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RAPID COMMUNICATION
Membrane-Bound Steel Factor Induces More Persistent Tyrosine Kinase
Activation and Longer Life Span of c-kit Gene-Encoded Protein
Than Its Soluble Form
By Keisuke Miyazawa, David A. Williams, Akihiko Gotoh, Jiroh Nishimaki, Hal E. Broxmeyer, and Keisuke Toyama
Alternative splicing of exon 6 results in the production of
two isoforms of Steel factor (SLFI: the membrane-bound
and soluble forms.To investigate differences in the kinetics
of c-kittyrosine kinase activated by thesetwo isoforms. we
(SIISF)established fromSIISlhomoused a stromal cell line
zygous murine embryofetal liver andits stable transfectants
containing .ither hSCp" cDNA (including exon6; secreted
form) or h S C p cDNA (lacking exon 6; membrane-bound
form) as the source of each isoform.
Interaction of factor
dependent myeloid cell
line M07e with stromal cells producing eitheriaoform resulted in activated c-kittyrosine kinase
and induction of
the same series of tyrosine phosphorylated
cellular proteins in M07e cells.However,S14-h220 Imembrane-bound form) induced more persistent activation of c-
kit kinase than S14-h248 (solubleform) did. Flow cytometric
analysisandpulse-chasestudiesusing
IsSlmethionine
showed that S14-h248 inducedrapid downmodulation
of cellsurface c-kit expression andits protein degradationin M07e
calls, whereas SI4-h220 inducedmore prolongedlife span of
c-kit protein. Addition of soluble recombinant human SLF
to S14-h220 cultures enhanced reduction of cell-surface
c-kit
expression andits protein degradation.Because the kinetics
of c-kit inactivation strikingly fitswith the protein degradation rates of c-kit under the conditionsdescribedabove,
rapidproteolysis of c-kit protein induced by solubleSLF
stimulation may function
as a "turn-off switch" for activated
okit kinase.
0 1995 by The American Society of Hematology.
S
of the membrane-bound SLF was required to mediate this
attachment." Adhesion of human megakaryocytes to BM
stromal fibroblasts has also been shown to mediate in part
the interaction between membrane-bound SLF and c-kit prot e i ~ ~In
. ' ~addition, using stable gene-transfected stromal cell
lines that selectively produce either the membrane-bound or
the secreted form of SLF, it has been shown that the membrane-bound form of SLF supports hematopoiesis in a longterm culture system longer than secreted pr~tein.'~
However,
the mechanism by which these biologic differences are induced by these isoforms remains tobeclarified
and the
physiologic role(s) of each isoforms are not yet known.
We and others have previously reported that treatment of
the factor-dependent myeloid cell line M07e with soluble
recombinant (r)SLF leads to transient tyrosine phosphorylation of cellular proteins including the 145-kDc-kit geneencoded
In addition, this c-kit protein was
rapidly polyubiquitinated, internalized, and degraded after treat-
IGNAL TRANSDUCTION initiated by the interactions
of growth factors with specific receptors is an important
mechanism of regulating normal cell growth and differentiation. The c-kit proto-oncogene encodes a 145-kDmembranespanning receptor that is structurally a member of the platelet-derived growth factor (PDGF) receptor family and
possesses intrinsic tyrosine kinase activity.I4 The gene for
Steel factor (SLF), the ligand for the receptor protein encoded by c-kit, has been cloned and shown to be expressed by
bone marrow (BM) stromal cell^."^ The soluble recombinant
SLF is a potent costimulatory cytokine that synergizes with
a number of cytokines to stimulate growth of hematopoietic
progenitors in vitro and blood cell production in vivo in
anirnal~.~
With regard to the initial signaling event leading
to a cellular response to SLF, some studies suggest that
interaction of SLF with the extracellular domain of the ckit-encoded protein induces receptor dimerization followed
by receptor transphosphorylation.1o3"This leads to enhanced
tyrosine kinase activity and the formation of phosphotyrosine
residues that serve as a coded template to direct substrate
association and subsequent
The SI gene encodes a primary translation product of 248
amino acids with a leader sequence and extracellular, transmembrane, and cytoplasmic domains. The resulting protein
contains a proteolytic cleavage site encoded by exon 6 sequences (between amino acids 149 and 177) and posttranslational processing at this site leads to the secretion of a biologically active protein (soluble SLF) of 165 amino acids.Ig An
alternative spliced cDNA codes for a smaller polypeptide of
220 amino acids that lacks exon 6 sequences including the
proteolytic cleavage site and, hence, results in a membranebound protein.*' Several lines of evidence support the physiologic importance of the membrane-bound form of SLF in
vivo: The viable SZ-Dickie mutant in which the genomic
regions encoding the transmembrane and cytoplasmic domains are deleted can only produce the secreted form of
SLF, but shows all the pleiotropic defects seen in SNSZ mutants.20*21
A study of mast cell attachment to fibroblasts derived from S I W mice showed that the extracellular domain
Blood, Vol85, No 3 (February 11, 1995: pp 641-649
From the First Department of Internal Medicine (Hematology and
Oncology), Tokyo Medical College,Tokyo, Japan; Howard Hughes
Medical Institute, Herman B Wells Center for Pediatric Research,
Riley Hospital for Children; Walther Oncology Center,and Indiana
University School of Medicine, Indianapolis.
Submitted July 22, 1994; accepted November 2, 1994.
Supported by a Grant-in-Aidfor Encouragement of Young Scientists, from the Ministry of Education, Science and Culture of Japan
to K.M. and by US Public Health Service Grant R 0 1 HL46528 to
D.A.W., and R37CA36464, R 0 1 HL46549, and R 0 1 HL49202 to
H.E.B. from the National Cancer Institute and National Institutes
of Health.
Address reprint requeststo Keisuke Miyazawa,MD, The First
6-7-1,
Depamnent of Internal Medicine, Tokyo Medical College,
Nishishinjuku, Shinjuku-ku, Tokyo 160, Japan.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with 18 V.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8503-0036$3.00/0
641
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642
MIYAZAWA ET AL
ment with soluble rSLF stimulation.25 This suggested that
ligand stimulation shortens the life span of c-kit protein and
this may function as a negative regulatory feedback loop for
activated c-kit protein. Therefore, we compared the effect of
the secreted and membrane-bound forms of SLF regarding
the kinetics of c-kir kinase and the degradation rate of c-kit
protein. These studies were done with M07e cells because in
many waysthis cell line responds to SLF as do normal
ce~~s9,'3,1"?h
and also because due to the very low frequency
of hematopoietic stem and progenitor cells in the main tissue
source of these cells, BM, and it is not yet possible to obtain
purified stemand progenitor cells to do these studies. The
results showed that the ligand-induced active state of c-kit
kinase persisted longer with the membrane-bound SLF than
with the soluble form in M07e cells. Additionally, the length
of the c-kit protein life span correlated well with the kinetics
of c-kit kinase activity after stimulation with each isoform of
SLF and the half-life of c-kit protein was longer after stimulation with the membrane-bound isoform. These data suggest
that ligand-induced proteolysis of the c-kit protein may function as a regulatory mechanism of c-kit lunase. The data presented may also explain at least some of the biologic differences between the membrane-bound and soluble forms of SLF.
MATERIALS AND METHODS
Cyfokines, unfihodies, und reugenfs. Highly purified recombinant human(rhu) SLF and rhu granulocytemacrophagecolonystimulating factor (GM-CSF: 4 x IO7 U/mL) were kindly provided
by Dr Douglas E. Williams (lmmunex Cop, Seattle, WA). Mouse
anti-c-kit monoclonal antibody (MoAb), YB5.B8, which recognizes
the extracellular epitope of the human c-kif gene product, was a gift
from Dr Leonie K. Ashman (Hanson Center for Cancer Research,
Adelaide, South Au~tralia).~'
Rabbit anti-c-kir polyclonal antibody
a sequence
raisedagainstthepeptideGSTASSSQPLLVHDDV,
found at the C-terminus, was obtained from Oncogene Science, Inc
10) and
(Uniondale, NY). MouseantiphosphotyrosineMoAb(4G
agarose-conjugated 4G10 were purchased from Upstate Biotechnology Inc (Lake Placid, NY). Alkaline phosphatase conjugated goatantimouse IgG (H + L) andgoat-antirabbit IgG (H + L) were
obtained form Zymed Laboratories. Inc (San Francisco, CA).
Cell lines. The human growth factor-dependent cell line, M07e.
was agiftfromAggieCiarletta(Genetic
Institute. Boston,MA).
This cell line responds to human GM-CSF and interleukin-3 (IL-3)
and human or murine SLF. The biologic characteristics of this cell
line and culture conditions have been previously described.'"'' The
cells were cultured in RPMI-1640 medium supplemented with 20%
fetal bovine serum (FBS) and100 UImL rhuGM-CSF. Exponentially
growing M 0 7 e cells were washed and then incubated for 18 hours
at 37°C in serum-free RPMI-1640 medium containing 0.5% bovine
serum albumin (BSA: Sigma, St Louis, MO). After "factor-starvation," cellswerewashedonce
with serum-freemediumandthen
used for further experiments.
Astromalcell
line SIISI'. which does not produceSLF,was
established from the liver ofa 14-day S I N homozygote murine fetus
obtained by heterozygote ( W + )crosses. The generation of S1/SI4
stable transfectants producing either membrane-bound SLF (SVSI4h220) or soluble SLF (S1/S14-h248) has been previously described.?4
Briefly, alternatively spliced forms of hSLF cDNAs inserted into
pJT-I (hSLF220) or pDSR (hSLF248) vectors and expressedoff the
SV40 early promoter were cotransfected with the p48 vector encoding hygromycin B using the Lipofectin method. The transfectants
were selected and expanded by culturing the cells with Dulbecco's
modified Eagle'smedium(DMEM:GIBCOIBRL,GrandIsland,
NY) containing 20% FBS and 200 pg/mL hygromycin. Thereafter,
SK'X' and SI/SI'-hSLF transfectants were maintained in DMEM ,upplemented with 1 0 % FBS.
sI/SI' clones were treated with mitomycin C (5 pg/mL; Sigma)
for 2 hoursat 37°C. washedthreetimes with phosphate-buffered
saline (PBS), treated with trypsin, and plated to confluence (1.5 x
IOs/well) on 24-well gelatin-coated tissue culture
plates (Corning,
Corning, NY) and further cultured for 48 hours. Underthese conditions,SIJ-h248secretesabout
15 ng/mL of rhuSLFand by SLF
immunoprecipitation and RNA analysis, S14-h220and Sld-h248both
produce equivalent amounts of protein."
Then the factor-starved M 0 7 e cells at 2.5 X IOh ceils/well were
loaded onto the stromal layers. The cell density of M 0 7 e cells was
adjusted to form a single cell layer over the confluent stromal cells.
Theculture plates weregentlycentrifugedfor
1 minuteat lO0g
using Tomy TS-4 swing rotor (Tomy, Tokyo, Japan)
to ensure direct
cell-cell contact, and further cocultured for various periods at 37°C.
Thereafter, the supernatants were removed, and cells were lysed in
lysis buffer (20 mmol/L Tris-HCI, pH 7.4, 150 mmol/L NaCI, 10%
glycerol. I % NP-40, 1 mmolL phenylmethylsulfonyl fluoride, 0.15
U/mL nprotinin. I O mmol/L EDTA, l0 ,ug/mL leupeptin, 100 mmol/
L sodium fluoride, 2 mmol/L sodium orthovanadate) at 4°C for 30
minutes. Cell lysates were clarified by centrifuging for 30 minutes
at 10,000g at 4°C. Total protein content of the lysate was determined
by a Bio-Rad protein assay kit (Bio-Rad, Richmond, CA). Cell lysates were used for immunoprecipitation and immunoblotting.
I~nmunc~~reci~~itution
und immunohloiting. Immunoprecipitation
andimmunoblottingwereperformedaspreviouslydescribed."
Briefly, cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were electrophoretically transferred onto Immobilon-P membranes (Millipore,
Bedford, MA). After blocking residual binding sites on the transfer
membrane by incubation with TBST (50 mmol/L Tris-HCI, pH 7.4,
150 mmol/L NaCI. 0.05% Tween-20 [Bio-Rad]) containing 5% dry
milk for 2 hours, the blots wereincubatedwiththeappropriate
primary antibody for 2 to 4 hours at room temperature. Immunoreactive proteins were visualized colorimetrically. Immunoprecipitations
were performed by incubating the cell lysates with anti-c-kit antibody for 3 hours at 4°C. Protein A-Sepharose beads (Pierce, Rockford, IL) were used to collect the antigen-antibody complexes.
Meruholic lubeling. For metabolic
labeling
with ["Slmethionine, exponentially growing M 0 7 e cells were washed and resuspended at IO6 cells/mL in methionine-free RPMI-1640 medium containing S% FBSandincubatedfor
1 hour. The cellswere then
resuspended ( I O6 cells/mL) in methionine-free RPMI- 1640 medium
containing 100 pCi/mL [7rS]methionine (specific activity >800 Ci/
mmollL; Amersham Corp, Buckinghamshire, UK), 1 0 % FBS,and
100 U/mL rhuGM-CSF and incubated for 3 hours at 37°C. After
metaboliclabeling,thecells
were washed threetimesandresuspended in complete medium containing a
10-fold molar excess of
for 1 hour to wash outfree
unlabeledmethionineandincubated
["S]methionine. Formetaboliclabeling
with ["Plorthophosphate,
M 0 7 e cell at 10' cells/mL were incubated in phosphate-free RPMI1640 medium containing 0.5% BSA for 2 hours. Then cells were
resuspended at 5 X 10'' cells/mL in phosphate-free medium equilibrated with carrier-free ["P]orthophosphate (Amersham) at 1 .0 mCi/
mL for 90 minutes at 3 7 T , and washed three times with PBS. The
radiolabeled cells were loaded onto stromal cell layers
that expressed
either soform of rhuSLF, as described above.
Immune-complex kinase ussuys. Immunoprecipitation with antic-kit antibody was performed as described above. The immunoprecipitates were washed three times with lysis buffer and three times
with kinase buffer (50 mmol/LTris-HCI, pH 7.4. 25 mmol/L pglycerophosphate. ?I mmol/Lsodiumorthovanadate, I mmol/Ldi-
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MEMBRANE-BOUNDANDSOLUBLEFORMSOFSTEELFACTOR
thiothreitol, 10 mmol/LMgCQTenmicrocurie
of ['*P-y] ATP
(Amersham) was addedfor each assay, and incubated
for 10 minutes
atroomtemperature.Thereactionwasterminated
by addition of
SDS-PAGE sample buffer containing 2-mercaptoethanoland boiled
for 5 minutes. The samples were analyzedby 7.5% SDS-PAGEand
transferredontolmmobilon-Pmembrane.Theblotsweresubsequently incubated in 1 moVL KOH for 1 hour at 55°C to selectively
remove the radioactive label from proteins phosphorylatedon serine
and threonine residues.
Flow cytomerric analysis. After coculture with stromal cell layof time,M07ecellswerecollected,
and
ersforvariousperiods
washedtwicewithPBScontaining
5% FBS. Cells, 1 X IO5,suspendedin0.5 mL PBScontaining 5% FBS wereincubated with
anti-c-kit MoAb, YB5.BS (1 pg) or with isotype-matched murine
IgG, (1 pg; Zymed) for 30 minutes at 4°C.Sampleswerethen
washed twice and incubated with fluorescein isothiocyanate (FITC)conjugated sheep-antimouse IgG secondary antibody (20 pg; Becton-Dickinson,Sunnyvale, CA) for 20 minutes at 4°C.Immunofluorescencewasanalyzed by flowcytometryusing a FACScan
(Becton-Dickinson) and a Consort 30 (Becton-Dickinson)FACS
data analysis program.
RESULTS
SVSr' transfectants producing either the membrane-bound
or the soluble form of SLF support growth of factor-dependent M07e cells. Sl/S14 stromal cell line lacking production
of SLF and its stable gene transfectants producing either the
membrane-bound SLF (S14-h220) or the secreted form of
SLF (S14-h248) were treated with mitomycin C, thoroughly
washed, then cultured for 48 hours as described in Materials
and Methods. Thereafter, factor-starved M07e cells were
loaded onto these mitomycin C-treated subconfluent stromal
cell layers and cocultured for another 48 hours. As shown
in Fig 1, both S14-h220 and S14-h248cells supported growth
of M07e cells. After 48-hour coculture with Sl/S14cells that
do not produce SLF, over 80% of M07e cells were not
viable as determined by trypan blue dye exclusion (data not
shown). It is noteworthy that in S14-h220cultures producing
the membrane-bound SLF, growing M07e cells were seen
adjacent to the cell-surface of S14-h220cells, whereas in S14h248 cultures secreting the soluble form of SLF, mostof
growing cells were observed in the open spaces left by S14h248 cells and only some of them were seen associated with
these stromal cells.
Induction of protein tyrosine phosphorylation in M07e
cells by interaction with either S14-h220or S14-h248stromal
cell layers. After treatment with mitomycin C, the confluent stromal cells were further incubated for 48 hours. Factorstarved M07e cells were loaded on these stromal layers and
cocultured for various periods of time. Since the recovery
of attached M07e cells from the stromal cell cultures by
pipetting induced cellular protein phosphorylation in M07e
cells, which resulted in high background in immunoblots
(data not shown), we removed the supernatants and directly
added lysis buffer into the cultures to make whole-cell lysates. The soluble cellular proteins consisting of M07e and
stromal cells were analyzed by immunoblotting with antiphosphotyrosine MoAb. Figure 2A shows induction of protein tyrosine phosphorylation in M07e cells after 15-minute
coculture with either S14-h220or S14-h248cells or treatment
with 50 ng/mL soluble rhuSLF. One prominent band with a
643
molecular weight of about 145 kD has been reported to
represent c-kit-encoded protein due to ligand-induced receptor transphospholylation in M07e cell^.'^.'^ Interaction of
M07e cells with SVS14 cells did not induce any change of
cellular protein tyrosine phosphorylation in M07e cells compared with the cells treated with control medium (Fig 2A,
lanes 2 and 7). To further confirm that these tyrosine phosphorylated protein bands were exactly induced in M07e
cells but not in the mitomycin C-treated stromal cell layers,
[3ZP]radiolabeledM07e cells were loaded on transfected
stromal cells and cocultured for 15 minutes. Then cells were
lysed and tyrosine phosphorylated cellular proteins were collected with agarose-conjugated 4G10, separated by SDSPAGE, and visualized by autoradiography (Fig 2B). The
same series of tyrosine phosphorylated bands were observed
among S14-h220-, S14-h248-, and rhuSLF-stimulated cells.
In addition, only a fewweak phosphorylated bands were
detected in cell lysates of the stromal cells after coculture
with M07e cells (Fig 2C). Therefore, most of phosphorylated protein bands in Fig 2A, lanes 4 and 6 represent cellular
phosphorylated proteins derived from M07e cells after interaction with S14-h220or S14-h248cells. These data also suggest that there are no significant differences in quality of
signals produced by the membrane-associated and the soluble forms of SLF. As for lower molecular weight proteins
of less than 50 k D , 10% SDS-PAGE showed no difference
regarding protein tyrosine phosphorylation pattern between
S14-h220 and S14-h248 stimulation (data not shown).
In contrast to the lack of difference in the quality of signals
induced by either SLF isoform, the kinetics of cellular protein phosphorylation in M07e cells was different after stimulation with the membrane-bound or the soluble SLF. As
shown in Fig 3, interaction with S14-h248cells induced transient tyrosine phosphorylation of cellular proteins in M07e
cells, and the protein phosphorylation level returned close
to that observed in the unstimulated state within 60 minutes.
This result was consistent with those of our previous report
in which we used
highly purified recombinant soluble murine
SLF as a c-kit activator in M07e cells." In contrast, after
60 minutes of interaction withS14-h220, cellular protein
phosphorylation was still high and remained elevated at 120
minutes.
The soluble SLF enhanced downmodulation of cell-surface c-kit expression more than the membrane-boundform.
We have previously reported that stimulation with recombinant soluble SLF at 37°C induced polyubiquitination of ckit-encoded protein and subsequent internalization of the
ligand-receptor complex resulting in rapid proteolysis of the
c-kit protein.25Therefore, we presumed that the difference
in kinetics of tyrosine phosphorylation shown in Fig 3 was
caused by enhanced degradation of the c-kit protein after
internalization of the ligand-receptor complex when M07e
cells were stimulated with soluble SLF. In this viewthe
membrane-bound SLF could not be internalized. To test this
hypothesis, we first examined cell-surface c-kit expression
in M07e cells by flow cytometry after interaction with the
soluble and the membrane-bound SLF. As shown in Fig 4,
coculture with S14-h248stromal cells for 15 minutes induced
downmodulation of cell-surface c-kit protein expression in
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644
MIYAZAWA ET AL
Fig 1. Coculture of the factor-dependent M07e cells with gene transfected SI/S14 cell lines producing either the membrane-bound or the
soluble form of SLF. Subconfluent stromal cells producing either the membrane-boundform of SLF (S14-h220)or the soluble form of SLF (SI4h2481 were treated with 5 pg/mL of mitomycin C for 2 hours at 37°C. thoroughly washed with PBS containing 5% FBS, and then cultured for
48 hours. Thereafter, factor-starvedM07e cells (5 x lo4 cellslmL) were cocultured for 48 hours with each type of stromal cells at 37°C. Upper
right and left photographsshow cultures of S14-h248 andSI4-h220cells alone, respectively. The lower right photograph corresponds to M07e
cells cocultured with S14-h248 cells;that on the left corresponds to M07ecells coculturedwith S14-h220 cells.
M07e cells compared with untreated cells. No downmodulation of c-kif was detected after coculture with SI/SI" stromal
cells. Interactionwith SIJ-h220 stromal cells resulted in a
slight but significant reduction of c-kif expression in M07e
cells compared withuntreated cells. However, addition of
15 ng/mL soluble rhuSLF toSI"-h220 cultures markedly
enhanced c-kit downmodulation in M07e cells. to levels
similar to cells cocultured with SI"-h248 stromal cells. which
are known to produce an equivalent amount of soluble
rhuSLF.'" These data suggest that the soluble form of SLF
plays an important role in cell-surface c-kif expression.
Kinetics of c-kit kinase inactivation m d ligand-irlduced
c-kit degradation rate @er inferaction wirh the memhranehound and soluble f o r m of SLF. To determine the degradation rate of c-kit-encoded protein after interaction with
each SLF-isoform, c-kit proteinin M07e cells was first metabolically labeledwith [ZsS]methioninethen chased during
coculture with the confluent layers of each SI/SI"transfectant
for various periods of time. After cell lysates were immunoprecipitated with anti-c-kif antibody and separated by SDSPAGE, autoradiography was performed. Interactionwith
S14-h248 cells induced rapid reduction of radiolabeled c-kit
protein in M07e cells (Fig S, B and C). In contrast, the life
span of c-kit protein was much more longer after interaction
with SIJ-h220 cells. Similar to its effect on cell-surface ckit expression (Fig 4). addition of soluble rhuSLF accelerated
c-kit degradation in M07e cells cocultured with the SIJ-h220
cell layer as much as in those cocultured with SIJ-h248cells.
A more pronounced c-kit degradation was observed when
the concentration of soluble rhuSLF was increased to S0 ng/
mL in SI"-h220 cultures (Fig SC). It is noteworthy that the
kinetics of c-kit degradation under these conditions strikingly
fit withboth the tyrosine phosphorylation levels andthe
immune-complex kinase activities of c-kit protein after interactionwitheach
SLF isoforms (Fig S, A and B andFig
6). In M07e cells stimulated with SIJ-h248 cells, tyrosine
phosphorylation of the c-kit protein wastransiently activated
and it returned to the unstimulated state within 90 minutes,
whereas it remained active even after 120 minutes of interactionwith SI4-h220 cells. Addition of S0 ng/mL soluble
rhuSLF to SI"-h220 cultures resulted in enhanced inactivation of c-kit kinase of M07e cells at 60-minute incubation,
though tyrosine phosphorylation of c-kit protein was more
enhanced at the initial period of incubation (Fig SA). Taken
together, these data show that the c-kit kinase remained activesignificantly longer after stimulation withthemem-
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MEMBRANE-BOUND AND SOLUBLEFORMSOFSTEELFACTOR
645
A
B
C
a,
kDa
205116.5-
koa
116.5-
80-
205 -
80-
-c-kit
1 16.5-
49.5-
49.5-
80 "
"
1
2
3
4
5
1
2
3
49.5-
1
2
3
4
5
6
7
8
Fig 2. Induction of protein tyrosine phosphorylation
in M07e cells after interaction with thegene transfected SIISI' stromal cell lines. (A)
Confluent stromal celllayers (1cm*/well) were treatedwith 5 pg/mL mitomycinC for 2 hours, thoroughly washed, and further incubated for
48 hours. Thereafter, factor-starved M07e cells (2.5 x 10' cellslwell) were loaded onto the stromal cell
layers. The culture plates were gently
centrifuged at l O O g for 1 minute t o ensure direct contact of M07e cells with the stromalcell layers. After incubation for 15 minutes at 37°C.
the supernatants were removed and
cellular proteins were lysed
with lysis buffer that contained sodium orthovanadate
as described in Materials
and Methods.Cellular proteins wereseparated by 7.5% SDS-PAGEand tyrosine phosphorylated proteins were
detected by immunoblotting with
antiphosphotyrosine MoAb.(B)Radiolabeled M07e cells with ['zPlorthophosphate were coculturedwith thestromal cell linesor treated with
15 ng/mL rhu SLF for 15 minutes at37°C. Tyrosine phosphorylated cellular proteins were collected using
agarose-conjugated 4G10, separated
by 7.546 SDS-PAGE, and visualized by autoradiography. Lane 1, M07e cells cocultured with SI/SI'; lane 2, cocultured with Sl'-h220; lane 3,
cocultured with Sl'-h248; lane 4, M07e cells treated with 15 ngImL soluble rhu SLF; lane 5, M07e cells cocultured with SI'-h220 and the cell
lysate was immunoprecipitated with isotype matched murine IgG,. (C) After coculture with M07e cells for 15 minutes at 37°C. M07e cells
were removed from the
adherent stromal cell layers by pipetting.Then the stromalcellular proteins werelysed, separated by 7.5% SDS-PAGE,
and immunoblotted with antiphosphotyrosineMoAb. Lane 1, SIISI'; lane 2, SI'-h220; lane 3, SI'-h248.
brane-associated SLF than when stimulated with the soluble
form. This appears to be caused, at least in part, by the
longer life span of c-kit protein after stimulation with the
membrane-bound SLF.
DISCUSSION
In this study, we have shown that activation of c-kit tyrosine kinase from M07e cells persists for a longer period
of time when M07e cells are interacting with membraneassociated SLF compared with soluble SLF. Soluble rhuSLF
added to the stromal cell cultures that produce themembrane-bound SLF accelerated inactivation of c-kit tyrosine
kinase. This suggests that the difference in the kinetics of ckit kinase is not caused by the amount of SLF produced by
each stromal cell line, but that the form of the ligand is more
crucial for this inactivation process. We and others have
rh220-1
7h248-1
koa
205
205 -
116.5
116.5-
Fig 3. Kinetics oftyrosinephosphorylation
in
M07e cells after
interaction
with the gene
80
transfected SIISI' stromal cells. Factor-starved
M07e cells were loaded onto S14-h220 or SI'-h248
stromal cell layers producing either themembrane49.5bound or the soluble forms
SLFofas described in the
legend for Fig 2 and cocultured forvarious periods of
time at 37°C. Cell lysates were separated by 7.5%
SDS-PAGE and immunoblotted with antiphosphotyrosine MoAb.This is a reproducible result from one
of five separate experiments.
80 -
"W----
0
15 30 45
49.5-
60 90 120
Time(rnin)
0
15 30 45
60 90 120
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MIYAZAWA ET AL
646
6
h220+SLF
(94)
(!j
_.
:’:
,.
!
i
Log Fluorescence Intensity
reported that stimulation of the cells expressing c-kit receptor
with soluble SLF induces rapid downmodulation of cellsurface c-kit expression and degradation of metabolically
radiolabeled c-kit p r ~ t e i n . ~ ~
We
. ~ ’have also reported that
soluble ~ ~ U [ ’ ~ ~ Iwas
]SL
promptly
F
internalized at 37°C after
ligand-receptor binding in M07e cells.25Degradation after
internalization of the ligand-receptor complex has been reported in many other receptor systems including the PDGF
and CSF-l receptors that are structurally related to c-kit.””’
Therefore, we would expect that theregulation of c-kit inactivation involved, at least in part, the proteolysis of c-kit protein mediated by accelerated internalization of the ligandreceptor complex after soluble ligand stimulation. In
contrast, because the immobilized membrane-bound ligandreceptor complex presumably cannot be internalized and degraded by this pathway, the activation of c-kit by the
membrane-bound SLF appears to be prolonged. Prolonged
activation of c-kit might be expected to continue producing
efficient signals for cell proliferation and differentiation.
This idea was supported by the lines of evidence presented
in this report: First, cell-surface expression of c-kit was
downmodulated after 15-minute interaction with the soluble
SLF, whereas downmodulation was much less after interactionwith the membrane-bound form (Fig 3). Second, the
radiolabeled c-kit protein was rapidly degraded after stimula-
Fig 4. Modulation of cell-surface c-kit expression
in M07ecells after stimulation with themembranebound or soluble forms of SLF. The factor-starved
M07e cells were loaded onto the layers of S14-h220
or S14-h248 cells, asdescribed in the legend for Fig 2
and incubated for 15 minutes at 37°C. The cell-surface c-kit-encoded protein was stained with antic-kit MoAb, YB5.B8, and the expression level was
1, M07e cells incubated
analyzed by flow cytometry.
in controlmedium; 2, cells cocultured with SI4-h248
cells; 3, cells cocultured with S14-h220 cells; 4, cells
cocultured with SI/S14 cells, 5, cells cocultured with
SI/S14 cells in the presence of 15 ng/mL soluble
rhuSLF; 6,cells cocultured with S14-h220in the presence of 15 ng/mL soluble rhuSLF. The figures in parentheses represent the mean channel number of
fluorescence intensity. This is one of representative
results from fiveseparate experiments.
tion with soluble SLF, whereas its half-life was much longer
after interaction with membrane-bound SLF. Moreover, soluble rhuSLF added to thestromal cultures that produce membrane-bound SLF enhancedc-kit degradation tothe same
extent observed in the case of the stromal cell cultures that
produce soluble SLF (Fig 5 , B and C). Third, the kinetics
of c-kit inactivation after stimulation with either SLF isoform
correlated well with the c-kit protein degradation rate under
either condition (Fig 5, A and B and Fig 6).
The potential role for protein tyrosine phosphatases in
growth regulation is becoming evident. Recently one novel
protein tyrosine phosphatase, termed hematopoietic cell
phosphatase (HCP) or PTPIC, has
been
identified.’’~3”
PTPIC encodes a cytoplasmic phosphatase that contains two
SH2 domains and has been shown to transiently associate
with ligand-activated c-kit protein through the SH2 domain
in vivo and dephosphorylate the ligand-induced autophosphorylated c-kit protein in v i t r ~ , ~implying
’
that this phosphatase might be involved in the negative feedback regulation of ligand-activated c-kit receptor. As shown in Fig 5A,
a slight but significant reduction of tyrosine phosphorylation
level of c-kit after a 90- to 120-minute interaction with the
membrane-bound SLF was observed, whereastheamount
of cellular protein was almost consistent until up to a 120minute incubation. This suggests that some protein tyrosine
+
Fig 5. Kinetics of the c-kit protein degradation rate
in M07e cells after interactionwith stromalcells producing either the membrane-bound
or the soluble forms ofSLF. (A) M07e cells were cocultured with S14-h248 cells, S14-h220 cells,or S14-h220cells in the presence of 50 ng/mL
soluble rhu SLF for various periods oftime at37°C. Then the cells were p r o c a d for immunoprecipitation usinganti-c-&& MoAb. The c-kit
immunoprecipitates wereseparated by 7.5% SDS-PAGE and probedwith antiphosphotyrosine MoAb. (B) The c-kit gene product was labeled
30 minutes t o wash out free-[%methionine. Thereafter,
for 4 hours with [86Slmethionineand then incubatedwith excem cold methionine for
separated by SDS-PAGE and
cells were loaded onto the stromal
layers and incubated as described above. The c-kit immunoprecipitates were
separated by SDS-PAGE and the proteins
detected by fluorography. (C) The immunoprecipitates of [“Slmethionine-labeled c-kit protein were
were transferred onto Immobilon-P membrane. Each lane in the molecular weight region that was greater than 100 kD was excised and
M07e calls cocultured with S14-hZ20 cells [membrane-bound SLF); (A) cells cocuktured with SI4radioactivity of each lane was counted. (0)
h220 cells plus 15 ng/mL soluble rhuSLF; 1 4 cells cocultured with S14-h220cells plus 50 ng/mL soluble rhuSLF; (0)cells cocuktured with SI4h248 cells (soluble SLF); ( 0 )cocultured with SI/S14 cells that do notproduce SLF. The data shown here are reproducible results from one of
three separate experiments.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
MEMBRANE-BOUND
AND
SOLUBLE FORMS
OF STEEL FACTOR
647
-
C Y
+
-
0
10 30 60 90 120
10 30 60 90 120
10 30 60 90 120
Time(min1
B
-c-kit
0
30 60 90 120
0 30 60 90 120
0 30 60 90
Time ( m i d
C
01
I
I
l
I
0
30
60
90
Time (rnin)
I
120
c-kit
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
MIYAZAWA ET AL
648
h 248
0
10
30
60
10
30
60
c-kit
-
c-kit
120
h 220
0
-
mouse which has a deletion the S I gene thatremovesthe
trnnsmembrane and intracellular domains and can only produce the secreted form of SLF. development of hematopoietic cells. melanocytes, germ
and
cells is
As
shown in Fig I . M07e cells proliferated only when directly
interacting with SIJ-h220 cells. whereas direct contact was
not required for its proliferation when cocultured with SIJh248 cells. Because SLF has pleiotropic effects on hematopoietic progenitors. further studies to determine biologic
differences between the two isoforms including cell differentiation and cell-cycle state should be pursued. Unfortunately,
the capacity to evaluate effects of membrane-bound and soluble SLF on receptor mediated and intracellular events including phosphorylation patterns of primary normal hematopoietic stemand progenitor cells mustawait advances in
technology thatwill allow one toisolate enough of these
rare cells in purified form to pursue such studies.
120
ACKNOWLEDGMENT
-
h220 +SLF
c-kit
We thank Dr Douglas E. Williams of Immunex Corporation for
providing rhu Steel factor and rhuGM-CSF. and Dr Leonie K. Ashman of Hanson Center for Cancer Research for the gift of anti-ckif MoAb YBS.BX.
REFERENCES
10
30
60
120
Time (min)
Fig 6. Kinetics of immune-complex kinase activity of c-kit protein
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1995 85: 641-649
Membrane-bound Steel factor induces more persistent tyrosine
kinase activation and longer life span of c-kit gene-encoded protein
than its soluble form
K Miyazawa, DA Williams, A Gotoh, J Nishimaki, HE Broxmeyer and K Toyama
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