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J Biol Chem, Vol. 274, Issue 37, 26361-26368, September 10, 1999
From the Departments of Affinity chromatography, employing the
extracellular domain of the Sea receptor, was used to enrich
Sea-binding proteins from chicken serum. One isolated protein bound
both a Sea-immunoglobulin fusion protein and an antisera raised against
murine macrophage stimulating protein. Amino-terminal sequencing of the
dual-reactive protein yielded sequences which were identical to the
predicted The receptor protein-tyrosine kinases are a structurally related
family of transmembrane proteins which regulate a wide variety of
cellular responses to extracellular stimulation (2). The binding of
ligand to the extracellular domain of these receptors results in rapid
intracellular autophosphorylation followed by the phosphorylation of
multiple downstream effector proteins (2).
The Met receptor protein-tyrosine kinase (3, 4) is the prototype of a
subfamily of receptor protein-tyrosine kinases. Other members of this
subfamily include the
RON1/Stk (5-7) and Sea
receptors (8). A common feature of this subfamily is that the mature
version of the receptor consist of a heterodimer between a small The Sea receptor was originally identified as the cellular homologue of
the avian retroviral oncoprotein v-sea. This avian erythroblastosis
virus (S13) induced sarcomas, erythroblastosis, and anemia in infected
birds (11, 12). Transformation of erythroid cells with the sea oncogene
rendered them erythropoietin independent (13, 14). The Sea receptor is
a heterodimeric protein composed of a 35-kDa A family of related proteins serve as ligands for these receptors.
Hepatocyte growth factor/scatter factor (HGF/scatter factor) is the
ligand for Met (17, 18), and macrophage-stimulating protein/hepatocyte
growth factor-like protein (MSP) is the ligand for RON/Stk (19, 20).
The ligand for the Sea receptor has not yet been identified. The known
ligands are part of an extended family of related proteins which
includes plasminogen. Like plasminogen, these growth factors require
proteolytic cleavage between homologous Arg-Val residues, which
generates heterodimeric disulfide-bonded subunits, for biological
activity (21). Both HGF and MSP are ~80-kDa heterodimeric proteins
which are found in plasma (22, 23). A number of studies indicated that
HGF has mitogenic, morphogenic, and motogenic effects on a variety of
cell types (reviewed in Ref. 24). Likewise, MSP has been shown to have
a variety of biological activities, particularly on cells of the
hematopoetic lineage. It induced macrophage shape change and
chemotactic migration (25), apoptosis of an erythroid cell line (26),
and megakaryocytopoiesis (27). MSP has also been shown to be active on
epithelia, bone, and neuroendocrine tissues (28).
In order to better understand the biological role of the Sea receptor,
we searched for a protein which would act as its ligand. As part of the
search, we tested whether chicken MSP (chMSP) could serve as a Sea
ligand. Although the gene encoding the chMSP has been cloned (9), there
are no reports of its recombinant expression. We obtained MSP from both
a natural source, chicken serum, and a recombinant source in order to
study its ability to activate the Sea receptor protein-tyrosine
kinases. The recombinant expression of activatible human or murine MSP
is reported to be problematic due to the presence of a specific Cys
residue which interferes with disulfide linkage of the nascent subunits
of MSP (1). Since the predicted sequence of chMSP, based on the cloned
gene, indicated the presence of a similar Cys residue, a Cys to Ala variant of chMSP was also studied. Reported here is our assessment of
chMSP as a potential ligand for Sea.
Isolation of Sea-Fc-binding Proteins from Chicken Serum--
The
Sea-Fc construct (14) was transfected into Chinese hamster ovary cells
and conditioned media produced as described for other recombinant
proteins (29). The Sea-Fc was purified with a protein G-Sepharose
HiTrap column (Mab Trap G II, Amersham Pharmacia Biotech) following the
manufacturer's instructions.
One liter of undialyzed chicken sera (Life Technologies, Gaithersburg,
MD), containing ~20 g of protein, was chromatographed on a 80-ml
heparin-Sepharose column (Amersham Pharmacia Biotech) that was
equilibrated with 0.15 M NaCl, 20 mM sodium
phosphate, pH 7.0 (PBS). The column was washed with PBS to remove
unbound proteins, and bound proteins were eluted with 2 M
NaCl, 20 mM sodium phosphate, pH 7.0. The 2 M
salt eluate, containing ~600 mg of protein in 150 ml, was dialyzed
overnight at 4 °C versus PBS, and then serine proteases
were inactivated with 1 mM Pefabloc (Roche Molecular
Biochemicals, Indianapolis, IN). Sea-Fc was coupled to cyanogen
bromide-activated Sepharose at 1 mg of Sea-Fc/ml gel, according to the
manufacturer's instructions (Amersham Pharmacia Biotech). The
heparin-Sepharose eluate was chromatographed on a 1-ml column of
Sea-Fc-Sepharose equilibrated with PBS. After sample application, the
column was washed with PBS until the A280 returned to baseline, then washed with 5 ml of PBS + 0.35 M
salt, followed by 10 ml of a high pH elution buffer consisting of 50 mM CAPS, pH 10.4, 0.5 M NaCl, 1 mM
CHAPS, 0.005% Tween 20. Both elution buffers eluted well defined peaks
of absorbtion at 280 nm containing 1-2 A280
units. Fraction 15 (Fig. 1) contained about 1 mg of protein. Unless
otherwise stated, chemical reagents came from Sigma, while the
molecular biology reagents came from Roche Molecular Biochemicals.
Gel Electrophoresis, Western Blot, and Sequence Analysis of
Purified and Recombinant Proteins--
SDS-PAGE gels were from Novex
(San Diego, CA). Nonreduced samples were mixed with sample buffer
containing 2% SDS but not
For Western blot analyses, separated proteins were transferred to
nitrocellulose or Immobilon-P membranes (Millipore, Bedford, MA).
Membranes were blocked with either 5% nonfat dry milk or 5% bovine
serum albumin and 1% ovalbumin (for anti-phosphotyrosine (anti-Tyr(P))
blots only) in 20 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature. For MSP detection, the membranes were incubated with 4-20 µg/ml anti-murine MSP
antibodies (raised against murine pro-MSP (1)) for 1 h at room
temperature, followed by donkey anti-rabbit IgG conjugated with
horseradish peroxidase (Amersham) for 20 min at room temperature. For
Sea-Fc detection, the membranes were incubated with 0.3-0.5 µg/ml
Sea-Fc for 1 h at room temperature, followed by goat anti-human
IgG (Fc)-conjugated with horseradish peroxidase (Pierce, Rockford, IL)
for 20 min at room temperature. For Sea detection, the membranes were
incubated with 4 µg/ml of the 4E10 antibody (15) for 1 h at room
temperature, followed by sheep anti-mouse IgG conjugated with
horseradish peroxidase (Amersham) for 20 min at room temperature. For
anti-Tyr(P) detection, the membranes were incubated with 1 µg/ml
anti-phosphotyrosine antibody (Upstate Biotechnology, Lake Placid, NY)
for 1 h at room temperature, followed by sheep anti-mouse IgG
conjugated with horseradish peroxidase (Amersham) for 20 min at room
temperature. For the Shc and ERK detection experiments, the membranes
were incubated either with 1 µg/ml rabbit anti-human Shc antibody
(Upstate Biotechnology, Lake Placid, NY) or with 1 µg/ml rabbit
anti-ERK 1/2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature, followed by donkey anti-rabbit IgG
conjugated with horseradish peroxidase (Amersham) for 20 min at room
temperature. Bound immune complexes were detected with ECL reagents
(Amersham). Amino-terminal sequencing was performed on an
Immobilon-PSQ (Millipore, Bedford, MA) blot using a Procise
494 sequencer (PE-ABD, Foster City, CA).
Detection of Tyrosine Phosphorylation in Sea-expressing 32D
Cells--
32DSea5-2 cells (15) were grown in RPMI 1640 media (Life
Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS) and 5% WEHI-3-conditioned medium
from WEHI-3 cells.
For phosphorylation studies, 32DSea5-2 cells were grown to the
exponential proliferative phase, and then switched to the above media
containing 0.5% fetal bovine serum (starvation media) and grown
overnight. A suspension of 1 × 107 cells were spun at
210 × g for 5 min at room temperature. Cells were
resuspended in 100 µl of conditioned (overnight) starvation media and
the cells and protein samples were prewarmed separately at 37 °C for
2 min. After the preincubation, cells and protein samples were
mixed together and incubated at 37 °C for 15 min.
To stop the phosphorylation reaction, 10 ml of cold 1× PBS was
added to each sample and the cells were lysed in 1 ml of Nonidet P-40
lysis buffer (50 mM Tris, pH 8.0, 1% Nonidet P-40, 2 mM EDTA, 150 mM NaCl, 400 µM
orthovanadate, and 10 µg/ml aprotinin). Lysates were
immunoprecipitated with either the monoclonal anti-Sea antibody 8C3
(14) or anti-Tyr(P) (Upstate Biotechnology, Lake Placid, NY) at 4 °C
overnight. Antibody-antigen complexes were then incubated with 50 µl
of protein G-Sepharose beads (Amersham Pharmacia Biotech) at 4 °C
for 30 min. Immune complexes were pelleted, washed, and resuspended in
sample buffer.
PCR Cloning of Chicken MSP Gene--
A full-length cDNA of
chMSP was constructed from three fragments generated by polymerase
chain reaction (PCR) using as template an adult chicken liver
5'-stretch cDNA library in Construction of C665A Chicken MSP--
Cys665 was
converted to Ala using an overlap amplification process. Wild type
chicken MSP plasmid and oligonucleotides 8 and 9 containing the Cys to
Ala change at nucleotides 2005 and 2006 were used to generate mutated
chicken MSP cDNA. The 67-bp product was generated by carrying out 5 cycles at 94 °C (15 s), 60 °C (15 s), and 72 °C (30 s),
followed by another 20 cycles at 94 °C (15 s), 64 °C (15 s), and
72 °C (30 s) with oligonucleotides 7 and 8. The 146-bp product was
generated by carrying out 5 cycles at 94 °C (15 s), 58 °C (15 s),
and 72 °C (30 s), followed by another 20 cycles of 94 °C (15 s),
68 °C (15 s), and 72 °C (30 s) with oligonucleotides 6 and 9. Both amplified fragments were isolated from a polyacrylamide gel and
eluted with MAXAM buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% SDS, pH
8.0). Equal molar ratios of the two isolated fragments were re-amplified with oligonucleotides 7 and 10 for 25 cycles at 94 °C
(15 s), 64 °C (15 s), and 72 °C (30 s) to generate the 192-bp mutated product. The isolated 192-bp fragment was digested with restriction enzymes ApaI and EcoRI to a 149-bp
fragment. Wild type chicken MSP plasmid was digested with restriction
enzymes EcoRI and BglII to generate a
4.6-kilobase fragment, and BglII and ApaI to
generate the 775-bp fragment. The mutated fragment was then ligated
with the 4.6-kilobase and 775-bp fragments to generate the mutated
chMSP cDNA in the pBJ5 expression vector.
Production of Recombinant MSP in COS Cells--
COS-7 cells were
transfected as described previously (30). Conditioned media of
transfected cells were concentrated by Centriprep-10 (Amicon, Beverly,
MA). In order to activate recombinant pro-MSP, concentrated conditioned
media was incubated at 37 °C for 1 h with human kallikrein
(Enzyme Research Laboratories, South Bend, IN) at a final concentration
of 20 µg/ml followed by addition of Pefabloc-SC (Roche Molecular
Biochemicals, Indianapolis, IN) to a final concentration of 0.1 mM to stop the proteolytic activation.
For some experiments, conditioned media was concentrated 100× by
chromatography on a 1-ml HiTrap heparin-Sepharose column (Amersham
Pharmacia Biotech) and eluted with 2 M NaCl. The buffer was
exchanged to PBS using Centricon-10 (Amicon Corp.) and activated by
kallikrein as above.
Sea-Fc Competition Studies--
30× concentrated, activated
conditioned media from COS cells transfected with C665A chMSP was
either mock-treated (PBS incubation) or preincubated with 10 or 100 µg of either the Sea-Fc protein or the leptin receptor-Fc protein (an
unrelated Fc fusion protein containing the extracellular domain of the
leptin receptor). The mixtures were incubated at 4 °C for 1 h,
then used to treat the 32DSea5-2 cells as above. The final
concentration of the C665A chMSP used to treat the cells represented
12× concentrated conditioned media.
During the course of our work with recombinant murine wild type
and C677A variant MSP (1), we observed that the Sea-Fc fusion protein
could be used as the primary probe in a Western blot
analysis.3 As such, it
detected active murine MSP with high sensitivity (see below), while
neither pro-MSP nor reduced MSP nor any species other than the intact
heterodimer were detected (data not shown). These observations
suggested that the Sea-Fc might be a useful reagent for affinity
purification of the Sea ligand. Since the known ligands for this family
of receptors are detected in normal sera (22, 23), we investigated
whether there were Sea-binding proteins present in chicken sera.
Both HGF and MSP bind strongly to heparin-Sepharose at neutral pH and
physiological salt concentration (0.15 M), and can be eluted with increasing salt concentrations of about 0.8 M
salt for MSP (31) and 1.2 M salt for HGF (32). Since it was
anticipated that the Sea ligand might have a similar tight
heparin-binding property, a heparin-Sepharose binding step with a
single high salt elution step was employed as an initial purification
step. One liter of chicken serum was chromatographed on a
heparin-Sepharose column and bound proteins were eluted with high salt.
The interaction of plasminogen-related growth factors with their
receptors requires proteolytic activation of the ligands (21).
According to literature reports, the heparin-Sepharose chromatography
step used here was expected to purify both plasma kallikrein (33), an
activator of human MSP (21), and hepatocyte growth factor activator
(34). Indeed, incubation of the high salt eluate from the
heparin-Sepharose column with the kallikrein substrate
benzoyl-Pro-Phe-Arg-nitroanilide (35) resulted in considerable
hydrolysis of the nitroanilide (data not shown), while the
unfractionated chicken serum was inactive. This observation suggested
that endogenous proteinase inhibitors had been removed and serum
proteases had become activated during purification and were present in
the eluate. Accordingly, the heparin-Sepharose eluate was incubated
overnight at 4 °C in order that the endogenous, active serum
proteases would activate potential plasminogen-related growth factors
that were present. After incubation, the endogenous proteases were
inactivated with Pefabloc, and the high salt heparin-Sepharose eluate
was dialyzed versus PBS and then chromatographed on a Sea-Fc
column. Bound proteins were eluted with a pH 10.5 buffer. Both the
heparin-Sepharose and the Sea-Fc column eluted material showed well
defined peaks of absorbtivity at 280 nm.
Analysis of the four peak fractions (numbers 13-16), as indicated by
A280 to contain proteins eluted from the
Sea-Fc-Sepharose column, is shown in Fig.
1. Staining with Coomassie Blue revealed that this two-step purified material contained multiple protein bands,
but the overall protein complexity was reduced considerably relative to
the starting sera (fraction 15 contains 1 mg of protein compared with
800 mg in the heparin pool, and 20 g in the starting serum). It
was expected that isolation of some of the proteins resulted from
nonspecific interactions with either the Sepharose resin or the Fc
portion of the Sea-Fc, a result observed with other affinity
purifications (data not shown). To characterize the proteins eluting
from the Sea-Fc column further, Western blot analyses were performed.
When the Sea-Fc fusion protein was used as the primary probe, a protein
band with an apparent mobility of 84 kDa was readily detected in all
the lanes, but especially in fraction 15 (Fig. 1B).
Although, several protein species were eluted from the Sea-Fc column
(Fig. 1A), the subsequent Western blot result implies a
particularly strong interaction between the Sea-Fc and the 84-kDa
species. For comparison, the same gel contained a standard of 100 ng of
mMSP, which also reacted with the Sea-Fc. When a similar blot was
probed with an anti-murine MSP antisera, only the lane containing the
largest amount of the 84-kDa protein was detected (Fig. 1C).
On the same gel, 1 ng of mMSP was readily detected. Comparison of Fig.
1, B and C, reveals that the 84-kDa band reacts
stronger with the Sea-Fc than 100 ng of mMSP; but reacts weaker with
the anti-murine MSP than 1 ng of mMSP. To summarize, the partially
purified preparation contained a discrete Coomassie-staining doublet
band of about 84 kDa that bound the Sea-Fc and anti-murine MSP
antisera.
To further characterize the reactive protein(s),
NH2-terminal sequencing was performed on the 84-kDa
reactive protein from fraction 15 of the Sea-Fc-Sepharose eluate and on
an additional preparation of chicken serum. The resulting residues and
yield, listed in Table II, identify the
84-kDa doublet band as chMSP. Sequences from both the
Chicken Macrophage Stimulating Protein Is a Ligand of the
Receptor Protein-tyrosine Kinase Sea*
§,
**,
,,,, and High Throughput Screening,
¶ Molecular Genomics, and §§ Mammalian
Genomics, Amgen Inc., Thousand Oaks, California 91320 and the
Department of Microbiology, Health Sciences Center, University
of Virginia, Charlottesville, Virginia 22908
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and 
subunits of chicken macrophage stimulating
protein. The partially purified chicken macrophage stimulating protein
caused autophosphorylation of the Sea receptor. Previous work showed that recombinant expression of fully activatible human or mouse macrophage stimulating protein required a specific Cys to Ala substitution (Wahl, R. C., Costigan, V. J., Batac, J. P., Chen, K., Cam, L., Courchesne, P. L., Patterson, S. D. Zhang, K., and Pacifici, R. E. (1997) J. Biol.
Chem. 272, 1-4). Therefore, we expressed both the wild type and
the specific Cys to Ala form of chicken macrophage stimulating protein
as recombinant proteins. After proteolytic activation, only conditioned
media from COS cells transfected with the C665A chicken macrophage
stimulating protein, but not from wild type chicken
macrophage-stimulating protein, or control vector, was detected by the
Sea-immunoglobulin fusion protein in Western blotting
experiments. Conditioned media containing the C665A chicken
macrophage-stimulating protein readily caused Sea phosphorylation,
while conditioned media containing the wild type chicken
macrophage-stimulating protein was only effective at inducing receptor
phosphorylation at high concentrations. In addition to receptor
phosphorylation, the C665A chicken macrophage-stimulating protein
induced phosphorylation of Shc, Erk1, and Erk 2. We conclude that
macrophage-stimulating protein is a ligand of the Sea receptor protein-tyrosine kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain (~35 kDa) and a larger chain (~160 kDa) which arise from
proteolytic cleavage of a precursor protein. The gene encoding Met has
been identified in both mammalian and chicken species (3, 4, 9).
However, RON/Stk has only been identified in mammalian species, while
Sea has only been found in chicken species, despite repeated efforts to
find orthologs in other species
(10).2
and a 160-kDa chain and is expressed at low levels in a variety of tissues including
kidney, intestine, liver, stomach, white blood cells, and
allantochorion (8, 15). Protein comparisons indicate that the
extracellular domain of the Sea receptor is more similar to RON (48%
identity) or stk (44% identity) than to Met (39% identity). Analysis
of the extracellular domain of chicken Met indicates 72% identity to
human MET, but only 37% identity to RON. Because a mammalian analog of
Sea has not yet been identified, and since a chicken Met gene but not a
chicken Ron gene has been identified, it is possible that RON and Sea are orthologous receptors. However, definitive data has not yet been
obtained to support or refute this hypothesis. Analysis of downstream
signaling events indicated that activation of the Sea intracellular
domain (fused to the extracellular domain of Trk and activated by nerve
growth factor) triggered similar biological responses to those of RON
or Met when these receptors were stimulated by their respective ligands
(16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, and were not heated.
gt10 (CLONTECH Laboratories, Palo Alto, CA). The primers listed in Table
I were designed according to the sequence
of GenBank accession number X84043 (9). Five µl (~5 × 105 plaque forming units) of phage library was used for
each PCR. Amplification was carried out for 35 cycles of 94 °C (15 s), 60 °C (15 s), and 72 °C (90 s) using AmpliTaq DNA Polymerase
with the GeneAmp kit (Perkin-Elmer, Norwalk, CT). The 354-bp product generated by oligonucleotides 1 and 2 was digested with the restriction enzymes XhoI and NarI to 270 bp; the 1245-bp
product generated by oligonucleotides 3 and 4 was digested with
restriction enzymes NarI and BglII to 936 bp; and
the 986-bp product generated by oligonucleotides 5 and 6 was digested
with restriction enzymes BglII and EcoRI to 975 bp. These three fragments were ligated into the mammalian expression
vector pBJ5 (30) pre-linearized with restriction enzymes
XhoI and EcoRI and the resulting construct was
transformed into DH10B cells (Life Technologies, Gaithersburg, MD). The
confirmed sequence of this full-length cDNA construct was identical
to the published sequence (9) except for a silent nucleotide change (C
instead of G) located at nucleotide 1981.
Sequences and positions of oligonucleotide primers (sequences written
in lowercase represent linkers that were added to the chMSP primers)
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
An 84-kDa protein in chicken sera binds to
both the Sea-Fc protein and an anti-murine MSP antisera. Chicken
sera proteins were eluted with a pH 10.5 buffer from a Sea-Fc-Sepharose
column and eluting fractions 13, 14, 15, and 16 were used for analysis.
In panels A, B, and C, identical sample fractions
were analyzed in parallel by 10% nonreducing SDS-PAGE. Panel
A, the SDS-PAGE gel was stained with Coomassie Blue. The end lane
contains 100 ng of purified, recombinant mMSP. Panel B,
parallel samples were separated by SDS-PAGE, transferred to
nitrocellulose, and probed with the Sea-Fc protein. The end lane
contains 100 ng of purified, recombinant mMSP. Panel C, same
as panel B except the nitrocellulose was probed with an
anti-murine MSP antisera. The end lane contains 1 ng of purified mMSP.
The isolated chMSP migrates slightly faster than the recombinant mMSP
as indicated on the right.
and subunits were detected. The presence of the amino acid residues His,
Arg, and Leu in the first, tenth, and eleventh position of the subunit sequence, and Met and Ile in the fifth and fifteenth position
of the subunit are characteristic of chMSP and differ from mouse
and human MSP (8). The NH2 terminus of the subunit of
chMSP is different from that predicted by Thery et al. (9).
By comparing the intensity of the 84-kDa band in fraction 15 to the
intensity of a known amount of mMSP, we estimate that about 50 µg of
chMSP was recovered from a liter of chicken serum. Rechromatography of
the Sea-Fc-Sepharose flow-through on the Sea-Fc-Sepharose, resulted in
the recovery of only 1/10 of the amount of chMSP found in the first
passage. This suggested that the amount of Sea-Fc-Sepharose was not
limiting in this purification and the majority of protein capable of
binding to Sea-Fc was recovered by the process. This overall
purification was repeated five times with similar results (data not
shown).
Edman sequencing of chicken serum MSP
and chains of chMSP, while in the second experiment, only one
sequence (labeled seq 2) corresponding to the chain of chMSP was
obtained.
Having recovered Sea-binding proteins which included chMSP, we
determined if they were capable of inducing Sea receptor
phosphorylation. A stably transfected 32D cell line expressing the Sea
receptor (15) was treated for 15 min with two amounts of the partially purified protein mixture from above. As shown in Fig.
2A, either 9 µl (lane
1) or 50 µl (lane 2) of the protein mixture resulted in phosphorylation of the Sea receptor. The ligand was present in
limiting amounts as an increased amount of protein resulted in an
increase in Sea phosphorylation. Treatment of the cells with PBS
containing 1% bovine serum albumin (lane 3) failed to induce phosphorylation. Analysis of a parallel set of samples by
Western blot using an anti-Sea antibody indicated that equivalent amounts of Sea receptor were present in all samples (Fig.
2B).
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The preceding results strongly implied that chMSP is the purified
protein responsible for Sea phosphorylation. However, since the protein
mixture was not homogeneous, it remained possible that another protein
was responsible for the observed phosphorylation. To directly address
this, we produced chMSP as a recombinant protein in a mammalian
expression system. Based on the published chMSP sequence (8), primers
were used to amplify the coding region from a chicken cDNA library.
Comparison of the isolated sequence to the published sequence revealed
identity except for a single conservative nucleotide change, a C
instead of a G, at position 1981. Analysis of a variety of clones from
several PCR experiments confirmed the isolated sequence. Previous work
on mouse and human MSP indicated that a Cys (residue 672 in the human
and 677 in the murine sequence) to Ala mutation was required for
maximal potency of the recombinant protein (1). The presence of a
homologous Cys residue in the chMSP (residue 665 in the chicken
sequence) indicated that a similar amino acid residue change would
likely be required, so the corresponding C665A MSP cDNA was
produced and expressed in parallel to the wild type chMSP. Based on
previous experiments with mouse or human MSP (1), it was anticipated that conditioned media would require treatment with kallikrein to
convert pro-MSP to active MSP. Therefore parallel batches of conditioned media were either untreated or treated with kallikrein and
incubated with Sea-expressing 32D cells. As shown in Fig. 3A, none of the nonactivated
conditioned media were able to induce Sea phosphorylation (lanes
1-9). In contrast, activation of the wild type or C665A MSP
containing conditioned media resulted in Sea phosphorylation
(lanes 15-18). Only the highest concentration of wild type
MSP-conditioned media led to detectable phosphorylation, while a clear
dose-response was obtained with the C665A chicken MSP-conditioned media
(lanes 16-18). Neither nonactivated nor activated
conditioned media from mock-transfected COS cells induced phosphorylation at any concentration (lanes 1-3 and
10-12). Analysis of similar blots probed with anti-Sea
antisera indicated that all treated cell samples contained equivalent
amounts of the Sea receptor (Fig. 3B). These results clearly
demonstrate that both wild type and C665A chicken MSP can induce Sea
phosphorylation.
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The ability of mMSP, purified natural chMSP, and conditioned media
containing recombinant chMSP (wild type and C665A) to interact with
either the Sea-Fc or the anti-MSP antisera in a Western analysis were
compared in Fig. 4. As shown previously,
Sea-Fc detected ~84-kDa bands in the murine C677A MSP and purified
chMSP preparations (Fig. 4A). Sea-Fc detected the C665A
chicken MSP only after activation (compare lanes 4 and
7). Therefore, the proteolytic cleavage-induced activation
is required for the interaction of chMSP with the Sea-Fc. Sea-Fc did
not detect bands in conditioned media from either wild type recombinant
chMSP nor mock-transfected COS cells. The inability to detect
recombinant chMSP with the Sea-Fc was likely a matter of sensitivity
because similar amounts of the protein were able to induce Sea
phosphorylation (Fig. 3A). The observation of similar results with the phosphorylation studies (Fig. 3) indicated that chMSP
activation is necessary for both binding to the Sea-Fc and inducing
receptor phosphorylation. The observed ~160-kDa reactive band(s) stem
from the kallikrein treatment and were present in the kallikrein
mixture alone used to treat the conditioned media (lane 3).
We have never observed Sea-Fc to detect any band other than the 84-kDa
heterodimer.
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Fig. 4B shows the results when the same set of proteins were probed with anti-MSP antisera. As shown previously, both the C677A mMSP and the purified chMSP were recognized by the antisera (lanes 1 and 2). Although the C665A chMSP was recognized by the antisera regardless of activation, activation of the wild type recombinant chMSP caused the disappearance of the ~84-kDa reactive band. Prolonged exposures of the lower panel showed a lower, ~50-kDa reactive band in the wild type chMSP-conditioned media after treatment (data not shown).
In order to directly compare C677A mMSP to C665A chMSP, both were
produced in COS cell-conditioned media. Varying amounts of activated
COS cell-conditioned media containing either C665A chicken or C677A
murine MSP were used to treat Sea-expressing cells. As before, the
chMSP-conditioned media readily caused Sea phosphorylation, while none
of the amounts of mMSP were able to induce phosphorylation (Fig.
5A). Analysis of parallel
blots probed with anti-Sea antisera indicated that all tested samples
contained the same amount of Sea receptor (Fig. 5B). A
separate Western blot analysis indicated that both proteins were
present in conditioned media, but because of their different reactivity
(murine greater than chicken) it was not possible to directly compare
expression levels. In a parallel experiment, purified, recombinant
C677A mMSP, at concentrations as high as 5 µg/ml, failed to induce
Sea phosphorylation (data not shown) even though this material was active on the murine receptor (1). Thus, Sea receptor phosphorylation induced by MSP is species specific.
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The Sea-Fc fusion protein was able to block the ability of recombinant C665A chMSP to stimulate autophosphorylation of the Sea receptor (Fig. 5, C and D). Preincubation with either 10 µg (Fig. 5C, lane 3) or 100 µg of Sea-Fc (Fig. 5C, lane 4) inhibited subsequent phosphorylation of the Sea receptor. However, neither 10 µg (Fig. 5C, lane 6) nor 100 µg (Fig. 5C, lane 7) of an unrelated Fc fusion protein (comprising the extracellular domain of the leptin receptor) had any effect on chMSP-induced receptor phosphorylation. Therefore, the chMSP is binding specifically to the Sea receptor, and inducing phosphorylation.
To begin to understand the biological significance of the interaction
of MSP with the Sea receptor, we examined whether potential downstream
targets of the Sea receptor were phosphorylated in a
ligand-dependent manner. Activated conditioned media from
COS cells either mock-transfected or transfected with the C665A chMSP were used to treat Sea-expressing 32D cells. Cells were lysed and
various proteins immunoprecipitated and analyzed by Western blot. Only
treatment of the cells with C665A chMSP-conditioned media led to the
specific phosphorylation of Erk 1 and Erk 2 (Fig. 6A), and Shc (Fig.
6B). Treatment with mock-transfected COS cell-conditioned media failed to cause an increase in phosphorylation above the level
seen with cells treated without COS cell-conditioned media (compare
lanes 1 and 3). Shown for comparison are results
from Sea-expressing 32D cells treated with interleukin 3, a cytokine known to cause phosphorylation of similar signal transduction molecules
and required for the survival and growth of 32D cells. Attempts to grow
Sea-expressing 32D cells with chicken C665A MSP-conditioned media in
the absence of interleukin 3 were not successful (data not shown),
however, purified, active recombinant hMSP was also unable to abrogate
the interleukin 3 dependence of 32D cells expressing the RON
receptor (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The biochemical purification that resulted in the isolation of chMSP was designed for the generic purification of plasminogen-related growth factors, as the known ligands in this family are detected in normal sera (22, 23), and bind strongly to heparin-Sepharose (31, 32). Likewise, the MSP-activating enzyme, kallikrein, and HGF activator, are also detected in normal sera and bind strongly to heparin-Sepharose (33, 34). We noted that our post-heparin purified pool was active in hydrolyzing a kallikrein substrate. Thus, since MSP was shown to interact with Sea-Fc by our Western blot procedure, it is not surprising that activated chMSP was purified from chicken serum by our two-step purification. It should be reiterated that this Western blot technique did not work when the disulfide bonds of MSP are reduced prior to SDS-PAGE.
Our results clearly demonstrate that chMSP is capable of acting as a ligand for the Sea receptor. Chicken MSP bound to a Sea-Fc protein, and caused both the autophosphorylation of Sea as well as the phosphorylation of downstream target proteins including Shc, Erk 1, and Erk 2. These activities were seen with chMSP derived from two different sources, either as a natural purified protein or as a recombinantly produced protein. It has been reported that a Cys to Ala variant of mMSP is the most active recombinant form, but the recombinant MSP with the complete wild type sequence showed activity at high concentrations (1). Similar results are shown here for the chMSP. The wild type sequence showed activity at high concentrations, while the C665A variant showed much greater activity. Preincubation of the C665A variant with the Sea-Fc soluble protein blocked the chMSP from inducing Sea receptor phosphorylation thus confirming receptor-specific binding.
Although mMSP was detected readily by Sea-Fc in a Western blot, it was unable to induce receptor phosphorylation, even using concentrations as high as 5 µg/ml of purified recombinant material (data not shown). This reinforces the concept that receptor binding is necessary, but not sufficient for causing receptor dimerization necessary for autophosphorylation. There is only 60% identity between chicken and mouse MSP. The species selectivity was also manifest in the better reaction of mMSP with the anti-murine antisera, and the better reativity of Sea-Fc with chMSP.
As shown previously for hMSP and mMSP (1), a conserved Cys residue has
a deleterious effect on recombinant expression of activatible chMSP.
Without activation, both the wild type and C665A variant are readily
detected as ~84-kDa proteins which bind anti-MSP antisera (Fig. 4).
However, after activation, the wild type recombinant protein is no
longer detected as an ~84-kDa protein. Long exposures detected the
presence of a much smaller ~50-kDa protein which could bind the
anti-MSP antisera. We interpret these results to indicate that the wild
type recombinant chMSP contained misfolded disulfide bonds which result
in a loss of the intersubunit disulfide, and an increased lability to
kallikrein. It has been reported that the RON-binding domain of MSP is
located in the chain (36). However, we never observed Sea-Fc
binding via the Western blot technique to any species other than the
84-kDa species. With regard to the partially purified naturally
occurring chMSP, if any disulfide bond variants are present in
vivo, the Sea-Fc affinity chromatography employed here presumably
selected for MSP in which the intersubunit disulfide was present.
Therefore, this material behaved more like the C665A variant. By
Western blot analysis, the Sea-Fc is quite efficient at discriminating between different forms of the chMSP. This is particularly evident in
the results shown in Figs. 3 and 4, where conditioned media containing
recombinant wild type chMSP failed to bind the Sea-Fc even though it
could weakly cause Sea phosphorylation. We interpret this to mean that
a small percentage of the recombinant wild type chMSP was folded
correctly, but was present in too low a concentration to be detected
with the Sea-Fc. It will be of interest to determine whether
non-intersubunit bonded species of MSP are present in vivo,
or whether additional unknown factors present in vivo cause the production of only properly folded MSP.
Although the current data clearly show that MSP is a ligand for the Sea receptor, it remains unclear if Sea and RON/Stk are corresponding orthologs. Protein comparisons indicate that the extracellular domain of the Sea receptor is more similar to RON (48% identity) than to Met (39% identity). Because a mammalian analog of Sea has not yet been identified, and since a chicken Met gene but not a chicken Ron gene has been identified, it has been suggested that indeed RON and Sea are orthologous receptors. Our finding that MSP can activate the Sea receptor provides further evidence that the Sea and RON/Stk receptors are functionally equivalent. However, proof that Sea and RON/Stk are orthologous receptors will require a much more extensive comparative analysis between the chicken and mammalian genomes.
Finally, it is interesting to note that Sea was originally isolated due
to its effect on hematopoiesis. The Sea receptor was identified as the
cellular homologue of the avian retroviral oncoprotein v-Sea. This
avian erythroblastosis virus (S13) induced sarcomas, erythroblastosis,
and anemia in infected birds (11, 12). Transformation of erythroid
cells with the sea oncogene rendered them erythropoietin independent
(13, 14). Likewise, MSP, through the RON/Stk receptor, has been shown
to have a variety of biological activities, particularly on cells of
the hematopoetic lineage. It induced macrophage shape change and
chemotactic migration (25), apoptosis of an erythroid cell line (26),
and megakaryocytopoiesis (27). The demonstration of the involvement of
MSP, Ron/Stk, and Sea in hematopoiesis strongly implies that chicken
MSP will likewise play a role in chicken hematopoiesis. To date, there
is limited information on the tissue distribution of Sea and chicken
MSP. Sea protein has been detected at low levels in several tissues
including the kidney, intestine, liver, stomach, white blood cells, and
allantochorion (15). Chicken MSP mRNA has been detected at all
stages of chicken development, with expression at particular stages
found in the neural tube, notochord, floor plate, myotome, and aortic
arches (9). Our purification studies indicate that adult chicken sera
is a rich source of MSP. Further work will be required to compare Sea
and MSP expression in development and adult chicken tissues to try to
gain clues as to where they may interact. Since MSP is a circulating ligand, it could interact with receptor(s) in a number of tissues, and
thus there may not be a one-to-one correspondence in the expression of
the Sea receptor and the chMSP. The production and purification of
significant quantities of active recombinant chMSP will allow for a
determination of the full range of its in vivo activities.
| |
ACKNOWLEDGEMENT |
|---|
We thank Rick Lindberg for invaluable technical and intellectual assistance.
| |
FOOTNOTES |
|---|
* 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.
§ Contributed equally to the results of this work.
** Current address: Dept. of Biological Sciences, University of Nevada, Las Vegas, 4505 Maryland Pkwy., Las Vegas, NV 89154.
Current address: Upstate Biotechnology, 199 Saranac Ave., Lake
Placid, NY 12946.
¶¶ To whom correspondence should be addressed: Amgen, Inc., MS 14-1-B, One Amgen Center Drive, Thousand Oaks, CA 91320-1789. Tel.: 805-447-2159; Fax: 805-447-1982; E-mail: awelcher@amgen.com.
2 A. A. Welcher, unpublished observations.
3 Based on sequences already in SWISS-PROT, the Cys residue number in human, murine, and chicken is 672, 677, and 665, respectively.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: RON, receptor d'origine nantaise; stk, stem cell tyrosine kinase; Sea, sarcoma, erythroblastosis, and anemia; HGF, hepatocyte growth factor; MSP, macrophage-stimulating protein; Sea-Fc, Sea receptor-immunoglobulin fusion; PBS, phosphate-buffered saline; anti-Tyr(P), anti-phosphotyrosine; PCR, polymerase chain reaction; chMSP, chicken MSP; mMSP, murine MSP; bp, base pair(s); CAPS, 3-(cyclohexylamino)propanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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RESULTS
DISCUSSION
REFERENCES
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) or kallikrein-treated (+),
electrophoresed on SDS-PAGE gels under nonreducing conditions, and
analyzed by Western blot. Panel A, transferred proteins were
detected with the Sea-Fc protein. Panel B, transferred
proteins were detected with an anti-murine MSP antibody. Lane
1A, 50 ng (lane 1B, 5 ng) previously activated C677A
murine MSP; lane 2, partially purified chicken serum MSP;
lane 3, PBS; lanes 4-9 contain concentrated
media from variously transfected COS cells. Lanes 4 and
7, recombinant C665A chMSP; lanes 5 and
8, recombinant wild type chMSP; lanes 6 and
9, conditioned media from mock-transfected cells. Note that
for the recombinant samples, activation with kallikrein is necessary
for reaction with Sea-Fc (lane 4 versus 7). However, for
reaction with the anti-MSP antisera, the activation with kallikrein
apparently destroys the wild type recombinant chMSP (lane 8 versus lane 5). Molecular weight markers (Amersham Pharmacia
Biotech) are shown on the right.
