From the Ludwig Institute for Cancer Research,
Biomedical Center, SE-751 24 Uppsala, Sweden, Sweden, the
** Department of Anatomy and Cell Biology, Monash University,
Clayton, Victoria 3800, Australia, the
Van
Andel Institute, Grand Rapids, Michigan 49503, and the
§§ School of Biomedical Sciences, University of
Newcastle, Callaghan, New South Wales 2308, Australia
Received for publication, November 18, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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In both mice and humans alternate splicing
results in isoforms of c-Kit characterized by the presence or the
absence of a tetrapeptide sequence, GNNK, in the juxtamembrane region
of the extracellular domain. Dramatic differences in the kinetics and magnitude of activation of the intrinsic tyrosine kinase activity of
c-Kit between the GNNK The receptor for stem cell factor, c-Kit, is a type III receptor
tyrosine kinase belonging to the same subfamily as the platelet-derived growth factor receptors, FLT3 (fms-like
tyrosine kinase 3), and the macrophage
colony-stimulating factor receptor (1). The c-Kit gene is
identical to the white spotting locus (W) in the mouse (2, 3). Partial or complete loss of function mutations in
c-Kit result in macrocytic anemia, aberrations in
pigmentation, decreased fertility, mast cell deficiency, reduction in
gastrointestinal motility, and impairment in learning function in the
hippocampus (reviewed in Refs. 4 and 5).
Ligand binding leads to activation of c-Kit, resulting in diverse
cellular responses such as differentiation, proliferation, growth,
survival, adhesion, and chemotaxis. These responses are the end result
of the activation of multiple signal transduction pathways. Signal
transduction molecules that bind to and become activated by c-Kit
include Src family kinases (binding to Tyr568), Grb2
(binding to Tyr703 and Tyr936), SHP-2 (binding
to Tyr568), SHP-1 (binding to Tyr570), and
phosphoinositide 3-kinase (binding to Tyr721) (6-10).
Alternative mRNA splicing results in the production of two isoforms
of c-Kit in the mouse and four in humans. In both mouse and human,
alternate splicing results in isoforms characterized by the presence or
absence of a four-amino acid sequence, GNNK, in the juxtamembrane
region of the extracellular domain (Fig. 1) (11, 12) This has been shown to be due
to the use of alternative 5' splice donor sites (13, 14). The two
splice forms, denoted GNNK and GNNK+ isoforms has previously been shown.
Here we report the analysis of downstream targets of receptor signaling, which revealed that the signaling was differentially regulated in the two splice forms. The kinetics of phosphorylation of
Shc, previously demonstrated to be phosphorylated by Src downstream of
c-Kit, was stronger and more rapid in the GNNK
form, whereas it
showed slower kinetics in the GNNK+ form. Inhibition of Src family
kinases with the specific Src family kinase inhibitor SU6656 altered
the kinetics of activation of the GNNK
form of c-Kit so that it
resembled that of the GNNK+ form. In cells expressing the GNNK
form,
SCF was rapidly degraded, whereas in cells expressing the GNNK+ form
only showed a very slow rate of degradation of SCF. In the GNNK+ form
the Src inhibitor SU6656 only had a weak effect on degradation, whereas
in the GNNK
form it dramatically inhibited degradation. In summary,
the two splice forms show, despite only a four-amino acid sequence
difference, remarkable differences in their signaling capabilities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and GNNK+, respectively, are co-expressed
in most tissues with the GNNK
form predominating (11, 12, 15).
Caruana et al. (16) demonstrated that NIH3T3 cells
expressing either isoform differed in their transforming activity. In
the presence of SCF,1 the
GNNK
form induced anchorage-independent growth, loss of contact
inhibition, and tumorigenicity. No difference in ligand binding
affinity was observed between the two isoforms. It was demonstrated
that upon ligand stimulation, the GNNK
isoform was more highly
tyrosine-phosphorylated, more rapidly internalized, and activated ERK
more strongly than the GNNK+ isoform.
View larger version (27K):
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Fig. 1.
Schematic outline of the architecture of
c-Kit. The extracellular part of c-Kit consists of five
immunoglobulin-like domains. Close to the plasma membrane, in the
extracellular part of c-Kit, resides the sequence GNNK, which is either
present or absent in the GNNK+ and GNNK splice forms of c-Kit,
respectively. The intracellular part of c-Kit contains the tyrosine
kinase domain, which is split into two parts by the kinase insert
sequence.
In this study, we have analyzed the molecular signaling mechanisms
activated by the GNNK and the GNNK+ isoforms. We have found that
dramatic differences in activation of particular signal transduction
pathways occur, whereas others remain identical between the two
isoforms. The differences in signaling between the two isoforms were
found to be to a large extent dependent on differential recruitment and
activation of Src family kinases.
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EXPERIMENTAL PROCEDURES |
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Antibodies, Antisera, Peptides, and Glutathione S-Transferase Fusion Proteins-- Recombinant human SCF was a kind gift of AMGEN, Inc. The Src inhibitor SU6656 was a kind gift of SUGEN, Inc. The rabbit antiserum Kit-C1, recognizing the C-terminal tail of c-Kit, was purified as described (17). A rabbit antiserum recognizing ERK2, EET, was raised against the C-terminal sequence of ERK2 (EETARFQPGYRS) (18). The Src antibody Ab-1 was from Oncogene Sciences. The anti-phosphotyrosine antibody PY99 and the Cbl antibody were from Santa Cruz Biotechnologies (Santa Cruz, CA), and affinity-purified anti-Shc antibodies were purchased from BD Transduction Laboratories; phospho-ERK (Thr202/Tyr204) was from Cell Signaling Technology. The glutathione S-transferase fusion protein of the c-Src SH2 domain was a kind gift from Dr. Tony Pawson.
Phosphospecific Antibodies--
Phosphospecific antibodies
against individual tyrosine phosphorylation sites in c-Kit were raised
by immunizing rabbits with the following synthetic peptides conjugated
to keyhole limpet hemocyanin by use of
m-maleimidobenzoyl-N-hydroxysuccinimide ester: CEEINGNNpYVYIDPTQ (Tyr(P)568), CSDSTNEpYMDMKPGV
(Tyr(P)721), CKNDSNpYVVKGA (Tyr(P)823), and
STNHIpYSNLANCS (Tyr(P)936). The antibodies were extensively
purified by chromatography over immobilized nonphosphorylated peptide,
over phosphotyrosine-Sepharose, and finally over immobilized
phosphopeptide. Purified antibodies were stored in 0.15 M
NaCl, 20 M HEPES, pH 7.4, 50% glycerol at 70 °C. The
specificity of each antibody was tested by Western blotting. COS7 cells
were transiently transfected using LipofectAMINE (Roche Molecular
Biochemicals) with either wild-type c-Kit or the corresponding
tyrosine-to-phenylalanine mutant of c-Kit in pcDNA3 (Invitrogen).
The cells were stimulated with 100 ng/ml of recombinant human SCF for
10 min, lysed, and subjected to immunoprecipitation with c-Kit
antibodies, followed by SDS gel electrophoresis and electrotransfer to
an Immobilon P membrane. All of the preparations of antibodies used in
this paper gave strong signals in wild-type receptors, whereas it gave
no signal or a very weak signal in the corresponding
tyrosine-to-phenylalanine mutant.
Cell Culture-- NIH3T3 cells stably expressing c-Kit have previously been described (16). Pools of cells expressing equal levels of c-Kit were used throughout the study. NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Immunoprecipitation and Western Blotting-- Immunoprecipitations and Western blotting were performed according to Ref. 17. Briefly, after stimulation of cells with 100 ng/ml SCF for the indicated period of time, the cells were washed once with ice-cold PBS, and lysed in a lysis buffer containing 1% Triton X-100, 25 mM Tris, pH 7.5, 1 mM Na3VO4, 1% Trasylol, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA. The lysates were centrifuged at 14 000 × g for 10 min at 4 °C, precleared for 30 min with 50 µl of a 1:1 slurry of protein A-Sepharose, and then incubated with the indicated antibody for 2 h at 4 °C. The immunoprecipitates were collected on protein A-Sepharose beads, washed three times in lysis buffer supplemented with 500 mM NaCl, and finally washed once with water. The samples were boiled for 3 min in reducing SDS sample buffer and separated by SDS-PAGE in 7% continuous gels, after which proteins were quantitatively electrotransferred to Immobilon P (Millipore) membranes. The membranes were blocked overnight in the cold with 0.2% Tween 20 in PBS, or in the case of phosphospecific antibodies, in 1% dry milk powder, 0.2% Tween 20 in PBS. The filters were incubated with primary antibody for 2 h at room temperature, followed by extensive washing with 0.5% Tween 20 in PBS. Incubation with secondary horseradish peroxidase-conjugated antibody was done for 1 h at room temperature, followed by extensive washing with 0.5% Tween 20 in PBS. The immunodetection was performed by enhanced chemiluminescence using the Super Signal Dura reagent from Pierce.
In Vitro Kinase Activity of c-Src and c-Kit--
In
vitro kinase assays of c-Src and c-Kit were performed as described
(8). In brief, subconfluent cells were starved overnight in serum-free
medium and stimulated with 100 ng/ml SCF at 37 °C for 5 min,
followed by lysis and immunoprecipitation with Kit-C1 antibody or Src
antibody Ab-1 (Oncogene Research), as outlined above. The
immunoprecipitates were incubated with 50 µM
[-32P]ATP in 40 µl of kinase buffer (10 mM MnCl2, 20 mM HEPES, pH 7.4, 1 mM dithiothreitol) for 10 min at room temperature before separation by SDS gel electrophoresis and exposure to x-ray film. Acid
denatured enolase was used as an exogenous substrate of Src (19).
Internalization Experiments--
Recombinant human SCF was
labeled with 125I as described (17). Internalization
experiments were performed essentially as described (20). In brief,
confluent cells in 12-well plates were incubated with 50,000 cpm of
125I-SCF/well for 60 min on ice. After washing twice with
binding medium (Dulbecco's modified Eagle's medium containing 1 mg/ml bovine serum albumin), the cells were incubated at 37 °C in binding medium for different time periods. The binding medium was removed and
precipitated with an equal volume of 10% trichloroacetic acid. The
amount of non-trichloroacetic acid-precipitable radioactivity was taken as an estimate of ligand degradation. After removal of the
medium, the cells were washed in binding medium and incubated for 5 min
on ice with phosphate-buffered saline containing 1 mg/ml bovine serum
albumin adjusted to pH 3.7 with acetic acid. This acid wash procedure
releases more than 90% of cell surface-bound ligand into the buffer.
After treatment with acidic buffer, the cells were lysed in 1% Triton
X-100. Radioactivity was determined using a Packard -counter.
[3H]Thymidine Incorporation Assay--
The assay
for incorporation of [3H]thymidine into trichloroacetic
acid-precipitable material was performed as described (21).
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RESULTS |
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Differences in Kinetics and Magnitude of Phosphorylation and the
Rate of Degradation between the Two Splice Forms--
NIH3T3 cells
stably expressing either the GNNK form or the GNNK+ form of human
c-Kit were stimulated with 100 ng/ml recombinant human SCF for
the indicated times at 37 °C, lysed in Triton X-100 lysis buffer,
and subjected to immunoprecipitation using the Kit-C1 antibody. The
immunoprecipitated proteins were separated by 7% SDS-PAGE, followed by
electrotransfer to Immobilon-P. The filter was first probed with
phosphotyrosine antibodies (PY-99), followed by stripping and reprobing
with Kit-C1 antibody. It could be seen that the GNNK
form was
strongly phosphorylated within minutes, followed by rapid degradation
(Fig. 2). In contrast, the GNNK+ form
showed a slow kinetics of rather weak autophosphorylation that
persisted over a long period of time with no apparent receptor degradation.
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Shc Is Phosphorylated More Rapidly and with Higher Stoichiometry by
the GNNK form than by the GNNK+ Form--
We have previously shown
that Src is activated downstream of c-Kit, leading to phosphorylation
of Shc, recruitment of Grb2-Sos, and activation of the
Ras/mitogen-activated protein kinase pathway (8). Therefore, we wanted
to investigate whether we could detect differences in Shc
phosphorylation by the two splice forms. Surprisingly, Shc was
phosphorylated more rapidly and with higher stoichiometry by the GNNK
form than by the GNNK+ form (Fig.
3A). Quantitation of data from
CCD camera detection of chemiluminescence revealed an almost 3-fold
higher magnitude of phosphorylation of Shc by the GNNK
form compared
with the GNNK+ form (Fig. 3B).
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Stimulation of the GNNK Form of c-Kit Leads to Association with
and Activation of Src--
Because we have previously shown that
phosphorylation of Shc by c-Kit is dependent on Src kinase activity
(8), we wanted to investigate whether Src was activated differentially
by the two c-Kit isoforms. NIH3T3 cells expressing either the GNNK
or the GNNK+ form of c-Kit were stimulated with SCF and lysed. The lysates
were incubated with a glutathione S-transferase fusion protein of the SH2 domain of c-Src prebound to glutathione-Sepharose beads. The beads were washed extensively, and the bound material was
separated on a SDS-PAGE, followed by electrotransfer to an Immobilon-P
membrane. Probing of the membrane with Kit-C1 antibody revealed that
the SCF-stimulated GNNK
isoform readily associated with the Src SH2
domain, whereas association of the GNNK+ form was very weak (Fig.
4A). To measure the kinase
activity of Src, lysates from NIH3T3 cells expressing either the GNNK
or the GNNK+ form of c-Kit were subjected to immunoprecipitation with
an antibody against c-Src and tested in an in vitro kinase
assay using acid-denatured enolase as a substrate. As a control for
specificity, the immunoprecipitates were incubated either with or
without the Src kinase inhibitor SU6656. The GNNK
isoform could be
demonstrated to induce a considerably stronger activation of c-Src than
the GNNK+ isoform (Fig. 4B). To verify that the kinase
activity of c-Kit was unaffected by SU6656, immunoprecipitates of c-Kit
from SCF-stimulated cells were incubated with
[
-32P]ATP in the presence or absence of SU6656 (Fig.
4C). As expected, the kinase activity of c-Kit, as judged by
autophosphorylation, was unaffected by SU6656. The lower activity seen
in the GNNK
form can be explained by the rapid ligand-induced
degradation of c-Kit, compared with the GNNK+ isoform. The lower band
seen corresponds to the immature form of c-Kit.
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Phosphorylation of ERK Induced by the GNNK Form of c-Kit Is
Stronger than by the GNNK+ Form and Dependent on the Activity of Src
Family--
When studying the signaling from the GNNK
and GNNK+
forms of c-Kit, Caruana et al. (16) demonstrated a dramatic
difference in ERK activation between the splice forms. Whereas the
GNNK
form was strongly and rapidly activated, the GNNK+ induced a
weak activation of ERKs. One possible explanation for this difference in ERK activation could be that different degrees of phosphorylation of
Shc by Src might lead to differences in ERK activation.
We showed that in NIH3T3 cells expressing the GNNK form of c-Kit, the
phosphorylation of ERK occurred much faster and reached a higher level
than in cells expressing the GNNK+ form (Fig.
5). Furthermore, activation of ERKs could
largely be inhibited by the Src inhibitor SU6656, suggesting a role of
Src family kinases in the activation of ERKs by c-Kit in these cells.
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The Src Inhibitor SU6656 Causes a Decrease in Phosphorylation and
Degradation of the GNNK Form of c-Kit, as Compared with the GNNK+
Isoform--
The finding that c-Src associated more strongly with the
GNNK
form than with the GNNK+ form, together with the finding that Shc, a substrate for c-Src, was phosphorylated to a higher degree by
the GNNK
form than the GNNK+ form, prompted us to investigate the
role of c-Src in the differential signaling seen by the two isoforms of
c-Kit.
Preincubation of NIH3T3 cells stably expressing the GNNK form of
c-Kit with the selective Src inhibitor SU6656 (22) caused slower
kinetics of autophosphorylation of c-Kit, as well as a lower magnitude
of autophosphorylation (Fig. 6).
Furthermore, degradation of c-Kit in the presence of SU6656 was not as
prominent as in the absence of inhibitor. When comparing the GNNK
form in the presence of Src inhibitor, the kinetics of phosphorylation and degradation resembled the pattern of the GNNK+ form, suggesting an
involvement of differential activation of c-Src as a mechanism of
splice-specific signaling.
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Kinetics of SCF-induced Internalization in the Two Splice Forms of
c-Kit--
To assess whether the decrease in c-Kit expression
following ligand stimulation of NIH3T3 cells expressing either the
GNNK form or the GNNK+ form was due to differences in
internalization, the cells were incubated with 125I-SCF on
ice for 1 h. The cells were washed extensively and transferred to
37 °C and incubated for the indicated period of time. The cell surface bound radioactivity was removed by washing with an acidic buffer. Ligand-induced internalization was only slightly faster in the
GNNK
form compared with GNNK+ form (data not shown), and by use of
the SU6656 Src kinase inhibitor, it could be shown to be partially
dependent on the activity of Src family kinases.
Degradation of SCF Internalized through c-Kit Occurs Very Rapidly
in Cells Expressing the GNNK Form, whereas It Is Degraded Slowly in
Cells Expressing the GNNK+ Form--
The cell lysates from the
experiment described above were treated with 10% trichloroacetic acid
to precipitate protein bound radioactivity. After centrifugation, the
supernatant was counted in a
-counter, as a measure of degradation.
It could clearly be seen that degradation of 125I-SCF bound
to the GNNK+ form (Fig. 7) occurred very
slowly and was slightly dependent on the activity of Src family
kinases. In contrast, degradation of 125I-SCF bound to the
GNNK
form was very rapid, and degradation was inhibited to about 50%
by the Src inhibitor SU6656.
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Cbl Is Phosphorylated following SCF Stimulation of NIH3T3 Cells
Expressing the GNNK Form of c-Kit, whereas the GNNK+ Form Mediates
Only a Weak Phosphorylation of Cbl--
The multifunctional adapter
protein Cbl has been shown to be involved in regulation of
polyubiquitination of proteins, which has been demonstrated to tag
proteins for degradation in either the lysosomes or in the proteasome
complex. Joazeiro and colleagues (23) demonstrated that Cbl
possesses a ubiquitin E3 ligase activity. Src has been demonstrated to
phosphorylate and positively regulate the ubiquitin ligase activity of
Cbl (24). Given the dramatic differences in degradation of the GNNK
and GNNK+ isoform following SCF stimulation, we wanted to test whether
differences in Src-mediated phosphorylation of Cbl could account for
the differences in degradation. NIH3T3 cells expressing either isoform
were stimulated with SCF in the presence or absence of SU6656, lysed,
and subjected to immunoprecipitation with an antibody against Cbl.
Immunoprecipitated proteins were separated by SDS gel electrophoresis,
electrotransferred to an Immobilon-P filter, and probed with
anti-phosphotyrosine antibodies followed by reprobing with Cbl
antibodies. It could be shown that phosphorylation of Cbl was
considerably stronger in cells expressing the GNNK
compared with
cells expressing the GNNK+ isoform and that the phosphorylation was
inhibited by the Src inhibitor SU6656 (Fig.
8).
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Mitogenic Response to SCF Is Stronger in NIH3T3 Cells Expressing
the GNNK Form as Compared with the GNNK+ Form--
It has previously
been shown that the GNNK
form is better than the GNNK+ form in
inducing anchorage-independent growth as well as focus formation of
NIH3T3 cells (16). Furthermore, several of the above mentioned signal
transduction pathways have been linked to transformation, such as the
Src family kinases and the Ras/ERK pathway. Therefore, we compared the
ability of the GNNK
and the GNNK+ forms to mediate an
SCF-dependent mitogenic response. NIH3T3 cells expressing
either isoform of c-Kit were starved for 24 h, followed by
stimulation with SCF for 48 h in the presence of
[3H]thymidine. Trichloroacetic acid-precipitable
radioactivity was counted in a scintillation counter. A difference in
mitogenic response to SCF was observed, where the GNNK
form was
better than the GNNK+ form (Fig. 9). This
is consistent with the observations that the Src family kinases and the
Ras/ERK pathway are more efficiently activated by the GNNK
form.
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Kinetics of Phosphorylation of Individual Tyrosine Residues in
c-Kit Follows Different Kinetics than the Overall
Phosphorylation--
To be able to study the phosphorylation of
individual tyrosine residues in c-Kit, a panel of phosphospecific
antibodies were produced recognizing phosphorylated Tyr568
(c-Src association site), Tyr721 (phosphoinositide 3-kinase
association site), Tyr823 (activation loop), and
Tyr936 (association site for Grb2 and Grb7). To verify the
specificity of the individual phosphospecific antibodies, COS7 cells
were transfected with either wild-type c-Kit (GNNK+) or the
corresponding tyrosine-to-phenylalanine mutation, stimulated with SCF
for 10 min at 37 °C, lysed, and immunoprecipitated with a c-Kit
antibody. After electrophoresis and transfer to Immobilon P membranes,
the filters were probed with either phosphospecific antibody. In all cases, wild-type c-Kit gave a strong signal, whereas the corresponding tyrosine-to-phenylalanine mutant gave a weak signal or no signal (Fig.
10A). To verify that the
tyrosine kinase activity of the mutant receptors was not impaired, the
filters were stripped and reprobed with anti-phosphotyrosine antibodies
(Fig. 10B).
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NIH3T3 cells expressing either the GNNK form or the GNNK+ form were
stimulated with 100 ng/ml SCF for the indicated time periods, lysed,
and subjected to immunoprecipitation with the Kit-C1 antibody.
Immunoprecipitated receptor was separated by SDS-PAGE and
electrotransferred to Immobilon-P membrane. The filter was then probed
with either phosphotyrosine antibodies (PY99), Tyr(P)568,
Tyr(P)721, Tyr(P)823, or Tyr(P)936
antibodies, respectively (Fig. 11). As
previously demonstrated, ligand stimulation of the GNNK
form led to
rapid phosphorylation, ubiquitination, and degradation of c-Kit,
whereas the GNNK+ showed a slower kinetics, weaker phosphorylation, but
persistent signaling (Fig. 11). When probing an identical filter with
the Tyr(P)568 antibody, rapid and transient phosphorylation
was seen in the GNNK
at the 2-min time point but then rapidly
declined. In contrast, the GNNK+ form showed a constant low level of
phosphorylation, even in the absence of SCF stimulation. The
Tyr(P)823 antibody against the activation loop tyrosine
thought to be of importance for activation of c-Kit kinase activity
followed the same kinetics as the overall tyrosine phosphorylation of
c-Kit; in the GNNK
form there was a rapid and strong increase in
phosphorylation, whereas in the GNNK+ form only a very weak signal was
detected. Probing with the Tyr(P)721 antibody, which
detects phosphorylation of the phosphoinositide 3-kinase association
site in c-Kit, revealed a rapid and strong phosphorylation in the
GNNK
form, whereas in the GNNK+ the phosphorylation was slow, but
persisted for a longer time, and was of the same magnitude as for the
GNNK
form. This is in agreement with previous observations by Caruana
et al. (16), demonstrating a similar magnitude in
association of phosphoinositide 3-kinase to c-Kit as well as
activation of Akt by the two splice forms. Phosphorylation of
Tyr936, constituting a docking site for both Grb2 and Grb7,
showed strong and rapid kinetics in the GNNK
form, whereas it was
even stronger and more persistent in the GNNK+ form (Fig. 11). In
summary, phosphorylation of individual sites did not follow the same
kinetics as the overall phosphorylation, which could explain why
different signal transduction pathways are affected to different extent
by the two splice forms.
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DISCUSSION |
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Several splice forms of human c-Kit have been described to date
(11, 12), but the physiological significance of these different forms
is unknown. Two of the existing splice forms differ in the presence or
absence of a four-amino acid stretch, GNNK. To investigate the function
of these isoforms, we have used NIH3T3 fibroblasts, which lack
endogenous c-Kit, transfected with either splice form of c-Kit (16). It
was previously shown that NIH3T3 fibroblasts expressing the GNNK form
exhibited SCF-dependent anchorage independent growth and
loss of contact inhibition and were tumorigenic in nude mice
(16). In contrast, NIH3T3 cells expressing the GNNK+ exhibited
anchorage-independent growth but relatively poor focus formation and
did not form tumors in nude mice. Despite similar affinity for
SCF, the GNNK
form displayed more rapid and extensive tyrosine
autophosphorylation and faster internalization.
In this paper, we have studied the molecular mechanisms behind the
differences in signal transduction from the two splice forms. We could
show that the kinetics of phosphorylation of c-Kit was much faster and
stronger in the GNNK form, compared with the GNNK+ form (Fig. 2).
This was followed by a rapid degradation of the GNNK
form, whereas
expression of the GNNK+ form remained stable. Yee et al.
(25) showed that in murine mast cells, ligand binding to c-Kit leads to
rapid internalization and ubiquitin-mediated degradation. Inactivation
of the receptor kinase resulted in reduced rate of internalization of
ligand-receptor complexes, and no ubiquitination took place. However,
Gommerman et al. (26) found, when studying the murine
lymphoma cell line DA-1 transfected with c-Kit, a dependence of an
intact Tyr721 site for internalization of ligand bound
c-Kit. A partial role for Src family kinases in ligand-driven
internalization of c-Kit was demonstrated by Broudy et al.
(27) using the inhibitor PP1. In our hands, inhibition of Src family
kinases caused a partial inhibition of c-Kit internalization, but a
dramatic effect was seen on ligand degradation (Fig. 8). A role for
c-Src in receptor degradation has been seen for platelet-derived growth
factor
-receptor (28). It was shown that receptor-mediated
phosphorylation of c-Cbl and polyubiquitination of the receptor
required the association site for Src family kinases on the receptor.
In other words, ligand-induced activation of Src family kinase activity
was a prerequisite for c-Cbl phosphorylation and ubiquitination. It has
recently been demonstrated that c-Cbl is a ubiquitin E3 ligase
(23).
We and others have previously shown (8, 29) that the main pathway of
c-Kit activation of the Ras/ERK pathway goes through Src-dependent phosphorylation of Shc. The Y568F/Y570F
mutant of c-Kit, which fails to activate Src family kinases, mediates
only a very weak activation of ERK. The two autophosphorylation sites, Tyr703 and Tyr936, that have been shown to bind
to Grb2 in vitro (10) seem to be of minor importance in the
in vivo situation. The stoichiometry of Tyr703
phosphorylation has been shown to be quite
low.2 Furthermore, the low
degree of direct association of Grb2 to c-Kit is lost if
Tyr703 is mutated to phenylalanine,2 suggesting
that Tyr936 plays a minor role in binding of Grb2 in the
in vivo situation. In this paper, we show that
c-Kit-mediated activation of ERK is stronger in the GNNK form
compared with the GNNK+ form, correlating with an increased degree of
activation of Src family kinases by the GNNK
form compared with the
GNNK+ form. Furthermore, activation of ERK by the two isoforms of c-Kit
is inhibited by the Src inhibitor SU6656 (Fig. 5). Thus, it is likely
that the differences in activation of the Ras/ERK pathway can be
accounted for by the differential regulation of Src family kinases by
the two splice forms. The role of ERK activation in the mitogenic
response to c-Kit stimulation is unclear. We have previously shown (8)
that when the two tyrosine residues responsible for activation of Src
family kinases, Tyr568 and Tyr570 in c-Kit, are
mutated and the level of ERK activation is very low, we still get a
reasonable mitogenic response. Other studies have also shown that,
under certain circumstances, c-Kit-mediated mitogenesis can be
independent of ERK activation (29).
The rapid and strong autophosphorylation of c-Kit seen in the GNNK
form could be due to c-Src-mediated phosphorylation of c-Kit at
Tyr823 in the activation loop of the tyrosine kinase
domain. Our data show that phosphorylation of Tyr823
parallels that of total receptor autophosphorylation.
Tyr823 in the activation loop is conserved in most tyrosine
kinases and in many cases found to be of importance for regulation of its activity in, for example, the insulin receptor and the fibroblast growth factor receptor (30, 31). However, cells expressing the Y823F
mutant of c-Kit still show a level of kinase activity similar to
wild-type receptors.2 Using cells overexpressing either
kinase active or inactive c-Src, Biscardi et al. (32) showed
that c-Src mediates phosphorylation of the epidermal growth factor
receptor at Tyr845 and Tyr1101.
Tyr845 is located in a position analogous to
Tyr823 in c-Kit, and phosphorylation of this site by c-Src
was found to contribute positively to epidermal growth factor-mediated mitogenicity.
It is striking that a stretch of only four amino acids in the
juxtamembrane region of the extracellular domain could make such a
dramatic difference in the signaling characteristics of a receptor.
However, recent data suggest that not only ligand-induced dimerization
is required for full activation of receptor tyrosine kinases but also
the steric orientation of the two receptor subunits (for review, see
Ref. 33). It has been shown that full activation of the erythropoietin
receptor requires not only dimerization of receptors but also the
correct orientation of the receptor subunit (34). Recently, Leibiger
et al. (35) described differences in the signal transduction
pathways activated and the repertoire of gene expression induced upon
stimulation of the two splice forms of the insulin receptor, INSR-A and
INSR-B. The INSR-A isoform was shown to induce expression of the
insulin gene, whereas the INSR-B isoform mediated induction of
glucokinase. Through differential use of exon11, the two splice forms
of the insulin receptor differ in only 12 twelve amino acids in the C
terminus of the -subunit. Furthermore, Bell et al. (36)
made a series of platelet-derived growth factor
-receptor mutants in
which they put a dimerization motif derived from the sequence of
oncogenic Neu at different positions in the transmembrane region. This
led to the creation of a series of constitutive receptor dimers in
which the intracellular parts were gradually rotated 103° for each
mutant. Interestingly, rotational linkage of the transmembrane domain
with the kinase domain was evidenced by a periodic activation of the
receptor because the dimerization motif was shifted across the
transmembrane domain. It might very well be that the four-amino acid
insert, which is located in a predicted
-helical region, leads to a
less favorable positioning of the intracellular parts of the receptor in a dimer than if those amino acids are absent. Interestingly, a
similar pair of splice variants affecting the juxtamembrane region of
the extracellular domain have been described for the ErbB2 receptor
(37). A novel transcript of ErbB2 was found in human carcinomas,
involving a deletion of 16 amino acids in the juxtamembrane region of
the extracellular domain. It was shown that this splice form showed
much stronger tyrosine kinase activity than the normal splice form, and
it was also much stronger in its transforming ability, as judged by
focus formation assay. Finally, Moriki et al. (38)
demonstrated the importance of correct orientation of the epidermal
growth factor receptors in a dimer for full kinase activity.
Future studies are aimed at understanding the precise mechanism by
which these two splice forms can activate Src family kinases and other
signal transduction pathways differently. By using cDNA microarray
technology, we will be able to dissect in greater detail the
differences and similarities in gene induction/repression by the
different splice forms of c-Kit and attempt to link these observations
to differences in biological responses induced by the two receptor forms.
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ACKNOWLEDGEMENTS |
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Recombinant human SCF was a kind gift of AMGEN, Inc., and the Src inhibitor SU6656 was kindly supplied by SUGEN, Inc.
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FOOTNOTES |
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* This work was in part supported by a grant from the Swedish Cancer Society.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.
§ These authors contributed equally to this work.
¶ Recipient of a postdoctoral fellowship from Wenner-Grenska Society (Stiftelsen Wenner-Grenska Samfundet). Present address: Dept. of Experimental Clinical Chemistry, Lund University, Wallenberg Laboratory, Malmö University Hospital, SE 205 02 Malmö, Sweden.
Present address: First Department of Surgery, Gunma University
School of Medicine, Gunma, Japan.
¶¶ Senior Researcher funded by the Swedish Research Council. To whom correspondence should be addressed. Present address: Dept. of Experimental Clinical Chemistry, Lund University, Wallenberg Laboratory, Malmö University Hospital, SE 205 02 Malmö, Sweden. Tel.: 46-40-33-72-22; Fax: 46-40-92-90-23; E-mail: Lars.Ronnstrand@klkemi.mas.lu.se.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M211726200
2 J. Lennartsson, E. Rollman, and L. Rönnstrand, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: SCF, stem cell factor; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Yarden, Y., Kuang, W. J., Yang-Feng, T., Coussens, L., Munemitsu, S., Dull, T. J., Chen, E., Schlessinger, J., Francke, U., and Ullrich, A. (1987) EMBO J. 6, 3341-3351[Abstract] |
2. | Chabot, B., Stephenson, D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988) Nature 335, 88-89[CrossRef][Medline] [Order article via Infotrieve] |
3. | Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988) Cell 55, 185-192[Medline] [Order article via Infotrieve] |
4. | Galli, S. J., Zsebo, K. M., and Geissler, E. N. (1994) Adv. Immunol. 55, 1-96[Medline] [Order article via Infotrieve] |
5. | Ashman, L. K. (1999) Int J Biochem. Cell Biol. 31, 1037-1051[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Linnekin, D.,
DeBerry, C. S.,
and Mou, S.
(1997)
J. Biol. Chem.
272,
27450-27455 |
7. |
Kozlowski, M.,
Larose, L.,
Lee, F.,
Le, D. M.,
Rottapel, R.,
and Siminovitch, K. A.
(1998)
Mol. Cell. Biol.
18,
2089-2099 |
8. | Lennartsson, J., Blume-Jensen, P., Hermanson, M., Pontén, E., Carlberg, M., and Rönnstrand, L. (1999) Oncogene 18, 5546-5553[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Serve, H.,
Hsu, Y. C.,
and Besmer, P.
(1994)
J. Biol. Chem.
269,
6026-6030 |
10. | Thömmes, K., Lennartsson, J., Carlberg, M., and Rönnstrand, L. (1999) Biochem. J. 341, 211-216[CrossRef][Medline] [Order article via Infotrieve] |
11. | Reith, A. D., Ellis, C., Lyman, S. D., Anderson, D. M., Williams, D. E., Bernstein, A., and Pawson, T. (1991) EMBO J. 10, 2451-2459[Abstract] |
12. | Crosier, P. S., Ricciardi, S. T., Hall, L. R., Vitas, M. R., Clark, S. C., and Crosier, K. E. (1993) Blood 82, 1151-1158[Abstract] |
13. | Hayashi, S., Kunisada, T., Ogawa, M., Yamaguchi, K., and Nishikawa, S. (1991) Nucleic Acids Res. 19, 1267-1271[Abstract] |
14. | Zhu, W. M., Dong, W. F., and Minden, M. (1994) Leuk Lymphoma 12, 441-447[Medline] [Order article via Infotrieve] |
15. | Serve, H., Yee, N. S., Stella, G., Sepp-Lorenzino, L., Tan, J. C., and Besmer, P. (1995) EMBO J. 14, 473-483[Abstract] |
16. | Caruana, G., Cambareri, A. C., and Ashman, L. K. (1999) Oncogene 18, 5573-5581[CrossRef][Medline] [Order article via Infotrieve] |
17. | Blume-Jensen, P., Siegbahn, A., Stabel, S., Heldin, C. H., and Rönnstrand, L. (1993) EMBO J. 12, 4199-4209[Abstract] |
18. | Leevers, S. J., and Marshall, C. J. (1992) EMBO J. 11, 569-574[Abstract] |
19. | Kypta, R. M., Goldberg, Y., Ulug, E. T., and Courtneidge, S. A. (1990) Cell 62, 481-492[Medline] [Order article via Infotrieve] |
20. |
Mori, S.,
Rönnstrand, L.,
Claesson-Welsh, L.,
and Heldin, C. H.
(1994)
J. Biol. Chem.
269,
4917-4921 |
21. | Blume-Jensen, P., Claesson-Welsh, L., Siegbahn, A., Zsebo, K. M., Westermark, B., and Heldin, C. H. (1991) EMBO J. 10, 4121-4128[Abstract] |
22. |
Blake, R. A.,
Broome, M. A.,
Liu, X.,
Wu, J.,
Gishizky, M.,
Sun, L.,
and Courtneidge, S. A.
(2000)
Mol. Cell. Biol.
20,
9018-9027 |
23. |
Joazeiro, C. A.,
Wing, S. S.,
Huang, H.,
Leverson, J. D.,
Hunter, T.,
and Liu, Y. C.
(1999)
Science
286,
309-312 |
24. | Levkowitz, G., Waterman, H., Ettenberg, S. A., Katz, M., Tsygankov, A. Y., Alroy, I., Lavi, S., Iwai, K., Reiss, Y., Ciechanover, A., Lipkowitz, S., and Yarden, Y. (1999) Mol. Cell 4, 1029-1040[Medline] [Order article via Infotrieve] |
25. |
Yee, N. S.,
Hsiau, C. W.,
Serve, H.,
Vosseller, K.,
and Besmer, P.
(1994)
J. Biol. Chem.
269,
31991-31998 |
26. |
Gommerman, J. L.,
Rottapel, R.,
and Berger, S. A.
(1997)
J. Biol. Chem.
272,
30519-30525 |
27. |
Broudy, V. C.,
Lin, N. L.,
Liles, W. C.,
Corey, S. J.,
O'Laughlin, B.,
Mou, S.,
and Linnekin, D.
(1999)
Blood
94,
1979-1986 |
28. |
Rosenkranz, S.,
Ikuno, Y.,
Leong, F. L.,
Klinghoffer, R. A.,
Miyake, S.,
Band, H.,
and Kazlauskas, A.
(2000)
J. Biol. Chem.
275,
9620-9627 |
29. |
Bondzi, C.,
Litz, J.,
Dent, P.,
and Krystal, G. W.
(2000)
Cell Growth Differ.
11,
305-314 |
30. | Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A., and Rutter, W. J. (1986) Cell 45, 721-732[Medline] [Order article via Infotrieve] |
31. | Mohammadi, M., Dikic, I., Sorokin, A., Burgess, W. H., Jaye, M., and Schlessinger, J. (1996) Mol. Cell. Biol. 16, 977-989[Abstract] |
32. |
Biscardi, J. S.,
Maa, M. C.,
Tice, D. A.,
Cox, M. E.,
Leu, T. H.,
and Parsons, S. J.
(1999)
J. Biol. Chem.
274,
8335-8343 |
33. | Jiang, G., and Hunter, T. (1999) Curr. Biol. 9, R568-71[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Remy, I.,
Wilson, I. A.,
and Michnick, S. W.
(1999)
Science
283,
990-993 |
35. | Leibiger, B., Leibiger, I. B., Moede, T., Kemper, S., Kulkarni, R. N., Kahn, C. R., Moitoso de Vargas, L., and Berggren, P. O. (2001) Mol. Cell 7, 559-570[Medline] [Order article via Infotrieve] |
36. |
Bell, C. A.,
Tynan, J. A.,
Hart, K. C.,
Meyer, A. N.,
Robertson, S. C.,
and Donoghue, D. J.
(2000)
Mol. Biol. Cell
11,
3589-3599 |
37. | Kwong, K. Y., and Hung, M. C. (1998) Mol Carcinog 23, 62-68[CrossRef][Medline] [Order article via Infotrieve] |
38. | Moriki, T., Maruyama, H., and Maruyama, I. N. (2001) J. Mol. Biol. 311, 1011-1026[CrossRef][Medline] [Order article via Infotrieve] |