From the
A unique protein with an apparent molecular mass of 96
kilodaltons (p96) was detected in the murine macrophage cell line,
BAC1.2F5. The murine cDNA encoding p96 was cloned and sequenced, along
with cDNAs representing two alternatively spliced forms of the protein.
All three proteins possessed identical amino-terminal domains with
significant similarity to the amino-terminal domain of the
Drosophila disabled gene product and carboxyl-terminal domains
containing proline-rich sequences characteristic of src homology region (domain 3) binding regions. BAC1.2F5 cells
predominately expressed the p96 protein, although mRNA and protein
corresponding to the p67 splice variant were also detected.
Electrophoretic gel retardation of p96 in response to stimulation of
the cells with colony-stimulating factor 1 was noticeable within 5 min
after growth factor addition and reached a maximum at 60 min. Metabolic
labeling experiments showed that the gel retardation of p96 was
associated with increased phosphorylation of the protein exclusively on
serine residues. These data identify a novel protein that is
phosphorylated in response to mitogenic growth factor stimulation.
CSF-1
Autophosphorylation of the CSF-1 receptor promotes its interaction
with cytoplasmic proteins that activate multiple signal transduction
pathways and culminate in a wave of immediate early gene expression
(for reviews, see Sherr(1991); Vairo and Hamilton(1991)). In addition,
CSF-1 is required throughout the G
As part of our investigation of proteins involved in
the signal transduction pathways in a murine macrophage cell line,
BAC1.2F5, initiated by PC-PLC and CSF-1, we have cloned the cDNA for a
unique 96-kDa protein related to the Drosophila disabled gene.
The 96-kDa protein, termed p96, is phosphorylated on serine residues
following mitogenic stimulation in the BAC1.2F5 cell line identifying
it as a component in the CSF-1 signal transduction cascade.
A second immunoscreen was used to
identify the
Plasmids were isolated using columns
purchased from Qiagen, and restriction enzyme digestions and agarose
gel electrophoresis were performed as described (Sambrook et
al., 1989). At least three independent plasmids derived from the
inserts of positive
Expression vectors were
constructed by cloning the p67 coding sequence from clone N10-2
into pBluescript downstream of the T7 promoter. The p93 and p96 vectors
were constructed by substituting the BclI-PpuMI
fragments from clones B21 and B7, respectively, for the
BclI-PpuMI fragment in N10-2. Plasmid DNA was
isolated, transcribed, translated, and labeled with
[
RT-PCR was
performed on total RNA isolated from 1
BAC1.2F5 cells were pelleted and resuspended in 200 µl of 0.6%
SDS and heated at 95 °C for 5 min to denature the proteins. The
lysate was diluted with 1 ml of Tris-buffered saline (pH 7.4), and the
antibody (either
Our results identify a novel protein, p96, that is rapidly
phosphorylated in macrophages in response to mitogenic stimulation of
CSF-1. The dephosphorylated form of the p96 protein accumulates in the
absence of the growth factor as cells arrest in the G
The p96 protein is most similar to
the Drosophila disabled gene product, the Dab protein
(Fig. 3B) (Gertler et al., 1989; Bennett and
Hoffmann, 1992; Gertler et al., 1993). The disabled gene is a second-site modifier of the abl mutant
phenotype. Flies defective in abl and disabled have
an embryonic lethal phenotype and fail to form proper axonal
connections in the central nervous system. Expression of either the
abl or disabled gene restores the embryonic axonal
architecture, illustrating the functional redundancy of the two gene
products. Furthermore, the ability of disabled to suppress the
abl mutant phenotype is dosage dependent, indicating that the
two proteins may be involved in a multimeric structure whose function
is sensitive to the stoichiometry of the individual components. The
expression of abl and disabled are closely
associated. The proteins are primarily localized in central nervous
system axon bundles but are also present in peripheral nervous system
cell clusters and the body wall musculature. The p96 and Dab proteins
have a common 140-amino acid domain in the amino terminus that is 46%
identical and 66% similar (Fig. 3B). Both proteins also
are expressed as two alternatively spliced forms with the lower
molecular weight form lacking an exon (221 residues in p96 or 214
residues in Dab) located in the center of the molecule. However, there
are also several significant differences between p96 and Dab. The Dab
protein consists of 2411 amino acids and is therefore much larger (264
kDa) than p96. Dab is phosphorylated on tyrosine, whereas there is no
indication that p96 is tyrosine phosphorylated in the murine macrophage
cell line (Fig. 6). Nonetheless, these data raise the possibility
that p96 is the mammalian homolog of the Drosophila disabled gene product; however, functional complementation experiments in
the Drosophila system are required to determine if this idea
is correct.
One clue to the function of p96 is that the disabled homology domain may represent a phosphotyrosine interaction
domain. This new protein motif was suggested from the findings that the
amino-terminal domains of Shc (Blaikie et al., 1994) and a
related protein, Sck (Kavanaugh and Williams, 1994), mediate
phosphotyrosine-dependent interactions. These regions were subjected to
data base search methods to identify proteins that might contain
homologous domains (Bork and Margolis, 1995). This search identified a
region of homology between p96 and Dab (Fig. 3B) that
was similar to residues 46-209 of Shc and thus constitutes a
potential phosphotyrosine interaction domain. These data suggest that
the Dab homology domain may mediate the interaction between p96 and
other proteins phosphorylated on tyrosine, an exciting hypothesis that
is currently being evaluated experimentally.
Mok et
al.(1994) isolated a cDNA fragment that is the human homolog of
the murine cDNA encoding p96. These investigators used a
DNA-fingerprinting approach to detect candidates for tumor suppressor
genes that are differentially expressed in human ovarian cancer cells.
They cloned a 767-bp cDNA (called DOC-2) that was highly expressed in
normal ovarian surface epithelial cells but consistently lacking in all
ovarian cancer lines examined. The DNA sequence of DOC-2 was 89%
identical to the p96 sequence between 346 and 1116 bp, and the amino
acid sequence predicted from the DOC-2 sequence was 94.5% identical and
96% similar to the predicted p96 protein sequence between residues 49
and 300. Like p96, two mRNAs that differed by approximately 500 bp were
detected by Northern analysis of normal ovarian epithelial cells.
Detection of p96 expression in human ovary by in situ hybridization using a
The
nucleotide sequence(s) reported in this paper has been submitted to the
GenBank
We thank Huong Nguyen, Margarita Pecha, and Jonathan
Powell for their technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is a growth factor that is
required for the differentiation, proliferation, and survival of cells
of the mononuclear phagocyte lineage (for review, see Stanley(1985)).
The biological effects of CSF-1 are mediated by its binding to a single
high affinity receptor (Guilbert et al., 1986) encoded by the
c-fms proto-oncogene (Sherr et al., 1985). The CSF-1
receptor is similar to the platelet-derived growth factor receptor and
the c-kit proto-oncogene in that the extracellular domain
consists of five immunoglobin-like loops linked by a single
transmembrane helix to the cytoplasmic tyrosine kinase domain that is
interrupted by a unique ``kinase insert'' sequence (Ullrich
and Schlessinger, 1990). CSF-1 binding triggers dimerization of the
receptor (Lee and Nienhuis, 1990; Li and Stanley, 1991; Ohtsuka et
al., 1990), autophosphorylation on tyrosine (Rettenmier et
al., 1985), and stimulation of its tyrosine kinase activity (Yeung
et al., 1987; Downing et al., 1989).
stage of the cell cycle
(Tushinski and Stanley, 1985), indicating that CSF-1-dependent
signaling pathways later in G
are required for the
production or activation of key proteins that ultimately determine the
commitment to DNA synthesis. The response to CSF-1 is very complex, and
describing the components in this signal transduction network is a
major challenge. CSF-1 elevates intracellular concentrations of
diglyceride derived from phosphatidylcholine in human monocytes
(Imamura et al., 1990), bone marrow-derived macrophages (Veis
and Hamilton, 1991), a fibroblast cell line expressing the CSF-1
receptor (Choudhury et al., 1991), and BAC1.2F5 cells (Xu
et al., 1993). The addition of exogenous
phosphatidylcholine-specific phospholipase C (PC-PLC) to BAC1.2F5 cells
mimics the mitogenic effects of CSF-1 pointing to a role for
phosphatidylcholine hydrolysis in CSF-1 signal transduction (Xu et
al., 1993). PC-PLC activates a signal transduction pathway that
triggers the transcription of the c-fos and junB
genes independent of the activation of protein kinase C and Ras and
that collaborates with c-myc in the mitogenic stimulation of
BAC1.2F5 cells.
Materials
Sources of supplies were as
follows: DMEM, Whittaker Bioproducts; fetal calf serum, HyClone
Laboratories; ECL kit for the detection of proteins in immunoblots,
Amersham Corp.; [S]methionine (specific
activity, 1,000 Ci/mmol), [
-
P]dCTP
(specific activity, 3000 Ci/mmol), and
[
P]orthophosphate (carrier-free), DuPont NEN;
goat antirabbit IgG conjugated to alkaline phosphatase, Jackson
Laboratories; phosphatidylcholine-specific phospholipase C and
sphingomyelinase from Bacillus cereus, Boehringer Mannheim;
mouse brain 5`-stretch
gt10 cDNA library, mouse brain
gt11
cDNA library, mouse macrophage
gt11 cDNA library, and PCR primers
flanking the
phage insert, Clontech; random primed DNA labeling
kit, Boehringer Mannheim; Immobilon-P membrane, Millipore; in vitro transcription/translation assay kit and restriction endonucleases,
Promega; plasmid purification kits, Qiagen, Inc.; and Bradford protein
assay reagent, Bio-Rad. Homogeneous CSF-1 was kindly provided by
Genetics Institute. L-cell conditioned medium was used as the source of
CSF-1 for the routine maintenance of BAC1.2F5 cells (Stanley and Heard,
1977). Polyclonal rabbit antisera were produced by Rockland
Laboratories, Inc. using synthetic peptides coupled to keyhole limpet
hemocyanin. The
M15 sera was raised against a synthetic peptide
(RTQNGVSERENQNGFHIKSS) corresponding to amino acids 376-394 of
p96. The
M2 antisera was raised against a synthetic peptide
(IDEKTGVIEHEHPVNKIS) corresponding to amino acid residues 105-122
of p96. The
M15 sera was raised against a sequence unique to p96
and p93, whereas the epitope in
M2 was a sequence that was present
in p96, p93, and p67. The rabbit antisera (
ERK1pep) was raised
against a synthetic peptide (LKELIFQETARFQPGAPEAP) corresponding to the
carboxyl-terminal region of ERK1 and was a generous gift from Dr. J.
Downing (St. Jude Children's Research Hospital). All other
chemicals were reagent grade or better.
Cell Culture
The BAC1.2F5 cell line was a
CSF-1-dependent clone (Schwarzbaum et al., 1984; Morgan et
al., 1987) that exhibits many of the properties of macrophages.
BAC1.2F5 cells were routinely maintained in DMEM supplemented with 15%
fetal calf serum, 25% L-cell conditioned medium, and 20 mM
HEPES, pH 7.4. BAC1.2F5 cells were growth-arrested in G phase of the cell cycle by the removal of CSF-1 for 18 h (Rock
et al., 1992) and restimulated by the addition of either human
recombinant CSF-1 (90 ng/ml), PC-PLC (1 unit/ml), sphingomyelinase (0.1
unit/ml), or TPA (10
M). Experiments were
replicated at least three times unless otherwise indicated.
Cloning, Sequence, and in Vitro
Transcription/Translation of p96, p93, and p67
A protein
with an apparent molecular mass of 96 kDa determined from its mobility
in SDS-gel electrophoresis whose electrophoretic gel mobility was
retarded following CSF-1 stimulation was detected with the ERK1pep
antisera directed against a peptide present in the carboxyl-terminal
domain of ERK1. This 96-kDa protein was designated p96, and the
unfractionated
ERK1pep antisera was used to screen a
gt11
murine brain cDNA expression library. Immunological screening for the
presence of p96 was carried out by incubating the phage plus
Escherichia coli host strain Y1090r (10,000 plaque-forming
units/150-mm plate) at 42 °C for 2 h. The plate was overlaid with a
nitrocellulose membrane soaked in Xgal. The proteins were adsorbed to
the nitrocellulose by incubating at 37 °C for 7-10 h.
Positive clones were identified by reaction of the nitrocellulose
membranes with
ERK1pep antibody essentially as described in the
Clontech manual except that the primary antibody was detected by
reaction with goat antirabbit IgG conjugated with alkaline phosphatase
(Harlow and Lane, 1988). The positive plaques were excised from the
agar and subjected to another 3-5 rounds of amplification and
plaque purification using immunoscreening to detect the positive
plaques. DNA was isolated from the plaque-purified phage, and the cDNA
inserts were amplified by PCR using flanking primers purchased from
Clontech and were cloned into a PCR cloning vector (pCRII, Invitrogen).
Plasmid pBluescript KS (Stratagene) was used as the vector for further
subcloning and DNA sequencing.
phages from the initial screen that specifically
expressed p96. The purified phages were plated with cells, and
nitrocellulose membranes containing adsorbed phage fusion proteins were
prepared as described above. The nitrocellulose membranes were
incubated with 2% dry milk in TBS for 1 h and then incubated in a
1/1000 dilution of
ERK1pep antisera in TBS containing 2% dry milk.
The membranes were washed three times with 100 ml of TBS containing
0.05% Triton X-100. The bound antibodies were eluted by soaking the
membrane in 10 ml of 100 µM glycine, pH 2.5, for 10 min.
The pH was neutralized by the addition of 1 ml of 1 M
Tris-HCl, pH 8.8. The resulting solutions were used as the primary
antibodies to test for reactivity against BAC1.2F5 cell proteins on
immunoblots. Most of the 24 clones reacted with ERK1, and DNA sequence
analysis of three of these clones verified that they expressed a fusion
protein that contained ERK1 carboxyl-terminal sequences. One clone,
N13, expressed a fusion protein that absorbed antibodies that
recognized p96 (but not ERK1) when used as the primary antibody in
immunoblotting total cell extracts of BAC1.2F5 cells. Antibodies were
affinity purified against the recombinant phage N13 fusion protein and
were used in a second immunoscreen of a macrophage
gt11 library.
Two clones (M15 and M7) were isolated. Sequence analysis of the insert
showed that the M7 and M15 clones were identical and that their
sequences overlapped with the N13 clone. Clone M15 was used to
synthesize a labeled probe to screen both the murine macrophage
gt11 and murine brain
gt10 cDNA libraries for clones that
hybridized with M15 sequences according to the methods outlined in
Sambrook et al.(1989). These experiments resulted in the
isolation of clones N10-1, N1-1, and N10-2 from the
macrophage
gt11 library and clones B4, B7, B14, and B21 from the
brain
gt10 library.
clones were mapped and their DNA sequence
determined on both strands. A combination of the universal M13 primers
or one of several dozen primers complementary to the predetermined
sequence were used to extend the sequence using the automated
sequencing instrumentation (Applied Biosystems Inc.) provided by the
St. Jude Molecular Resource Center.
S]methionine using the Promega T7-coupled
transcription/translation kit according to the manufacturer's
instructions. The labeled proteins were analyzed by SDS-gel
electrophoresis either before or after immunoprecipitation with
M15 and
M2 antisera. The labeled proteins were electroblotted
onto nitrocellulose membrane and visualized by autoradiography.
RNA Analysis
BAC1.2F5 cell cultures were
harvested by centrifugation, and total RNA was isolated by a guanidine
isothiocyanate lysis procedure followed by pelleting RNA by CsCl
gradient centrifugation (Chirgwin et al., 1977). RNA pellets
dissolved in 10 mM Tris-HCl, pH 7.5, 5% -mercaptoethanol,
0.5% Sarkosyl, 0.5% SDS, and 5 mM EDTA were extracted with
phenol:chloroform:isoamyl alcohol (24:24:1) and precipitated with 2
volumes of ethanol. Poly(A)
RNA was isolated by
passing the total RNA through an oligo(dT) spin column (Clontech) as
described by the manufacturer. Poly(A)
RNA (10 µg,
determined from A
) was denatured for 10 min at
55 °C in electrophoresis buffer (20 mM MOPS, pH 7.0, 5
mM sodium acetate, 1 mM EDTA, 6% formaldehyde)
containing 50% deionized formamide. The samples were then quickly
chilled on ice and fractionated by electrophoresis in 1.0% agarose
gels. Blotting, prehybridization, hybridization with
P
probes, and washing of blots was performed as described by
Thomas(1980).
P-Labeled probes used for analysis of RNA
levels were prepared by random priming using restriction enzyme
fragments isolated by agarose gel electrophoresis.
10
BAC1.2F5
cells. The cell pellet was lysed by adding 450 µl of 50 mM
Tris-HCl, pH 8.0, 1% Nonidet P-40 and incubated on ice for 5 min. The
cell debris was removed by centrifugation at 12,000
g for 2 min. The cell lysate (450 µl) was transferred into a
clean eppendorf tube and mixed with 8 µl of 10% SDS and 3 µl of
proteinase K (20 mg/ml), and the mixture was incubated at 37 °C for
15 min. The RNA was extracted by adding an equal volume of
phenol/chloroform (1:1, pH 8.0), and the extraction was repeated twice.
Total RNA was precipitated with ethanol. The RNA pellet was rinsed once
with 70% ethanol and resuspended in 10 µl of
diethylpyrocarbonate-treated H
O. The final concentration of
total RNA was 0.76 µg/µl. The Stratascript RT-PCR kit
(Stratagene) was used for reverse transcription to synthesize the first
strand of cDNA with random primers. The procedure was performed as
described in the manufacturer's manual except that 3 µg of
total RNA was used in each reaction. 15 µl of the first strand cDNA
synthesis mixture was used for PCR amplification of p96 sequences. The
forward primer was 5`-GCTGGTCGCTCTCAGGGACAA-3` corresponding to
460-480 bp of the p96 sequence, and the reverse primer was
5`-AAGGACTGTAGACAACAGGCG-3` corresponding to 1750-1771 bp of the
p96 sequence (see Fig. 1). The PCR was performed in a 100-µl
reaction volume with 40 thermocycles at 94 °C for 1 min, 50 °C
for 2 min, and 72 °C for 2 min. The PCR products were precipitated
with ethanol and separated by agarose-gel electrophoresis. The bands
were excised from the gel, purified individually, and cloned into the
PCR cloning vector, pCRII (Invitrogen).
Figure 1:
Schematic
representation of the eight completely sequenced clones isolated from
mouse macrophage and brain libraries. The M15 clone was isolated using
the ERK1pep antisera to detect fusion protein production. The
remaining clones were isolated by hybridization with a labeled
oligonucleotide probe derived from M15. The designations in
parentheses indicate the proteins that were predicted to be
encoded by these cDNAs. In the case of M15 and N10-1, the
sequences isolated were common to both the p96 and p93 isoforms. The
solidbar indicates the sequences common to p96 and
p93 that were absent from p67. The twoarrows indicate the position of the PCR primers used in the RT-PCR
experiment described in Fig. 5C. The abbreviations for the
restriction enzymes are: A, AccI; B,
BclI; K, KpnI; M, PvuMI;
P, PstI; V, EcoRV; and X,
XhoI.
Immunoblotting and
Immunoprecipitation
Cells were washed with cold
phosphate-buffered saline, scraped into 0.5 ml of SDS sample buffer,
and immediately boiled for 5 min. The amount of protein present was
determined by the method of Bradford(1976). The cell lysate (100
µg) was separated by SDS-gel electrophoresis on 8% polyacrylamide
gels and electroblotted onto nitrocellulose membranes. The membranes
were then blocked with 1% dry milk for 1 h, washed with Tris-buffered
saline (150 mM NaCl, 10 mM Tris-HCl, pH 8.0), and
exposed to the primary antibody for 2 h. The dilutions of the primary
antibodies were M2, 1/500;
M15, 1/5000. The blots were washed
with Tris-buffered saline containing 0.05% Triton X-100 and incubated
with the second antibody conjugated with peroxidase for 1 h. After five
rinses with Tris-buffered saline containing 0.05% Triton X-100, the
Amersham ECL detection kit was used to locate the secondary antibody.
M15 or
M2) was added. The immune complexes
were isolated by the addition of protein A-Sepharose beads (50 µl
of a 50% solution), which were collected by a brief centrifugation and
washed twice with Tris-buffered saline. The beads were boiled in SDS
sample buffer and separated by electrophoresis using an 8% separating
gel. The proteins were transferred onto Immobilon-P membranes and
visualized by autoradiography following metabolic labeling or detected
by immunoblotting using the ECL assay kit.
Metabolic Labeling
To measure the
incorporation of phosphate into p96, 100-mm dishes of BAC1.2F5 cells
were deprived of CSF-1 for 18 h in phosphate-free DMEM plus dialyzed
FBS, and during the last 2 h, 0.1 mCi/ml
[P]orthophosphate was added. Cells were
harvested at zero time or after a 30-min stimulation with 90 ng/ml
recombinant CSF-1. The cells were then washed with cold
phosphate-buffered saline and lysed in 1 ml of 50 mM HEPES, pH
7.4, 150 mM NaCl, 1.5 mM MgCl
, 10%
glycerol, 1% Triton X-100, 1 mM EGTA, 0.2 mM sodium
orthovanadate, 10 mM sodium pyrophosphate, 100 mM
sodium fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1
mM phenylmethylsulfonyl fluoride. The cell lysate was
immunoprecipitated, and the immune complexes were separated by SDS-gel
electrophoresis on 8% polyacrylamide gels. The
P-labeled
p96 band was located by autoradiography, excised, and hydrolyzed with
5.7 N HCl for 1 h at 110 °C. The hydrolyzate was
lyophilized and was resuspended in 5-10 µl of pH 1.9 buffer
(88% formic acid/glacial acetic acid/H
O; 50:156:1794 (v/v))
containing 0.2 mg/ml of each phosphoserine, phosphothreonine, and
phosphotyrosine and then spotted onto a cellulose thin layer plate. The
phosphoamino acids were separated by two-dimensional electrophoresis
according to Cooper et al.(1983). The first dimension was
developed in pH 1.9 buffer at 1500 V for 20 min and the second
dimension in pH 3.5 buffer (pyridine/glacial acetic
acid/H
O; 10:100:1890 (v/v)) at 1300 V for 25 min. The
standard phosphoamino acids were visualized by staining with 0.2%
ninhydrin. The
P-labeled phosphoamino acids were
visualized by autoradiography and identified by comparing their
locations with the standard phosphoamino acids.
Isolation of p96 cDNA Clones
An
ERK1pep antiserum raised against a peptide corresponding to the
carboxyl-terminal region of ERK1 also detected a protein with an
apparent molecular mass of 96 kDa determined from SDS-gel
electrophoresis of total lysates of BAC1.2F5 cells and that exhibited
slower electrophoretic mobility following CSF-1 stimulation. This
96-kDa protein was designated p96, and the unfractionated
ERK1pep
antisera was used to screen a brain
gt11 expression library. 24
immunoreactive clones were obtained and plaque purified. Plate lysates
of the purified clones were adsorbed onto nitrocellulose membranes, and
the membranes were used to affinity purify antibodies from the
ERK1pep antisera pool (see ``Experimental Procedures'').
Antibodies eluted from the individual discs were used to immunoblot
BAC1.2F5 cell lysates. Most of the clones adsorbed antibodies that
detected a 44-kDa protein, but not a 96-kDa protein, on the
immunoblots, and several of these clones were confirmed to encode
murine ERK1 by DNA sequence analysis. The antibodies absorbed by a
single clone(N13) detected the p96 protein on immunoblots but did not
detect the 44-kDa ERK1 protein. This clone was used to prepare
affinity-purified antisera using the phage fusion protein as the
ligand, and the purified antisera were then used to screen a macrophage
gt11 expression library. Two positive clones (M15 and M7) were
obtained and were found to be identical by DNA sequence analysis. The
M15 clone was between 1224 and 1987 bp of the p96 sequence, and the
original N13 clone extended from 1347 to 2136 bp of the p96 sequence.
Radiolabeled DNA probes were synthesized from the M15 clone and used to
screen both a macrophage
gt11 and a brain
gt10 library for
complementary sequences. Clone M15 and the seven largest clones arising
from these screens were sequenced completely and are schematically
shown in Fig. 1. The sequence analysis of the cDNA fragments
suggested the existence of three alternately spliced forms of the
protein.
Analysis of the p96 Sequence
The DNA
sequences from the clones in Fig. 1were aligned, and the
composite sequence is shown in Fig. 2. The boxedareas in Fig. 2indicate the sequences that were
absent in the p93 (residues 209-229) and p67 (residues
230-447) spliced forms of p96. The longest open reading frame
corresponding to the p96 protein detected in BAC1.2F5 cell lysates was
predicted to encode a protein with 837 amino acids and a molecular
weight of 90,764 and a pI of 6.62. The AUG at 217 bp was preceded by
stop codons in all three reading frames and flanked by bases consistent
with its function as an initiator codon (Kozak, 1987). The p93 protein
was predicted to contain 816 amino acids with a molecular weight of
88,545 and a pI of 7.95. The p67 protein was predicted to have 619
amino acids with a molecular weight of 67,232 and a pI of 7.25. In all
cases, the most abundant amino acid residues in all of the splice
variants were proline comprising between 10.9 and 11.5 mol % and
serine, which comprised between 10.4 and 11.6 mol %. The p96 protein
was hydrophilic, and there was no indication of hydrophobic domains in
p96 that would suggest interaction with membranes.
Figure 2:
DNA and predicted amino acid sequence of
p96. The complete cDNA sequence was assembled by bidirectional sequence
analysis of multiple clones isolated from both a murine macrophage
gt11 library and a murine brain
gt10 library as outlined in
Fig. 1 and described under ``Experimental Procedures.'' The
two ATG start codons in the open reading frame were located at 217 and
418 bp and are highlighted in bold. The firstbox indicates the sequences that were not present in the clone
encoding p93 (amino acids 209-230), and the secondbox indicates the sequences absent from the clone
encoding p67 (amino acids 230-447).
Analysis of the
p96 predicted amino acid sequence revealed three areas with
similarities to known protein structural motifs
(Fig. 3A). First, there was a sequence beginning at
residue 25 (KKEK) that was identical to the sequence in villin that has
been identified as critical for the interaction of villin with actin
(Friedrich et al., 1992). The second domain in the amino
terminus had a high degree of similarity to the amino-terminal domain
of the Drosophila disabled gene product
(Fig. 3B). These two proteins were 46% identical and 66%
similar in this region. The third region was a domain rich in proline
and serine residues that contained five proline-rich sequences
(Fig. 3A) that represent potential binding sites for
proteins containing SH3 domains (Viguera et al., 1994; Yu
et al., 1994; Feng et al., 1994; Lim et al.,
1994).
Figure 3:
Analysis of the p96 protein sequence.
Panel A, a schematic representation of the domain structure of
p96, p93, and p67. The predicted open reading frames (bars)
contained an actin-binding motif (KKEK) located between residues 25 and
29 of p96, a region between residues 40 and 180 of p96 with similarity
to the Drosophila disabled gene product (Dab), and a
serine/proline-rich region between residues 447 and 837 of p96. The
proline-rich sequences that could potentially associate with SH3
domains were: residues 502-510, PVTPPQAGP; residues
559-567, PASPPPP; residues 617-625, PPQPPP; residues
661-669, PPLVP; and residues 712-720, PPKPAP. The location
of the peptides used to generate the M2 and
M15 antisera are
indicated above the p96 protein. Panel B, the similarity
between the predicted amino acid sequence of p96 and the Drosophila
disabled gene product. Alignment of the amino-terminal regions of
p96 (residues 40-180) and Dab (residues 43-179) showed that
the two proteins were 46% identical and 66% similar in this region.
Identical residues are indicated by
, and similar residues are
indicated by :. Amino acid groups are defined as P, A, G, S, T; Q, N,
E, D; H, K, R, C; V, L, I, M; and F, Y, W.
Expression of p96 Isoforms in BAC1.2F5
Cells
The full-length coding sequences for all three p96
isoforms were cloned into pBluescript KS and expressed in vitro (Fig. 4). The largest proteins synthesized by each of these
clones corresponded to the predicted size of the protein according to
the DNA sequence. Two peptide antisera were generated in rabbits to
characterize the expression of p96 isoforms in BAC1.2F5 cells. The
M15 antiserum was raised against a peptide corresponding to
residues 376-394, which were within the sequences present in p96
and p93 but were absent from p67. The
M2 antiserum was raised
against residues 105-122 that were common to p96, p93, and p67.
The
M15 antiserum immunoprecipitated the p96 and p93 proteins
expressed in vitro but did not precipitate the p67 protein
(Fig. 4). The
M2 antiserum precipitated all three proteins
expressed in vitro. We have also expressed the p96 cDNA clone
in COS-7 cells and detected a single protein band with the
M15
antiserum at the correct molecular weight (data not shown). The in
vitro expression results confirmed the isolation of cDNAs encoding
proteins of the predicted sizes and showed that the proteins reacted
with the
M15 and
M2 antibodies with the anticipated
specificity.
Figure 4:
Translation of p96, p93, and p67 cDNAs
in vitro. The assembled p96, p93, and p67 coding sequences
were cloned into pBluescript KS downstream of the T7 promoter. The
proteins expressed by these constructs were identified using a coupled
transcription/translation reticulocyte lysate system (Promega)
containing 40 µCi/50 µl [S]methionine
(1,000 Ci/mmol). The products were analyzed by SDS-gel electrophoresis
either before (left lanes) or after (right lanes)
immunoprecipitation (intraperitoneal) with either
M15 or
M2
antisera, and the bands were visualized by autoradiography. A plasmid
expressing luciferase was used as a positive
control.
The expression of p96 isoforms in BAC1.2F5 cells was
examined at the protein and mRNA level. BAC1.2F5 cells expressed p96
protein that was electrophoretically retarded following CSF-1
stimulation (Fig. 5A). Both antisera also faintly
detected another protein of approximately 86 kDa. This faint band did
not correspond to any of the cDNAs and was not investigated further. It
is possible that the 86-kDa protein was an undetected splice variant of
p96, a translation product derived from the second initiator methionine
in the sequence, or perhaps a degradation product, although protease
inhibitors were present in all the samples. These data indicated that
BAC1.2F5 cells expressed p96 (or p93) and p67. Northern blots were used
to analyze mRNA expression in BAC1.2F5 cells (Fig. 5B).
Using a labeled probe derived from a sequence unique to p96 and p93, a
single 3.7-kb mRNA was detected (Fig. 5B, lane1). Northern analysis with a labeled probe containing
cDNA sequences common to all three splicing variants revealed two mRNA
species of 3.7 and 2.9 kb. These data supported the idea that BAC1.2F5
cells expressed both the p96 (or p93) protein and the p67 protein. We
performed a reverse transcriptase PCR analysis (Fig. 5C)
since analysis of either protein or mRNA expression could not
unambiguously distinguish between the expression of p96 or p93 in
BAC1.2F5 cells. Primers located outside of the splice junctions were
synthesized (Fig. 1), and PCR products derived from random
priming of mRNA from BAC1.2F5 cells were amplified and cloned. Two PCR
reaction products of 1.3 and 0.66 kb were detected consistent with the
expression of p96 and p67. To conclusively establish the identity of
these bands, they were cloned and digested with AccI
(Fig. 5C). Digestion with this restriction enzyme
distinguished between p96 and p93 because p96 had an AccI site
at nucleotide 873 (Fig. 2) that was missing in p93. Taken
together, these expression data showed that p96 was the most abundant
form of the protein expressed in BAC1.2F5 cells, although the
expression of p67 was also significant. There was no indication that
the p93 isoform was expressed in BAC1.2F5 cells.
Figure 5:
Expression of both p96 and p67 isoforms in
BAC1.2F5 cells. Panel A, antibody specificity and detection of
protein expression in BAC1.2F5 cells by immunoblotting. BAC1.2F5 cells
were starved for CSF-1 for 18 h and either harvested or stimulated for
30 min with CSF-1. A sample of the total cell lysate (150 µg of
protein) was loaded into each lane, and the blots were probed
with either the M15 antibody (left lanes) or the
M2
antibody (right lanes) as described under ``Experimental
Procedures.'' Panel B, analysis of p96/p67 mRNA
expression by Northern blotting. Poly(A)
mRNA was
isolated from exponentially growing BAC1.2F5 cells and, 10 µg of
mRNA per lane was fractionated on a 1.0% agarose gel, transferred to
nitrocellulose, and probed as described under ``Experimental
Procedures.'' Lane 1, the mRNA was detected with a
labeled probe derived from DNA between 1760 and 2136 bp of the p96
sequence, which was present in the p96 and p93 isoforms but not the p67
isoform. Lane 2, the mRNA was detected with a labeled probe
derived from DNA between 1225 and 1440 bp of the p96 sequence. This
probe contained sequences common to p96, p93, and p67. The sizes of the
detected bands were calculated from a standard curve generated from an
RNA ladder run on the same gel. Panel C, RT-PCR was used to
amplify p96 sequences from the mRNA pool from BAC1.2F5 cells as
described under ``Experimental Procedures.'' The two products
(1.3 and 0.66 kb) were cloned and analyzed by restriction enzyme
digestion. Digestion with EcoRI confirmed the presence of the
appropriate insert in the clones. Digestion of the 0.66-kb clone with
AccI yielded a single 0.23-kb band diagnostic for p67, whereas
digestion of the 1.3-kb clone yielded 0.32- and 0.56-kb fragments
diagnostic for p96.
Phosphorylation of p96 in CSF-1-stimulated
Cells
The preliminary immunoblotting experiments with
M15 showed that p96 was retarded in its electrophoretic mobility
following the stimulation of cells with CSF-1 (Fig. 5A).
To determine if this change could be accounted for by phosphorylation,
BAC1.2F5 cells were labeled with
[
P]orthophosphate and stimulated with CSF-1
(Fig. 6). The p96 protein was immunoprecipitated and separated by
SDS-gel electrophoresis. There was a pronounced increase in the
labeling of the protein following CSF-1 stimulation concomitant with a
slower electrophoretic mobility. Two-dimensional phosphoamino acid
analysis before and after CSF-1 stimulation showed that p96 was
exclusively phosphorylated on serine residues. The kinetics of p96
modification following CSF-1 stimulation was examined by immunoblotting
(Fig. 7). The electrophoretic gel shift was detected at 2 min and
was clearly evident by 5 min after CSF-1 stimulation. The
electrophoretic retardation of p96 continued to increase up to 30 min
after CSF-1 stimulation and remained at this level through 120 min (not
shown). Mitogenic stimulation with PC-PLC, sphingomyelinase, and TPA
also triggered a retardation in the electrophoretic mobility of p96
(Fig. 7). Analysis of p96 electrophoretic mobility in
asynchronously growing BAC1.2F5 cell populations showed that the
protein was detected in the slower migrating, phosphorylated molecular
form in growing cells continuously exposed to CSF-1 (data not shown).
Figure 6:
Correlation between retarded
electrophoretic mobility and serine phosphorylation of p96. BAC1.2F5
cells were deprived of CSF-1 for 18 h and metabolically labeled with
[P]orthophosphate (0.166 mCi/ml) in
phosphate-free DMEM for the last 2 h of the CSF-1 starvation period.
The cells were then stimulated by the addition of 90 ng/ml CSF-1 for 30
min. The control and stimulated cells were harvested, lysed, and
immunoprecipitated with
M15 antisera as described under
``Experimental Procedures.'' The immune complexes were
separated by SDS-gel electrophoresis and transferred to an Immobilon-P
membrane. The labeled proteins were visualized by autoradiography
(panel A). The p96 bands were excised from the membrane, and
the phosphoamino acid composition of the hydrolyzed protein was
determined by two-dimensional electrophoresis (panel
B).
Figure 7:
Phosphorylation of p96 phosphorylation
following mitogenic stimulation. BAC1.2F5 cells were arrested in
G by the removal of CSF-1 for 18 h. The cells were then
stimulated with CSF-1 (90 ng/ml) for the indicated times or stimulated
with either PC-PLC (1 unit/ml), sphingomyelinase (0.1 unit/ml), or TPA
(10
M) for 15 min. The cells were lysed,
and 150 µg of total cellular protein per lane was separated by
SDS-gel electrophoresis; the p96 protein was detected by immunoblotting
with
M15 antiserum as described under ``Experimental
Procedures.''
stage
of the cell cycle and then becomes progressively phosphorylated
following incubation of the cultures with CSF-1, PC-PLC,
sphingomyelinase, or TPA. Both PC-PLC and TPA, and also
sphingomyelinase to a lesser extent, are capable of initiating
progression through the first cell cycle (Xu et al., 1993),
and these agents elicit phosphorylation of p96 with the accompanying
gel mobility shift ( Fig. 6and Fig. 7). The detection of
increased p96 phosphorylation within 5 min after the addition of CSF-1
and the exclusive phosphorylation of the protein on serine residues
indicates that p96 is an early, but not an immediate, component in
CSF-1 signal transduction. There are three sites in the carboxyl
terminus of p96 that are potential ERK kinase phosphorylation sites
(Pro-X-Ser/Thr-Pro) (Thomas, 1992) beginning at residues 502,
510, and 559, suggesting that p96 may be an ERK kinase substrate. The
protein also contains several proline-rich sequences that are potential
binding sites for proteins containing SH3 domains. The rules for the
interaction between SH3 domain proteins and proline-rich sequences (Yu
et al., 1994; Viguera et al., 1994; Feng et
al., 1994; Lim et al., 1994; Sparks et al.,
1994) indicate that all five of the sequences found in p96
(Fig. 3A) possess the minimum structural features
required to interact with SH3 domain proteins, although p96 does not
possess a proline-rich sequence that will bind with high affinity to
well characterized SH3 domains. However, the idea that p96 interacts
with proteins possessing SH3 domains should not be discarded since the
SH3 domain is a common structural motif found in many cytoskeletal and
cytoplasmic proteins (Musacchio et al., 1992). The p96 protein
was found primarily in the soluble fraction of detergent cell lysates,
although up to 15% was associated with the cytoskeletal
fraction.
(
)
S-labeled DOC-2 antisense
riboprobe shows that the hybridization signal is restricted to the
surface epithelial cells of the ovary, the cell type thought to give
rise to ovarian carcinomas. Mok et al.(1994) suggest that the
expression pattern of p96 (DOC-2) indicates that this gene may be
involved in the development of ovarian carcinomas; however,
considerably more information on the function and expression of p96 is
required to substantiate this interesting idea.
/EMBL Data Bank with accession number(s) U18869.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.