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HEMATOPOIESIS
From The Arthritis and Immune Disorder Research Centre,
The Toronto Hospital and Department of Immunology, University of
Toronto, Toronto, Ontario, Canada; Department of Pediatrics, Herman B. Wells Center for Pediatric Research, James Whitcomb Riley Hospital for
Children, Howard Hughes Medical Institute, Indiana University
School of Medicine, Indianapolis, IN.
The authors investigated the roles of PI3-kinase and PLC- c-Kit, the gene product of the W locus,
is a receptor tyrosine kinase that regulates the survival, growth, and
differentiation of hemopoietic stem cells, mast cells, germ cells, and
melanocytes.1-4 Steel Factor (SLF), the gene product of
the Sl locus, is the ligand for c-Kit. Mutations in either
gene exhibit similar defects in their target tissues, highlighting the
complimentary nature of the ligand-receptor
interaction.5-8
SLF is expressed on stromal cells or fibroblasts as a membrane-bound
molecule. Soluble SLF (sSLF) is generated by chymase cleavage of the
extracellular portion of the growth factor.9-12 Increasing
evidence indicates that the 2 forms of the ligand stimulate qualitatively different responses.13 For example, in
vitro, combinations of other factors with membrane-bound SLF (mSLF) but not sSLF support the survival of long-term hemopoietic progenitors and
erythroblastic leukemia cells14-17 and stimulate
erythropoietic development.18 Stromal cells and
fibroblasts from Steel-Dickie mice (Sld) secrete sSLF but
do not express mSLF. Mice with this mutation exhibit hemopoietic, coat
color, and germ cell defects, suggesting that mSLF plays a unique
biologic role in these tissues.19 In vivo, the
concentration of sSLF in serum is such (3 ng/mL) that it is likely in a
monomeric form, and thus limited in its mitogenic potential.20 Therefore, mSLF is presumably the relevant
physiologic form of the ligand in vivo.
Although the biochemical basis for the differences between sSLF and
mSLF are not completely understood, some signaling differences between
the 2 ligand isoforms have been observed. After ligand binding, signal
transduction cascades are initiated by the stimulation of receptor
autophosphorylation on key tyrosine residues.21 In the
case of sSLF, tyrosine phosphorylation of c-Kit is rapid (within
minutes), followed by a decline in phosphorylation. This decline in
phosphorylation coincides with receptor internalization and
endocytosis,22 leading ultimately to receptor degradation. In contrast, phosphorylation of c-Kit by mSLF persists over much longer
periods.23 This persistence in tyrosine phosphorylation was attributed to the enhanced stability of the c-Kit receptor on the
cell surface after mSLF stimulation, likely because of the prevention
of receptor internalization by this form of ligand.
Autophosphorylation of receptor tyrosine kinases generates binding
sites that recruit SH2-containing proteins to the receptor. For c-Kit,
stimulation with sSLF results in recruitment of PI3-kinase, PLC- Valius and Kazlauskas43 tested the functional relevance of
PI3-kinase and PLC- Given the apparent redundancy between PI3-kinase and PLC- Cell culture and transfection
The complementary DNAs (cDNAs) (obtained from Dr R. Rottapel) for
wild-type (wt) murine c-kit or mutants in which either tyrosine 719 was
replaced with phenylalanine (YF719) or tyrosine 728 was replaced with
phenylalanine (YF728) were cloned into LXSN retroviral expression
vectors. This retroviral vector also contains the neo gene that confers
resistance to the antibiotic G418. The LXSN YF728 construct was used to
generate a YF719/YF728 double mutant by using a directed mutagenesis by
the polymerase chain reaction (PCR) method. Briefly, synthetic primers
containing both the YF719 mutation and an EcoRI site were
combined with flanking primers and amplified to produce 2 fragments,
overlapping in the region of the mutating primer. The 2 fragments were
isolated and reamplified with the flanking primers. The resulting 1 kilobase (kb) fragment was then TA cloned and verified by
restriction digest, followed by digestion with ApaI and
SalI to produce a 630-base pair (bp) fragment for subsequent
ligation and subcloning into the LXSN Kit YF728 vector. Both junctions
and mutations were verified by sequencing.
These vectors were transfected into gp + a NIH 3T3 packaging cells
by lipofectamine treatment (Gibco, Grand Island, NY) and selected in 1 mg/mL G418 (Gibco). The 32Ds were cocultured with a confluent layer of
pooled, irradiated (20 Gy) gp + a transfectants for 24 hours. The
nonadherent 32Ds were removed and cultured for an additional 2 to 5 days in IL-3, followed by selection for c-Kit positive cells in 1 mg/mL
G418 and IL-3. The c-Kit-positive 32D cells were further enriched by
cell sorting (FacStar Plus, Becton Dickinson, Franklin Lakes,
NJ). Surface expression of c-Kit was detected using biotinylated-SLF
and streptavidin-conjugated phycoerythrin (Jackson, Westgrove, PA) as
described previously.22 Stable populations of
c-Kit-expressing 32Ds were then cloned by limiting dilution. The
c-Kit-negative 32D-neo cells that express only the neo gene were generated with a similar process that used the empty LXSN vector.
Reagents
Western blotting For the analysis of receptor autophosphorylation and recruitment of p85, 5 × 106 32D infectants were starved overnight in RPMI + 0.5% FBS. Cells were then washed in phosphate-buffered saline containing FBS (PBS-FBS) 3 times, followed by stimulation with 500 ng/mL SLF for 2.5 minutes at 37°C. Cells were immediately washed in ice-cold PBS-FBS, then lysed in lysis buffer containing 50 mmol/L Tris (pH 7.0), 1% NP-40, 20 mmol/L EDTA with the following inhibitors: 200 µmol/L sodium orthovanadate, 20 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin (all Sigma). Lysates were then spun at 10 000 rpm for 20 minutes, and supernatants were incubated with 50 µL of a 20% Protein A slurry (Pharmacia Biotech Inc, Baie D'Urfe, Quebec) and 5 µL of Rb125, a polyclonal rabbit antibody raised against the intracellular portion of the c-Kit protein (gift from Dr Herman Ziltener, Vancouver, British Columbia). Lysates were incubated for 2 hours at 4°C, and the beads were washed 3 times in lysis buffer with inhibitors. Beads were resuspended in loading buffer with 2-mercaptoethanol and boiled for
5 minutes. Released proteins were resolved on a 7.5% acrylamide gel,
transferred to nitrocellulose, and blocked in TBS plus 3%
gelatin and 0.5% Tween 20. Blots were incubated with 4G10
antiphosphotyrosine antibody (UBI, Lake Placid, NY) in tris-buffered saline (TBS) plus 1% gelatin and 0.5% Tween 20 at a dilution of 1:1000 for 1 hour, followed by goat antimouse-conjugated horseradish peroxidase secondary antibodies27 at a dilution of 1:5000
and visualized with chemiluminescence (NEN Life Science Products). Blots were stripped by acid treatment and reprobed with rabbit polyclonal anti-c-Kit antisera at a dilution of 1:500 or with rabbit
polyclonal anti-p85 antisera (Santa Cruz Biotechnology, Santa Cruz,
CA), followed by incubation with protein-A-horseradish peroxidase
(Amersham Corp, Mississauga, Ontario) at a dilution of 1:30 000.
Visualization was again by chemiluminescence. For the analysis of
PLC- phosphorylation, cells were starved in RPMI + ITS Liquid
Media Supplement (Sigma) + 0.05% BSA for 6 hours. Cells were spun
down and resuspended at a concentration of 2 × 107/mL. A
2x solution of RPMI + ITS ± SLF (2 µg/mL) preheated to 37°C was added to an equal volume of cells and incubated for 5 minutes in a 37°C waterbath. After the incubation, samples were removed and placed on ice. Cells were washed in ice-cold PBS twice and
lysed by freezing and thawing in a buffer containing 50 mmol/L Tris (pH
7.0), 1% NP40, 50 mmol/L EDTA, protease inhibitor cocktail (Roche,
Laval, Quebec), phosphatase inhibitor cocktail (Sigma), 200 µmol/L
sodium orthovanadate, 20 mmol/L NaF, and 1 mmol/L PMSF. Cells were
frozen in a dry ice ethanol bath, thawed on ice, and pelleted in a
microfuge at 10 000 rpm for 20 minutes at 4°C. The supernatant was
recovered and 50 µL of a 50% protein A slurry, precoated with
anti-PLC1 antisera (Pharmingen, Mississauga, Ontario) was added. The
samples were incubated 2 hours at 4°C with rotation and the beads
were washed 5 times in lysis buffer. After the final wash, the beads
were resuspended in gel-loading buffer that contained 2-mercaptoethanol and boiled, and the samples were resolved on a 6%
polyacrylamide gel. The proteins were transferred to nitrocellulose that was blocked in TBST containing 1% gelatin (Bio-Rad, Mississauga, Ontario), incubated with antiphosphotyrosine antibody (4G10; Upstate Biotechnology) at 0.5 µg/mL in a 1% gelatin solution overnight at
room temperature. Secondary antibody, development of the blot, stripping, and reprobing with anti-PLC1 antibody was performed as
described previously.
Calcium mobilization measurements The 32D infectants were starved in RPMI with no FBS for 2 hours at 37°C, washed 3 times in Ca++ mobilization buffer, (140 mmol/L NaCl, 4 mmol/L KCl, 1.8 mmol/L CaCl2 · 2H2O, 0.8 mmol/L Mg2SO4 · 7H2O, 1 mmol/L KH2PO4, and 10 mmol/L glucose, pH 8) and then incubated with 10 µmol/L Indo-1 AM ester dye (Molecular Probes, Eugene, OR) plus 0.03% pluronic acid (Molecular Probes) for 1 hour at 37°C. Cells were then washed in the same buffer at pH 7.4 and resuspended at a volume of 1 × 107 cells/mL. Cells were kept at room temperature and, for each Ca++ measurement, warmed to 37°C and kept at this temperature for the duration of the 400-second run. SLF was added at a concentration of 200 ng/mL after 30 seconds, and Ca++ mobilization was monitored using flow cytometry by following the ratio of bound-to-unbound fluorophore over time.Stimulation assays For bioassays with soluble SLF, 32D infectants were washed 3 times in RPMI + 0.5% FBS and then plated in the same medium with sSLF at a density of 2.5 × 104 cells per well in 96-well flat-bottom plates. The X9/D3 cells are Sl/Sl4 cells that have been transfected with an expression vector that produces only the membrane-bound form of SLF. For Sl/Sl4 or X9/D318 coculture assays, the adherent cells were treated with mitomycin C (1.8 µg/mL) for 2 hours at 37°C, washed 3 times with PBS, trypsinized, counted, and then plated in 96-well plates that had been precoated with 0.1% gelatin (Sigma) at a concentration of 1 × 104 cells per well. The cells were allowed to adhere for 4 to 6 hours. The 32D cells were washed 3 times in RPMI + 0.5% FBS and starved of growth factor for 5 hours. The 32D cells were then added to the wells at a density of 2 × 104 cells per well in medium containing 2% FBS final concentration. For the immobilized anti-c-Kit antibody assays, 96-well plates (Nunc) were coated with 10 µg/mL of a mouse antirat monoclonal antibody (Jackson) overnight at 4°C. Excess antibody was then washed from the plate, the plate was blocked with PBS plus 1% FBS, followed by the addition of varying concentrations of ACK-2 (gift from Dr S. Nishikawa) in PBS-FBS.48 Plates were incubated for 2 hours at 4°C and washed several times, then 2 × 104 32D infectants were added to each well. Neomycin sulfate, when used, was preincubated with the 32D cells for 20 minutes. In all cases, after 18 hours of stimulation, 0.037 MBq (1 µCi) of 3H-thymidine was added to each well for 6 hours. Cells were then harvested and incorporated radioactivity was determined by scintillation counting. The degree of stimulation was determined by calculating the ratio of radioactivity incorporated by the infectants in the presence of the SLF-expressing stromal cells to radioactivity incorporated by the infectants in the presence of stromal cells not expressing SLF after subtracting off counts obtained with the stromal cells alone, according to the formula:
Leukemic potential The 5 × 106 32D infectants in PBS were injected intravenously into 5- to 7-week-old female C3H/He mice. Six to 7 weeks later, the mice were killed, and spleen cells and bone marrow cells from both femurs were removed. Single cell suspensions were prepared and counted with a hemocytometer. To determine the 32D-Kit cell content, 1 × 105 cells were plated in RPMI containing 0.3% agar plus 10% FBS, 4% IL-3, and 0.5 mg/mL G418. The plates were incubated for 10 days, after which the number of colonies was determined. Control experiments have determined that the plating efficiency of 32D cells and the various infectants was approximately 30%.
Expression of c-Kit on 32D cells To evaluate the role of PI3-kinase and PLC- activation by c-Kit
after stimulation by sSLF or mSLF, we transferred WT and c-Kit
receptors mutated at recruitment sites for these enzymes into 32D
cells. The 32D cells are a c-Kit negative, IL-3-dependent myelomonocytic cell line that have been shown previously to express c-Kit on infection with retroviral vectors containing the
c-kit gene.47 The 32D cells were infected with
KitWT, KitYF719, and KitYF728 retroviral constructs, selected, and
sorted for c-Kit-positive cells by FACS. Single clones were generated
and tested for c-Kit expression by flow cytometry. As shown in Figure
1A, 32D cells infected with either KitWT,
KitYF719, KitYF728, or a double mutant, KitYF719/YF728, express
detectable levels of c-Kit on their surface. The mean fluorescence
intensities are approximately the same, indicating equivalent levels of
receptor on the cell surface of all infectants.
Tyrosine phosphorylation of 32D c-Kit and recruitment of p85 We evaluated the ability of the WT and mutant c-Kit receptors to undergo autophosphorylation on ligand binding by exposing the infectants to sSLF, followed by immunoprecipitation with anti-Kit antisera and immunoblotting with antiphosphotyrosine antibodies. As shown in Figure 1B, KitWT, KitYF719, and KitYF728 were tyrosine phosphorylated on stimulation with sSLF (middle panel). When this blot was stripped and reprobed with antibodies to the p85 regulatory domain of PI3-kinase, it was found that p85 coimmunoprecipitated with KitWT and KitYF728 receptors but not KitYF719 receptors. This is in accordance with previous results which demonstrated that Y719 is part of the consensus binding site for PI3-kinase.49,50 The amount of precipitated c-Kit (bottom panel) was found to be similar for all 3 infectants as determined by stripping and reprobing the blot with anti-c-Kit antibodies (Rb 125). We failed to observe immunoprecipitated c-Kit protein or tyrosine phosphorylation in uninfected 32D cells. Therefore, all 3 of these infectants demonstrate kinase activity as exhibited by tyrosine phosphorylation on stimulation with sSLF, and only KitWT and KitYF728 receptors coimmunoprecipitate p85.KitYF728 receptors do not induce Steel Factor-stimulated
PLC- SH2 binding domain.51,52 It has been previously demonstrated that PLC- is recruited to the c-Kit receptor and becomes tyrosine phosphorylated after stimulation by
sSLF.53,54 To confirm the involvement of Y728 in
activating PLC-, we investigated the tyrosine phosphorylation of
PLC-1 in response to SLF stimulation. Preliminary experiments
confirmed that PLC-1 was expressed in both 32D cells and bone
marrow-derived mast cells (BMMCs). We also observed that in contrast to
BMMCs, 32D cells expressed very low levels of PLC-2. Therefore,
phosphorylation of this isoenzyme was not analyzed. The 32D cells
expressing KitWT or KitYF728 were stimulated with sSLF for 5 minutes,
PLC-1 was immunoprecipitated and then analyzed by Western blotting
with antiphosphotyrosine antibodies. As an additional control, PLC-1
from sSLF-stimulated BMMCs was analyzed in a similar fashion. As shown
in Figure 2B PLC-1 tyrosine
phosphorylation was increased in response to sSLF in both BMMCs and
32DkitWT cells. The degree of phosphorylation was considerably greater
in BMMCs than in 32D-kitWT cells likely because of the higher c-Kit
receptor levels on BMMCs (Figure 2A). In contrast, no additional
tyrosine phosphorylation of PLC-1 was observed in lysates from
32D-KitYF728 cells in response to sSLF.
To further investigate the effect of the YF728 mutation, we followed
Ca++ mobilization, a downstream consequence of PLC-
KitWT, KitYF719, and KitYF728 but not KitYF719/YF728 32D cells respond to soluble Steel Factor Both PI3-kinase and PLC- have been implicated in growth factor
receptor-mediated mitogenesis,35-37,39,40,56 and we have previously demonstrated that PLC--stimulated Ca++
influx is critical for SLF-dependent cell survival.42 We
determined the effect of the KitYF719 and KitYF728 mutations on
SLF-stimulated mitogenesis by incubating KitWT-, KitYF719-, and
KitYF728-expressing 32D cells with varying concentrations of sSLF and
measuring thymidine incorporation. In addition to these 3 cell types,
32D cells expressing the KitYF719/YF728 double mutant were also tested.
As shown in Figure 4A, the 3 cell lines
32D-KitWT, KitYF719, and KitYF728 responded equally well to sSLF (empty
circles, filled circles, and empty squares, respectively). In contrast,
the KitYF719/YF728 double-mutant 32D cells (filled squares) failed to
respond to sSLF, exhibiting counts that were not significantly
different than those obtained with the uninfected 32D c-Kit-negative
cells (crosses). A failure to incorporate thymidine may represent
either loss of cell viability or simple growth arrest. To distinguish between these 2 possibilities, we determined the proportion of viable
cells in the cultures after incubation in the presence or absence of
sSLF. As shown in Figure 4B, SLF maintains the viability of 32D cells
expressing the WT receptor or receptors with either the YF719 or YF728
mutations. In contrast, cells expressing the double mutant do not
remain viable in sSLF. These data are therefore consistent with the
requirement for either PI3-kinase or PLC- activation for survival
and mitogenic signals. These results are also in agreement with those
of Valius and Kazlauskas43 who demonstrated that either
the PI3-kinase or the PLC- binding sites were sufficient to restore
PDGF-mediated mitogenesis, but that receptors bearing mutations at both
of these sites were mitogenically inert.
Neomycin sulfate inhibits stimulation of KitYF719- but not KitWT- or KitYF728-expressing cells by soluble Steel Factor Our observation that the YF719/YF728 double c-Kit mutant does not mediate a survival or mitogenic signal in response to sSLF suggests that in the absence of PI3-kinase recruitment, PLC- activation is
required for cell support. To further confirm this requirement, cells
expressing either the WT or mutant receptors were stimulated in the
presence or absence of neomycin sulfate, an antagonist of PLC
activity.57 As shown in Figure
5, neomycin concentrations as high as 500 µmol/L have little effect on stimulation by sSLF of 32D cells
expressing the KitWT or KitYF728 receptor. In contrast, 32D cells
expressing KitYF719 are inhibited by neomycin sulfate. The
IC50 is approximately 100 µmol/L, a concentration previously reported to be inhibitory for PLC-dependent
processes.57-61 These data therefore support the
conclusion that, in the absence of PI3-kinase recruitment, PLC
activation is required for a full mitogenic signal by sSLF.
Response of KitYF728 receptors to membrane-bound Steel Factor is impaired We next determined the ability of c-KitWT and mutant receptors to be stimulated by mSLF. We used X9/D3 stromal cells as a source of mSLF.18 These cells were generated by transfecting SLF-negative Sl/Sl4 cells with an expression vector encoding a form of murine SLF that produces only the membrane bound and not the soluble form of the ligand. X9/D3 cells were treated with mitomycin C, plated, and then cocultured with KitWT, KitYF719, KitYF728, or uninfected 32D cells. As shown in Figure 6, both KitWT and KitYF719 32D infectants are stimulated 7- to 8-fold by coculture with X9/D3 cells above stimulation obtained with SLF-negative Sl/Sl4 cell cocultures. This pattern of stimulation has been observed in a number of experiments that used other mSLF-expressing cell lines as well (not shown). In contrast, the KitYF728 32D infectants were stimulated at most 3-fold by X9/D3 cells above that seen using SLF-negative Sl/Sl4 cells as the stimulus. This experiment revealed that, although 32D-KitYF728 cells are fully stimulated by sSLF, these cells are poorly stimulated by mSLF, suggesting that PLC- activation
may be critical for responding to the membrane-bound isoform
of SLF.
KitWT and KitYF719 receptors but not KitYF728 receptors respond to plate-bound anti-c-Kit antibodies An alternate method that mimics stimulation by mSLF is the use of plate-bound c-Kit-specific antibodies.62 We therefore used this form of stimulation, which is not complicated by the presence of other cellular factors, to investigate the response of cells bearing WT and mutant receptors. We found that c-Kit-positive 32D cells failed to be stimulated by rat antimouse c-Kit-specific monoclonal antibody ACK-2 alone or mouse-antirat IgG alone (data not shown). However, when both plate-bound antirat antibodies, followed by ACK-2, were used together, both 32D-KitWT and KitYF719 cells responded to the plate-bound antibodies in a concentration-dependent fashion with maximal stimulation of 8- to 9-fold above background (Figure 7). In contrast, KitYF728 cells exhibited stimulation no more than 2- to 3-fold above background. Therefore, although 32D-KitYF728 cells respond to sSLF, they fail to fully respond to plate-bound anti-Kit antibodies. Given the previous observation that plate-bound anti-c-Kit antibodies have been shown to mimic mSLF,62 this experiment is consistent with a requirement for PLC- activation after stimulation with mSLF or
immobilized ligand.
Neomycin sulfate inhibits stimulation by membrane-bound Steel Factor or immobilized anti-c-Kit antibodies Our results indicate that PLC- activation may be critical for
responding to the membrane-bound isoform of SLF or to immobilized agonists such as anti-c-Kit antibodies. To further investigate this
possibility, we determined the effect of the PLC antagonist neomycin
sulfate on stimulation of c-Kit-positive cells by mSLF, sSLF, or
immobilized anti-c-Kit antibodies. As shown previously in Figure 5,
addition of neomycin sulfate has only a minimal effect on the ability
of sSLF to stimulate 32D-KitWT cells. In contrast as shown in Figure
8, neomycin sulfate specifically inhibits
the ability of fibroblasts or immobilized anti-c-Kit antibodies to support these cells. Furthermore, the IC50 in both cases is
approximately 100 µmol/L, a concentration consistent with previously
reported concentrations required to inhibit PLC activation
58,59 and the concentration required to inhibit stimulation
of YF719 cells by sSLF (Figure 5). These results therefore provide
further evidence that PLC activation is important for cells stimulated
by mSLF but not sSLF.
32D-KitYF728 cells have impaired leukemic potential Our results indicate that PLC- activation is particularly
important for supporting Kit-positive cells by mSLF. Because mSLF is
likely the more important form of the growth factor in vivo, our
results predict that cells dependent on c-Kit-SLF interactions for in
vivo growth may also be dependent on PLC- activation in vivo. The
32D cells are normally nonleukemogenic. However, transfer and
expression of c-kit in these cells renders them
responsive to SLF and leukemogenic.47 We therefore tested
the leukemic potential of the 32D cells expressing Kit-WT, YF719, and
YF728 receptors. The 5 × 106 cells were injected
intravenously into unirradiated syngeneic C3H/He mice. Six to 7 weeks
later, the presence of 32D cells in the spleen and bone marrow was
quantitated using colony formation in agar as an assay. The presence of
cells in these organs has been found to be a reliable indicator for the
eventual development of lethal leukemia47 (Sittaro and
Berger; unpublished data, June 2000). As summarized in Tables
1 and
2, 32D-KitWT and KitYF719 cells
were readily detected in the spleen and bone marrow of inoculated mice
(P values of .001 for WT vs control, and
P = .034 and .002 for KitYF719 vs control). Tumor load for
YF719-expressing cells appeared to be reduced compared with
WT-expressing cells; however, this difference was not statistically
significant. Furthermore, mice inoculated with these cells will develop
massive splenomegaly, extensive infiltration of liver and lymph nodes,
and eventual death (Sittaro and Berger; unpublished data, June
2000). In contrast, recovery of 32D-KitYF728 cells from the spleen and
bone marrow was severely diminished (P values of .03, .004, and .001 vs KitWT in spleen; P values of .012 and .004 vs WT
in bone marrow). Diminished recovery of cells was observed for 2 independent 32D-KitYF728 clones as well as an uncloned, heterogenous
population of 32D-KitYF728 cells derived by sorting for Kit-positive
cells after large scale infection with the KitYF728 virus. It is
therefore unlikely that the diminished recovery of KitYF728 cells in
vivo is due to the variation in leukemic potential among different
clones. Furthermore, we have extended these observations for as long as
12 weeks after inoculation and have observed that even at this late
time point, recovery of YF728 cells is severely diminished (Berger and
Sittaro; unpublished data, June 2000). Taken together, these
results therefore suggest that tyrosine 728 is of particular importance
in supporting 32D cells in vivo, likely because of the contribution of
this residue in activating PLC-.
In this study, we have investigated the role of 2 c-Kit-associated signaling molecules, PI3-kinase and PLC- We extended these observations by investigating the response of
these c-Kit mutants to mSLF. We observed that, although both KitWT and
KitYF719 transmitted mitogenic signals in response to mSLF, KitYF728,
the mutant that does not activate PLC- A number of studies have demonstrated cross-regulation of PLC- Our results highlight the importance of PLC- Although this study has focused on the roles of PI3-kinase and PLC-
We wish to thank Drs R. Rottapel, J. Greenberger, and H. Ziltener for cell lines and reagents. We also wish to acknowledge the excellent assistance of R. Kapur, D. Bouchard with the Ca++ measurements, and R. Chow with the vector production and sequencing.
Submitted November 8, 1999; accepted August 1, 2000.
Supported by grants from the National Cancer Institute of Canada and the Leukemia Research Fund of Canada to S.A.B. and from the NIH (2RO1 DK 48605) to D.A.W.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stuart A. Berger, Arthritis and Immune Disorder Research Centre, 620 University, Suite 700, Toronto, Ontario, Canada, M5G 2M9; e-mail: berger{at}oci.utoronto.ca.
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Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking. Mol Biol Cell. 1995;6:1145-1158. 27. Wennstrom S, Hawkins P, Cooke F, et al. Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr Biol. 1994;4:385-393. 28. Thelen M, Uguccioni M, Bosiger J. PI 3-kinase-dependent and independent chemotaxis of human neutrophil leukocytes. Biochem Biophys Res Commun. 1995;217:1255-1262. 29. Derman MP, Cunha MJ, Barros EJ, Nigam SK, Cantley LG. HGF-mediated chemotaxis and tubulogenesis require activation of the phosphatidylinositol 3-kinase. Am J Physiol. 1995;268:F1211-1217. 30. Hazeki O, Hazeki K, Katada T, Ui M. Inhibitory effect of wortmannin on phosphatidylinositol 3-kinase-mediated cellular events. J Lipid Mediat Cell Signal. 1996;14:259-261. 31. Kundra V, Escobedo JA, Kazlauskas A, et al. 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Gonzalez GA, Merida I, Martinez AC, Carrera AC. Intermediate affinity interleukin-2 receptor mediates survival via a phosphatidylinositol 3-kinase-dependent pathway. J Biol Chem. 1997;272:10220-10226. 38. Catt KJ, Hunyady L, Balla T. Second messengers derived from inositol lipids. J Bioenerg Biomembr. 1991;23:7-27. 39. Kamat A, Carpenter G. Phospholipase C-gamma1: regulation of enzyme function and role in growth factor-dependent signal transduction. Cytokine Growth Factor Rev. 1997;8:109-111. 40. Alimandi M, Heidaran MA, Gutkind JS, et al. PLC-gamma activation is required for PDGF-betaR-mediated mitogenesis and monocytic differentiation of myeloid progenitor cells. Oncogene. 1997;15:585-593. 41. Sette C, Bevilacqua A, Bianchini A, Mangia F, Geremia R, Rossi P. Parthenogenetic activation of mouse eggs by microinjection of a truncated c-kit tyrosine kinase present in spermatozoa. Development. 1997;11:2267-2274. 42. Gommerman JL, Berger SA. Protection from apoptosis by Steel factor but not interleukin-3 is reversed through blockade of calcium influx. Blood. 1998;91:1891-1900. 43. Valius M, Kazlauskas A. Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signals. Cell. 1993;73:321-334. 44. Kinashi T, Escobedo JA, Williams LT, Takatsu K, Springer TA. Receptor tyrosine kinase stimulates cell-matrix adhesion by phosphatidylinositol 3 kinase and phospholipase C-gamma 1 pathways. Blood. 195;86:2086-2090. 45. Moriya S, Kazlauskas A, Akimoto K, et al. Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A. 1996;93:151-155. 46. Derman MP, Chen JY, Spokes KC, Songyang Z, Cantley LG. An 11-amino acid sequence from c-met initiates epithelial chemotaxis via phosphatidylinositol 3-kinase and phospholipase C. 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J Cell Sci. 1994;18:121-126. 53. Rottapel R, Reedijk M, Williams DE, et al. The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor. Mol Cell Biol. 1991;11:3043-3051. 54. Huber M, Helgason CD, Scheid MP, Duronio V, Humphries RK, Krystal G. Targeted disruption of SHIP leads to Steel factor-induced degranulation of mast cells. EMBO J. 1998;17:7311-7319. 55. Columbo M, Botana LM, Horowitz EM, Lichtenstein LM, MacGlashan DJ. Studies of the intracellular Ca2+ levels in human adult skin mast cells activated by the ligand for the human c-kit receptor and anti-IgE. Biochem Pharmacol. 1994;47:2137-2145. 56. Wang Z, Gluck S, Zhang L, Moran MF. Requirement for phospholipase C-gamma1 enzymatic activity in growth factor-induced mitogenesis. Mol Cell Biol. 1998;18:590-597. 57. Gabev E, Kasianowicz J, Abbott T, McLaughlin S. Binding of neomycin to phosphatidylinositol 4,5-bisphosphate (PIP2). 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© 2000 by The American Society of Hematology.
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  S. Mithraprabhu and K. L Loveland Control of KIT signalling in male germ cells: what can we learn from other systems? Reproduction, November 1, 2009; 138(5): 743 - 757. [Abstract] [Full Text] [PDF]
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 L. Hong, V. Munugalavadla, and R. Kapur c-Kit-Mediated Overlapping and Unique Functional and Biochemical Outcomes via Diverse Signaling Pathways Mol. Cell. Biol., February 1, 2004; 24(3): 1401 - 1410. [Abstract] [Full Text] [PDF]
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 G.-Q. Yao, J.-J. Wu, B.-H. Sun, N. Troiano, M. A. Mitnick, and K. Insogna The Cell Surface Form of Colony-Stimulating Factor-1 Is Biologically Active in Bone in Vivo Endocrinology, August 1, 2003; 144(8): 3677 - 3682. [Abstract] [Full Text] [PDF]
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 B. L. Tan, L. Hong, V. Munugalavadla, and R. Kapur Functional and Biochemical Consequences of Abrogating the Activation of Multiple Diverse Early Signaling Pathways in Kit. ROLE FOR Src KINASE PATHWAY IN KIT-INDUCED COOPERATION WITH ERYTHROPOIETIN RECEPTOR J. Biol. Chem., March 21, 2003; 278(13): 11686 - 11695. [Abstract] [Full Text] [PDF]
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 R. Kapur, S. Chandra, R. Cooper, J. McCarthy, and D. A. Williams Role of p38 and ERK MAP kinase in proliferation of erythroid progenitors in response to stimulation by soluble and membrane isoforms of stem cell factor Blood, July 30, 2002; 100(4): 1287 - 1293. [Abstract] [Full Text] [PDF]
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S. Maddens, A. Charruyer, I. Plo, P. Dubreuil, S. Berger, B. Salles, G. Laurent, and J.-P. Jaffrezou Kit signaling inhibits the sphingomyelin-ceramide pathway through PLCgamma 1: implication in stem cell factor radioprotective effect Blood, July 30, 2002; 100(4): 1294 - 1301. [Abstract] [Full Text] [PDF]
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J. Soboloff and S. A. Berger Sustained ER Ca2+ Depletion Suppresses Protein Synthesis and Induces Activation-enhanced Cell Death in Mast Cells J. Biol. Chem., April 12, 2002; 277(16): 13812 - 13820. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) or stimulated (+) with SLF for 2.5 minutes
at 37°C. Receptors were then precipitated and resolved by 7.5%
SDS-PAGE, followed by transfer to nitrocellulose and blotting with
antiphosphotyrosine antibodies (middle panel). Blots were then stripped
and reprobed with anti-p85 antibodies (upper panel). Equal levels of
Kit were precipitated in each sample as evidenced by stripping and
reprobing with anti-c-Kit antibodies (bottom panel).
), KitYF719 (
), KitYF728
(
), KitYF719/YF728 (
), or uninfected (×) were incubated with
various concentrations of sSLF for 18 hours at 37°C, followed by a 6 hours 3H-thymidine pulse. Cells were then harvested and
incorporated counts were determined by scintillation counting. Error
bars represent the standard error determined from triplicate
measurements. (B) 32D infectants were incubated with sSLF overnight.
Percentage (%) viability was determined by scoring ability to exclude
trypan blue. Viability of all cells in the presence of WEHI cm
was 93% to 97%.
)
were incubated with 150 ng/mL sSLF and varying concentrations of
neomycin sulfate for 18 hours at 37°C, followed by a 6 hours
3H-thymidine pulse. Cells were then harvested and
incorporated counts were determined by scintillation counting.
Percentage control refers to incorporated counts observed in the
absence of neomycin sulfate. Error bars represent the standard error
determined from triplicate measurements.
