSF-1-stimulated Protein Tyrosine Phosphorylation-- To assess the importance of the actin cytoskeleton for the normal CSF-1 response, we examined the effect of cytochalasin D-mediated depolymerization of cytoskeleton on CSF-1-stimulated protein tyrosine phosphorylation. Preincubation of BAC1.2F5 cells with 5 µM cytochalasin D significantly decreased CSF-1-stimulated protein tyrosine phosphorylation of most proteins in the Nonidet P-40 soluble cell lysate, although the kinetics of stimulation was not affected (Fig. 9). The effect of cytochalasin D was more marked for some proteins than for others. Tyrosine phosphorylation of proteins at 190, 165 (CSF-1R), 75, 70, and 37 kDa was substantially reduced, whereas the tyrosine phosphorylation of 85- and 47-kDa proteins was completely inhibited (Fig. 9). Tyrosine phosphorylation of 66 and 36.5 kDa proteins was not affected. The effect of cytochalasin D on cell shape and protein tyrosine phosphorylation was reversible. Cells treated with 5 µM cytochalasin D for 3 h rounded up within 1 h. When the medium was replaced with normal growth medium, the cells rapidly spread again and continued to grow normally. This result implies that efficient and specific CSF-1-stimulated protein tyrosine phosphorylation depends on the integrity of the actin cytoskeleton.
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DISCUSSION |
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This study has focused on the characterization of the cytosolic
PY-RF which contains the majority of non-CSF-1R PY proteins in
CSF-1-stimulated macrophages. Several PY proteins and associated non-PY
proteins have been identified and their organization and reorganization
in response to CSF-1 described.
Identification of Proteins in the Cytosolic -PY-RF--
Among
the PY proteins identified in the cytosolic
PY-RF were several known
signaling proteins, including Cbl (22, 38), STAT3, STAT5a, STAT5b (27,
28), SHP-1 (20, 39), Shc (40), and Vav (41, 42). Despite an earlier
report demonstrating activation of STAT1 by CSF-1 (27), we were unable
to demonstrate the presence of STAT1 in the cytosolic
PY-RF by
anti-STAT1 Western blotting. Previous studies have shown that all seven
of the PY proteins detected are rapidly tyrosine-phosphorylated in the
response of macrophages to CSF-1 (20, 22). Apart from these known
signaling proteins, we also obtained internal peptide sequences of two
novel PY proteins, pp37 and pp57. cDNA clones encoding pp37 have
recently been isolated and predict a novel member of a protein tyrosine phosphatase substrate family that plays an important role in
cytoskeletal regulation.3 The
fact that seven of the nine PY proteins identified are known to have
important roles in growth factor signal transduction suggests that pp37
and pp57 will have important signaling roles and validates this
approach as one likely to lead to the identification of additional novel signaling proteins.
Organization of Proteins in the Cytosolic PY-RF and the Effect
of CSF-1 Stimulation--
Two-dimensional gradient gel electrophoresis
of 32P-labeled cytosolic
PY-RF showed that CSF-1 not
only stimulated a dramatic increase in phosphorylation of proteins in
this fraction but also an increase in the apparent
Mr of many phosphorylated proteins. Consistent
with these observations, in higher molecular weight fractions of the
non-denaturing S6 size exclusion chromatography of the cytosolic
PY-RF, many of the PY and non-PY proteins were present at a higher
level in the CSF-1-stimulated fractions than in the corresponding
fractions from unstimulated cells. However, both results also show that
most of the PY and non-PY proteins in the cytosolic
PY-RF from
unstimulated cells have higher apparent molecular weights on
non-denaturing separation than on denaturing separation. These findings
indicate that protein complexes composed of different PY and non-PY
proteins pre-exist in the unstimulated cells and that CSF-1 stimulation
causes significant increases in their size. Furthermore, the fact that
the total protein of the
PY-RF only increases by ~20% with CSF-1
stimulation, whereas the PY content increases by more than 5-fold, is
also consistent with the pre-existence of complexes in which protein
components are tyrosine-phosphorylated and to which PY proteins are
differentially recruited, upon CSF-1 stimulation. Although we have
demonstrated the existence of protein complexes in the
PY-RF, we may
not have detected low affinity complexes which dissociate in the
detergent-containing buffer system we have used.
Complexes Involving Actin--
As actin is a major silver-stained
protein present in all fractions in the non-denaturing S6 size
exclusion chromatography, this protein may be the common component of
several complexes of the cytosolic PY-RF. We were unable to
demonstrate the presence of any DNase I-binding G-actin in this
fraction. Although the total amount of F-actin in the cytosolic
PY-RF was usually slightly decreased by CSF-1 stimulation, CSF-1
stimulation led to an increase in its concentration in the higher
molecular weight fractions from the non-denaturing S6 column and a
decrease in its concentration in the lower molecular weight fractions.
Should these changes involve the actin component of the complexes, it
is possible that the CSF-1-induced increase in F-actin in the higher
molecular weight fractions is generated by the severing of cytoskeletal actin filaments (57) and that the decrease in F-actin in the lower
molecular weight fractions is due to the translocation of some of the
lower molecular weight actin complexes to the membrane or cytoskeleton.
However, de novo assembly and disassembly of the F-actin
complexes cannot be excluded as an explanation of these
observations.
Functional Significance of PY Complexes in Cellular
Signaling--
As shown by confocal microscopy in the present study
(Fig. 1), the tyrosine-phosphorylated proteins accumulate in close
proximity to the plasma membrane suggesting that tyrosine
phosphorylation and complex formation/restructuring following CSF-1
stimulation occur at the plasma membrane. The complexes formed are
diverse and should have specific functions. It is likely that in the
resting cells, multimers of cytosolic PY-RF proteins of varying
complexity are maintained via different types of interactions
(e.g. SH3, pleckstrin homology, calponin homology, SH2, and
phosphotyrosine-binding domain-mediated) and are positioned in the
cytoplasm close to the receptor on the plasma membrane. When the
receptor kinase is activated by ligand binding, tyrosine
phosphorylation of proteins within and outside these complexes occurs
rapidly. This covalent modification changes their conformation and/or
binding properties, causing them to associate with other signaling
proteins and complexes. These complexes, in turn, mediate cytoskeletal
changes, the activity of specific enzyme systems, and gene expression.
The present findings demonstrate that CSF-1R signal transduction
involves the integrated participation of cytoskeletal and other
proteins in organized, pre-existing complexes. The analysis of these
complexes and their novel components, e.g. pp57 and pp37,
should significantly increase our understanding of this process.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. Backer and J. Condeelis for reviewing the manuscript and various members of the laboratory (past and present) who assisted in the preparation of cell extracts. The sequencing was performed at the Laboratory of Macromolecular Analysis of the Albert Einstein College of Medicine. We thank Yuan Shi for handling of the samples and for help in the interpretation of the data.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA 26504 and the Albert Einstein Cancer Center Grant P30-13330.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.
Supported by a Betty and Howard Isermann Scholarship and
National Institutes of Health Medical Scientist Training Grant
T32-GM07288.
§ To whom all correspondence should be addressed: Dept. of Developmental and Molecular Biology, 1300 Morris Park Ave., Albert Einstein College of Medicine, Bronx, New York 10461. Tel.: 718-430-2344; Fax: 718-430-8567; E-mail: rstanley{at}aecom.yu.edu.
1
The abbreviations used are: CSF-1,
colony-stimulating factor 1; PY, anti-phosphotyrosine;
PY-RF,
anti-phosphotyrosine reactive fraction; PY, phosphotyrosine; EF-1
,
elongation factor 1-
; G3BP, Ras-GTPase-activating protein SH3
domain-binding protein; Hsp40, heat shock protein 40 kDa; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; MRLC, myosin regulatory light
chain; IAA, iodoacetic acid; PAGE, polyacrylamide gel electrophoresis;
S6, Superose 6; RP-HPLC, reverse phase high pressure liquid
chromatography; PI 3-kinase, phosphatidylinositide 3-kinase; p85,
regulatory subunit of PI 3-kinase; PIPES,
piperazine-N,N'-bis[2-ethanesulfonic acid];
GdnHCl, guanidine hydrochloride; FITC, fluorescein isothiocyanate; CSF-1R, CSF-1 receptor.
2 K. Berg, personal communication.
3 Y. G. Yeung, S. Soldera, and E. R. Stanley, manuscript in preparation.
4 D. B. Einstein, unpublished data.
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REFERENCES |
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