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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1319-1329
By
From The Departments of Pathology and Hematology/Oncology, Osaka
University Medical School, Suita, Osaka, Japan; and the First
Department of Pathology and Institute for Advanced Medical Sciences,
Laboratory of Host Defenses, Hyogo College of Medicine, Hyogo, Japan.
The c-kit receptor tyrosine kinase (KIT) is constitutively
activated by naturally occurring mutations in either the juxtamembrane domain or the kinase domain. Although the juxtamembrane domain mutations led to ligand-independent KIT dimerization, the kinase domain
mutations (Asp814
THE PROTO-ONCOGENE c-kit encodes
a receptor tyrosine kinase (RTK) that is a member of the same RTK
subfamily (type III RTK) as the receptors for platelet-derived growth
factor and macrophage colony-stimulating factor
(M-CSF)/colony-stimulating factor-1.1,2 This RTK subfamily
is characterized by the presence of five Ig-like repeats in the
extracellular domain and an insert that splits the cytoplasmic kinase
domain into the adenosine triphosphate (ATP)-binding and
phosphotransferase regions.1-6 The ligand for c-kit
receptor tyrosine kinase (KIT) has been identified and variously designated as stem cell factor (SCF), mast cell growth factor, kit ligand, or steel factor7-10 (hereafter, we
refer to the ligand as SCF). The binding of SCF promotes dimerization
of KIT and activates intrinsic tyrosine kinase of KIT, resulting in
transphosphorylation at critical tyrosine residues. The
tyrosine-phosphorylated KIT can then bind a unique array of
intracellular signaling molecules, including phosphatidylinositol
3'-kinase, phospholipase C- Although the enzymatic activity of KIT is tightly regulated by the
binding of SCF, we have found that KIT is constitutively activated by
point mutations of c-kit gene in human, rat, and murine mast
cell lines. A human mast cell leukemia cell line (HMC-1) carried two
types of constitutively activating mutations of c-kit gene: the
Val560 to Gly mutation in the juxtamembrane domain and the
Asp816 to Val mutation in the phosphotransferase
domain.17 Constitutively activating mutation in the
corresponding Asp of the phosphotransferase domain was also detected in
a rat mast cell leukemia cell line (RBL-2H3; Asp817
In addition to hematopoietic cell lines, the Asp816
mutation in the phosphotransferase domain of human c-kit gene
has also been found in peripheral blood mononuclear cells from patients
with myelodysplastic disorders accompanying mastocytosis, in mast cells from patients with urticaria pigmentosa and aggressive mastocytosis, and in leukemia cells from patients with acute myelocytic
leukemia.24-26 Furthermore, we have recently demonstrated
that activating mutations of c-kit are detected in
gastrointestinal stromal tumors (GISTs), the most common mesenchymal
tumors in the human digestive tract, that may originate from ICCs
expressing both KIT and CD34.27 However, all of the
c-kit activating mutations detected in GISTs were located only
in the juxtamembrane domain. The occurrence of the activating mutations
at the same Asp codon in human hematologic disorders as well as mouse,
rat, and human tumor mast cell line implied that the Asp codon may be a
hot spot for activating mutation in hematopoietic systems and suggested
that the Asp mutation may be involved in neoplastic transformation of
mast cells and hematopoietic stem cells. However, the precise
mechanisms by which the c-kit mutations activate KIT tyrosine
kinase and transmit oncogenic signals are not fully understood.
The ligand-stimulated receptor dimerization is known to be a key event
in the activation of intrinsic protein kinase activity and signal
transduction of RTK,4,5,28,29 and constitutive activation
of RTK oncogenes tends to involve changes that mimic ligand-stimulated
activation, such as receptor dimerization. We have previously shown
that activating mutations within the juxtamembrane domain of
c-kit, such as the murine Val559
(Val560 in human) Reagents.
Recombinant murine (rm) SCF and rmIL-3 were generous gifts of Kirin
Brewery Co Ltd (Tokyo, Japan). Recombinant human (rh) M-CSF was a
generous gift of Yoshitomi Pharmaceutical Industries Ltd (Osaka,
Japan). Rat antimouse c-kit (ACK2) monoclonal antibody (MoAb)33 and full-length of murine c-kit cDNA were
kindly provided by Dr S.-I. Nishikawa (Kyoto University, Kyoto, Japan).
The plasmid, pSMc-fms, containing the human c-fms cDNA
that encodes FMS/M-CSF receptor, was kindly provided by Dr C.J. Sherr
(Howard Hughes Medical Institute Research Laboratories, Memphis,
TN).34,35 Rabbit antiserum against the kinase domain of
murine KIT (anti-KITKinase serum)36 was kindly
provided by Dr A. Bernstein (Samuel Lunenfeld Research Institute,
Toronto, Ontario, Canada) and rabbit antiserum against a C-terminal
peptide corresponding to the last 10 amino acids of murine KIT
(anti-KITC-terminal serum)8 by Dr D.E. Williams
(Immunex Corp, Seattle, WA). Rabbit polyclonal antibody (Ab-1) against
a synthetic peptide of C-terminal of human KIT was purchased from
Oncogene Science, Inc (New York, NY); this antibody reacted with both
murine and human KITs. Antiphosphotyrosine MoAb, a murine MoAb
generated against phosphotyramine, was generously supplied by Dr B.J.
Druker (Oregon Health Sciences University, Portland, OR). Rat MoAb
(3-4A4) against the extracellular domain of human FMS/M-CSF receptor
was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).
Cell lines.
The 293T cell line was derived from human embryonic kidney cells
transformed by DNA from human adenovirus type 5,37 and 293T
cells were maintained in Dulbecco's modification of Eagle's medium
(DMEM; ICN Biomedicals, Costa Mesa, CA) supplemented with 10% fetal
bovine serum (FBS; Irvine Scientific, Santa Ana, CA). The Ba/F3 murine
IL-3-dependent cell line was cultured in Construction of transgene and transfection.
Bluescript I KS( Metabolic labeling by [35S]-methionine.
Metabolic labeling was performed as previously described.41
Briefly, cells were incubated in methionine-free DMEM (Life Technologies, Grand Island, NY) containing
[35S]-methionine (DuPont/NEN Research Products, Boston,
MA; 100 mCi/mL), 5 mmol/L glutamine, 1 mmol/L sodium pyruvate, and 10%
dialyzed FBS for 5 hours. Radiolabeled KIT was precipitated with
protein-G Sepharose beads (Pharmacia, Uppsala, Sweden) and either rat
ACK2 MoAb, rabbit anti-KITKinase serum, or rabbit
anti-KITC-terminal serum. The immunoprecipitates were
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) with 5% to 20% gradient polyacrylamide. The gel was dried,
and radioactive proteins were detected by autoradiography.
Flow cytometry.
To detect cell surface expression of KIT, cells were incubated with
ACK2 MoAb at 4°C for 30 minutes, stained with fluorescein isothiocyanate (FITC)-conjugated rabbit antirat Ig antibody (DAKO A/S,
Glostup, Denmark), and analyzed on a FACScan (Becton Dickinson, Los
Angeles, CA). To detect the expression of KITDel-Wild and
KITDel-Tyr814, cells were fixed with ice-cold
methanol-aceton (1:1) for 10 minutes, incubated with rabbit
anti-KITKinase serum, stained with FITC-conjugated donkey
antirabbit Ig antibody (Jackson Immuno Research Laboratories, West
Grove, PA), and analyzed on a FACScan.
Immunoblotting.
The procedures of cell lysis, SDS-PAGE, and immunoblotting were
performed according to the methods described previously.42 Briefly, after depletion of serum and factors, cells were treated with
or without either rmSCF (100 ng/mL) or rhM-CSF (10,000 U/mL) for 15 minutes at 37°C. The cells were then washed with cold PBS and lysed
in lysis buffer (20 mmol/L Tris-HCl, 137 mmol/L NaCl, 10% glycerol,
1% Nonidet P-40, pH 8.0, and protease and phosphatase inhibitors).
After removal of insoluble materials by centrifugation, cell lysates
were incubated with rabbit anti-KITKinase serum and
protein-G Sepharose beads. The immunoprecipitates were subjected to
SDS-PAGE with 5% to 20% gradient polyacrylamide, and proteins were
electrophoretically transferred from the gel onto a polyvinylidene
difluoride membrane (Immobilon; Millipore Corp, Bedford, MA).
Immunoblotting was performed with either antiphosphotyrosine MoAb or
rabbit Ab-1.
Immune complex kinase assay.
The immune complex kinase assay was performed as previously
described.17-20,43 Briefly, cell lysates were incubated
with rabbit anti-KITKinase serum, ACK2 MoAb, or 3-4A4 MoAb
followed by the addition of protein-G Sepharose beads to collect the
antigen-antibody complexes. After washing, the immune complexes were
incubated in kinase buffer (10 mmol/L MnCl2, 20 mmol/L
Tris-HCl, pH 7.4) containing 1 µL of Cell proliferation assays.
Proliferation of cells was quantified by [3H]-thymidine
incorporation as previously described.44 The exponentially
growing cells were washed twice with Statistical analysis.
The Student's t-test was used to evaluate the significance of
the [3H]-thymidine incorporation and the number of viable cells.
Preparation of mutant c-kit genes lacking the extracellular
domain and introduction into 293T and murine IL-3-dependent Ba/F3
cells.
The Ig-like repeats in the extracellular domain of KIT are known to be
indispensable for ligand-binding and receptor dimerization. To examine
the role of extracellular domain in constitutive activation of mutant
KIT bearing the Asp814 mutation, we constructed the
truncated c-kitWild and
c-kitTyr814 cDNAs, in which the extracellular
Ig-like repeats of c-kit were deleted
(Fig 1A). The expression vector pEF-BOS
containing c-kitDel-Wild and
c-kitDel-Tyr814 cDNAs was first transfected into
293T cells to determine if KITDel-Wild and
KITDel-Tyr814 were efficiently expressed in
cells and were detected with anti-KIT antibodies. The transfected 293T
cells were radiolabeled with [35S]-methionine, and cell
lysates were subjected to immunoprecipitation experiments with either
ACK2 MoAb against the KIT extracellular domain (ECD), rabbit antiserum
against the KIT kinase domain (anti-KITKinase serum;
Kinase), or rabbit antiserum against the KIT C-terminal domain
(anti-KITC-terminal serum; C-terminal) (Fig 1B). As
expected, both KITDel-Wild and KITDel-Tyr814
were immunoprecipitated with anti-KITKinase serum and
anti-KITC-terminal serum, but not with ACK2 MoAb.
Activation state of KITWild and KITTyr814
after removal of their extracellular domains.
To examine the state of KIT-tyrosyl phosphorylation in the transfected
Ba/F3 cells, the cells were deprived of serum and growth factors for 12 hours and stimulated with or without rmSCF (100 ng/mL) for 15 minutes.
KIT was then immunoprecipitated with anti-KITKinase serum
and subjected to immunoblotting with either antiphosphotyrosine MoAb or
anti-KIT polyclonal antibody (Ab-1). In accord with our previous
findings, immunoblotting with an antiphosphotyrosine MoAb showed that
KITTyr814 was constitutively phosphorylated on tyrosine
residues, whereas tyrosine phosphorylation of KITWild was
induced in a ligand-dependent manner (Fig 2B). In the case of deletion
mutants, KITDel-Wild was not phosphorylated on tyrosine
before and after stimulation with rmSCF; KITDel-Tyr814 was
found to be significantly phosphorylated on tyrosine regardless of
rmSCF stimulation (Fig 2B). We further examined the kinase activity of
KIT by immune complex kinase assay. KIT was immunoprecipitated with
anti-KITKinase serum from cell lysates of Ba/F3,
Ba/F3Wild, Ba/F3Tyr814,
Ba/F3Del-Wild, and Ba/F3Del-Tyr814 cells that
were not stimulated with rmSCF, and the kinase activity was examined.
Almost identical levels of KIT proteins were immunoprecipitated from
the cell lysates of Ba/F3Wild, Ba/F3Tyr814,
Ba/ F3Del-Wild, and Ba/F3Del-Tyr814 cells
(Fig 2C, upper panel). Consistent with the data on immunoblotting analysis, both KITTyr814 and KITDel-Tyr814
exhibited a striking kinase activity, whereas little kinase activity was detected in KITWild and KITDel-Wild (Fig
2C, lower panel).
The effects of KITDel-Wild and KITDel-Tyr814
on the growth of Ba/F3 cells.
To determine if KITTyr814 could induce factor-independent
growth of Ba/F3 cells after removal of its extracellular domain, Ba/F3, Ba/F3Wild, Ba/F3Tyr814,
Ba/F3Del-Wild, and Ba/F3Del-Tyr814 cells were
cultured in the presence of 0 to 100 ng/mL rmIL-3 or 0 to 1,000 ng/mL
rmSCF for 72 hours, followed by measurement of cell proliferation using
a [3H]-thymidine incorporation assay
(Fig 3). The parental Ba/F3 cells showed
rmIL-3-dependent proliferation, but did not proliferate in response to
rmSCF. As in the case of parental Ba/F3 cells, both
Ba/F3Wild and Ba/F3Del-Wild cells proliferated
in response to rmIL-3 in a dose-dependent manner, and rmSCF-dependent
proliferation was observed in Ba/F3Wild cells, but not in
Ba/F3Del-Wild cells that lacked SCF-binding region of KIT.
By contrast, Ba/F3Tyr814 and, albeit to a lesser degree,
Ba/F3Del-Tyr814 cells proliferated in the absence of
exogenous rmIL-3 or rmSCF. These findings suggested that the
extracellular region required for ligand-binding and receptor
dimerization may not be involved in the constitutive activation and
growth-promoting signaling of KITTyr814.
Effect of dominant-negative KITW42 on the
factor-independent growth of Ba/F3Del-Tyr814 cells.
The Asp790
Self-association of KITTyr814.
The dominant-negative effect of KITW42 on the proliferation
of Ba/F3Del-Tyr814 cells raised the possibility that
KITTyr814 may yield homodimeric and heterodimeric
association of KIT not in the extracellular region. To test this
possibility, 293T cells were transfected with
c-kitWild, c-kitW42,
c-kitDel-Wild, or
c-kitDel-Tyr814 genes or with the combinations of
c-kitWild and c-kitDel-Wild
genes, c-kitW42 and
c-kitDel-Wild genes, c-kitWild
and c-kitDel-Tyr814 genes, or
c-kitW42 and c-kitDel-Tyr814
genes, and then the expression and association of KIT was examined. Immunoblotting with anti-KIT polyclonal antibody (Ab-1), followed by
immunoprecipitation with anti-KITKinase serum (Kinase),
showed that each type of transfectant expressed full-length KIT (KIT:
KITWild and KITW42) and/or deleted-form
KIT (Del-KIT: KITDel-Wild and KITDel-Tyr814) at
a nearly similar level (Fig 5A, upper
panel). When KIT was immunoprecipitated with ACK2 MoAb (ECD) and
subjected to immune complex kinase assay before treatment with rmSCF
(Fig 5A, lower panel), phosphorylation bands of KITWild and
KITW42 were barely detectable. In addition, because
KITDel-Wild and KITDel-Tyr814 were not
recognized by ACK2 MoAb, phosphorylation bands of
KITDel-Wild and KITDel-Tyr814 were
undetectable. When the cells expressing KITDel-Wild
together with KITWild or KITW42 were used,
phosphorylation bands of Del-KIT and KIT were also barely detected. By
contrast, when the cells expressing KITDel-Tyr814 together
with KITWild or KITW42 were used,
phosphorylation bands of both Del-KIT (KITDel-Tyr814)
and KIT (KITWild or KITW42) were
detected, suggesting that KITDel-Tyr814 could be
coimmunoprecipitated with KITWild or KITW42.
It is known that enzymatic activity of RTKs is deregulated by a myriad
of structural alterations, named loss-of-function and gain-of-function
mutations. We have previously shown that KIT can be constitutively
activated by gain-of-function mutations in either the juxtamembrane
domain or the phosphotransferase (kinase) domain.17-20,27
However, the molecular mechanism regulating constitutive activation
appears to be different between the juxtamembrane domain and the kinase
domain mutants, because the juxtamembrane domain mutants of KIT were
organized in the plasma membrane in a dimerized form without the
addition of exogenous rmSCF, whereas a dimeric form of the kinase
domain mutants (KITAsp814 The authors thank Dr D. Baltimore (Rockefeller University, New York,
NY) for providing the 293T cell line, Dr S. Nagata (Osaka University, Osaka, Japan) for pEF-BOS expression vector,
Dr S.-I. Nishikawa (Kyoto University, Kyoto, Japan) for
ACK2 MoAb and full length of murine c-kit cDNA, Dr C.J. Sherr
(Howard Hughes Medical Institute Research Laboratories, Memphis,
TN) for pSMc-fms, Dr D.E. Williams of Immunex Corp
(Seattle, WA) for rabbit antiserum against a C-terminal peptide
corresponding to the last 10 amino acids of the murine KIT, Dr A. Bernstein (Samuel Lunenfeld Research Institute, Toronto, Ontario,
Canada) for rabbit antiserum against a kinase domain of
the murine KIT, Dr B. Druker (Oregon Health Sciences University,
Portland, OR) for anti-phosphotyrosine MoAb, Kirin Brewery
Co Ltd for rmIL-3 and rmSCF, and Yoshitomi Pharmaceutical Industries
Ltd for rhM-CSF.
Submitted April 17, 1998; accepted October 5, 1998.
Supported in part by grants from the Japanese Ministry of Education,
Science and Culture, the Japanese Ministry of Health and Welfare, the
Mochida Memorial Foundation for Medical and Pharmaceutical Research,
and the Ryoichi Naito Foundation for Medical Research.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Yuzuru Kanakura, MD, PhD, The
Department of Hematology and Oncology, Osaka University Medical School,
2-2, Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail:
kanakura{at}bldon.med.osaka-u.ac.jp.
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Top
Abstract
Introduction
Val or Tyr) did not.
In an effort to determine if the kinase domain mutant could transfer
oncogenic signaling without receptor dimerization, we have constructed
the truncated types of c-kitWild and
c-kitTyr814 cDNAs
(c-kitDel-Wild and
c-kitDel-Tyr814 cDNAs, respectively), in which
ligand-binding and ligand-induced dimerization domains were deleted.
When c-kitDel-Wild and
c-kitDel-Tyr814 genes were introduced into a murine
interleukin-3 (IL-3)-dependent cell line Ba/F3,
KITDel-Tyr814 was constitutively phosphorylated on tyrosine
and activated, whereas KITDel-Wild was not. In addition,
Ba/F3 cells expressing KITDel-Tyr814
(Ba/F3Del-Tyr814) grew in suspension culture without the
addition of exogenous growth factor, whereas Ba/F3 cells expressing
KITDel-Wild (Ba/F3Del-Wild) required
IL-3 for growth. The factor-independent growth of
Ba/F3Del-Tyr814 cells was virtually abrogated by
coexpression of KITW42 that is a dominant-negative form
of KIT, but not by that of KITWild, suggesting that
KITDel-Tyr814 may not function as a monomer but may require
receptor dimerization for inducing factor-independent growth.
Furthermore, KITDel-Tyr814 was found to be
coimmunoprecipitated with KITWild or KITW42 by
an ACK2 monoclonal antibody directed against the extracellular domain
of KIT. Moreover, KITW42 was constitutively associated with
a chimeric FMS/KITTyr814 receptor containing the
ligand-binding and receptor dimerization domain of c-fms
receptor (FMS) fused to the transmembrane and cytoplasmic domain of
KITTyr814, but not with a chimeric FMS/KITWild
receptor even after stimulation with FMS-ligand. These results suggest
that constitutively activating mutation of c-kit at the Asp814 codon may cause a conformation change that leads to
receptor self-association not in the extracellular domain and that the receptor self-association of the Asp814 mutant may be
important for activation of downstream effectors that are required for
factor-independent growth and tumorigenicity.
1, and protein tyrosine phosphatase
SHP1, thereby initiating a signaling cascade that leads to various
cellular responses.
Tyr cDNA, in which both
ligand-binding and ligand-induced dimerization sites in the
extracellular domain were deleted,
-minimal essential medium
(
) plasmids (Stratagene, La Jolla, CA) containing
the whole coding regions of wild-type c-kit cDNA
(c-kitWild) and activating mutant
(Asp814 
) or negative control antibody (---). To detect
the whole expression of KIT, cells were fixed with methanol-aceton
(1:1) and then incubated with either rabbit anti-KITKinase
serum (Kinase) (
