(Received for publication, October 24, 1995; and in revised form, December 20, 1995)
From the
The c-kit receptor tyrosine kinase (KIT) is
constitutively activated in three different types of neoplastic mast
cell lines by naturally occurring mutations that result in
substitutions of Val or Tyr for Asp in the
phosphotransferase domain. In an effort to characterize the role of the
Asp
residue, we have investigated the properties of
mutant KITs in which the Asp
residue was deleted or
mutated to a series of other amino acids. With the exception of rare
instances, mutant KITs with substitutions of Asp
were
found to be constitutively phosphorylated on tyrosine and activated in
the absence of the ligand, stem cell factor (SCF), whereas a deletion
mutant lacking Asp
(KIT
) did not
exhibit tyrosine phosphorylation and activation even after treatment
with SCF. In addition to constitutive activation, furthermore, both
highly activated substitution mutants (KIT
and
KIT
) and modestly activated substitution mutants
(KIT
and KIT
) were continuously
degraded in the absence of SCF, whereas wild-type KIT
(KIT
) required SCF stimulation to undergo degradation.
These results suggested that the Asp
residue may play a
crucial role in regulating enzymatic activity and expression of KIT and
that various types of mutations at the Asp
residue may
generate oncogenic protein with constitutive activation and
degradation.
The c-kit proto-oncogene encodes a receptor tyrosine
kinase (RTK) ()that is a member of the same RTK subfamily as
the receptors for platelet-derived growth factor and colony-stimulating
factor-1 (CSF-1)(1, 2) . This RTK subfamily is
characterized by the presence of five immunoglobulin-like repeats in
the extracellular domain and an insert that splits the cytoplasmic
kinase domain into an ATP binding region and the phosphotransferase
domain(1, 2) . The enzymatic activity of RTKs is
tightly regulated by the binding of their ligands. The binding of
ligands promotes receptor dimerization and phosphorylation at specific
tyrosine residues, which can serve as docking sites for downstream
signal transduction molecules containing Src homology 2
domains(3) . The tyrosine-phosphorylated and activated RTKs act
as a center for the assemblage of a multiprotein complex that transmits
a series of biochemical signals. The activated RTKs are then rapidly
internalized and targeted to lysosomes, where both receptors and
ligands are degraded.
The c-kit RTK (KIT) is encoded by the W locus on mouse chromosome 5, whereas its ligand, stem cell
factor (SCF), is encoded by the Sl locus on mouse chromosome
10. A variety of loss-of-function mutations at either the KIT/W or SCF/Sl locus have been described, and these mutations
have provided insights into KIT function and site of action. The
phenotypes of mice bearing the KIT or SCF mutations include melanocyte
deficiency, macrocytic anemia, mast cell deficiency, and sterility,
emphasizing how essential SCF-regulated enzymatic activity of KIT is
for normal hematopoiesis, melanogenesis, and gametogenesis(4) .
In contrast to loss-of-function mutations, information about
gain-of-function mutations of KIT has been very limited. However, we
have recently found the presence of constitutively activating mutations
of c-kit gene in three different types of neoplastic mast cell
lines, the human mast cell leukemia cell line (HMC-1)(5) , the
rat mast cell leukemia cell line (RBL-2H3)(6) , and the murine
mastocytoma cell line (P-815) (7) . The c-kit gene of
HMC-1 cells was found to carry two constitutively activating mutations,
the Val to Gly mutation in the juxtamembrane domain and
the Asp
to Val mutation in the phosphotransferase
domain(5) . In addition, both RBL-2H3 and P-815 cells possessed
the constitutively activating mutation of the c-kit gene at
the corresponding Asp codon in the phosphotransferase domain, resulting
in the substitution of Tyr
for Asp in RBL-2H3 cells and
that of Tyr
for Asp in P-815 cells,
respectively(6, 7) .
The occurrence of the
activating mutations at the same Asp codon in the three neoplastic mast
cell lines suggested that the Asp codon may be a hot spot for
activating mutation of c-kit. Furthermore, since the Asp lies
near the highly conserved Asp-Gly-Phe sequence and occupies the
equivalent position in other members of RTKs such as receptors for
platelet-derived growth factor, CSF-1, insulin, and hepatocyte growth
factor(8) , the Asp region might be important in function and
regulation of RTKs, including KIT. To better understand the role of the
Asp, we have investigated the properties of murine KITs with the
various substitutions and deletion of Asp.
Each expression vector was transfected into 293T cells by the calcium phosphate method as described previously(14) . Two days after transfection, the cells were used for further analysis.
For in vitro kinase assay, the
cell lysates were prepared by lysis buffer and incubated for 45 min at
4 °C with a rabbit polyclonal antibody against the whole murine KIT
and Protein G-Sepharose beads to collect antigen-antibody complexes.
The immune complexes were washed and incubated in kinase buffer
containing -[
P]ATP (DuPont NEN; 20
µCi/ml) for 20 min at 25 °C as described
previously(15) . The immune complexes were then washed and
separated by SDS-PAGE with 5-20% gradient polyacrylamide. The gel
was dried, and radioactive proteins were detected by
autoradiography(5, 6, 7) .
Figure 1:
Tyrosine phosphorylation and activation
of mutant-type KIT with substitutions (A) and deletion (B) of Asp. A, KITs were
immunoprecipitated with an ACK2 mAb from the lysates of transfected
cells with wild- and mutant-type c-kit cDNAs and subjected to
immunoblotting with an anti-phosphotyrosine mAb or an anti-KIT
polyclonal antibody. In vitro kinase assay for autokinase
activity was performed after the immunoprecipitation with a rabbit
polyclonal antibody to the whole murine KIT. All samples were not
stimulated with rmSCF. B, KIT
and
KIT
were also subjected to immunoblotting and in vitro kinase assay before and after treatment with rmSCF
(100 ng/ml) for 15 min. This experiment was repeated three times
producing similar results. P-Tyr,
phosphotyrosine.
In addition to substitution mutants, we also
made a deletion mutant lacking the Asp (KIT
). In accordance with previous
findings, SCF treatment led to a marked increase in tyrosine
phosphorylation and kinase activity of
KIT
(5, 6, 7) . By contrast,
tyrosine phosphorylation and kinase activity of KIT
were only minimal or absent even after stimulation with rmSCF,
suggesting that the deletion of Asp
results in impairment
of KIT function (Fig. 1B).
Figure 2:
Time course of SCF-induced degradation of
KIT. A, the
S-labeled KIT in 293T
cells are immunoprecipitated with ACK2 mAb after non-treatment or
treatment with rmSCF (100 ng/ml) for 15 min and following 0-90
min of incubation at 37 °C. B, the amounts of KIT
quantified by densitometric analysis are expressed as a percentage of
the value at the starting point. The figure shows the representative
results of three experiments.
Figure 3:
Time course of SCF-independent degradation
of KIT, KIT
, KIT
and KIT
. A,
S-labeled KIT
in 293T cells are immunoprecipitated with ACK2 mAb after 0-90 min
of incubation at 37 °C in the absence of rmSCF. B, the
amounts of mutant KITs quantified by densitometric analysis are
expressed as a percentage of the value at the starting point. This
experiment was repeated three times producing similar
results.
We have previously found that KIT is constitutively activated
by naturally occurring mutations of an Asp residue in the
phosphotransferase domain of the c-kit proto-oncogene,
resulting in substitutions of the nonpolar amino acid Val or the
uncharged polar amino acid Tyr for the acidic amino acid
Asp(5, 6, 7) . When the
c-kit
mutant was introduced into cells of
murine IL-3-dependent cell lines, Ba/F3 (pro-B type), FDC-P1 (myeloid
type), and IC-2 (mast cell type), the cells expressing the activated
KIT
were found to show a factor-independent growth in vitro and to produce large tumors at the injection sites in
nude mice(17, 18) . Furthermore, IC-2 cells expressing
KIT
also produced large tumors at the injection sites
in nude mice. (
)These results suggest that the
constitutively activating mutations of c-kit such as
KIT
and KIT
could induce a
factor-independent and tumorigenic phenotype. In this study, we found
that constitutive activation of KIT was also generated by the
conversion of Asp
to a wide variety of amino acids other
than Val and Tyr, including uncharged polar (Asn, Gln), basic (Arg,
His), and nonpolar (Leu, Ile, Phe, Trp, Met, Pro) amino acids. In
contrast to substitutions, the deletion of Asp
was found
to abolish tyrosine kinase activity of KIT even after stimulation with
rmSCF. These results indicate that the Asp
may play a
crucial role in regulating enzymatic activity of KIT and suggest that a
variety of mutations of the c-kit gene at the Asp
codon may yield aggressive oncoproteins capable of inducing cell
transformations.
Our data also suggest that Asp may be
important in regulating expression of KIT. Our previous studies
demonstrated that the immature intracellular KIT precursor of
125
kDa was predominantly observed in cells transfected with
c-kit
or c-kit
cDNA, whereas the mature (
145 kDa) form of KIT protein was
predominant in cells transfected with c-kit
cDNA(5, 6, 7) . Furthermore, surface
expression of the activating KIT
was significantly
lower than that of KIT
after retroviral infection of
c-kit
and c-kit
cDNAs into murine IL-3-dependent cell lines, Ba/F3, FDC-P1 and
IC-2(17, 18) . These results suggested that activating
mutations of Asp
affected intracellular transport of
mutant-type KIT. In addition to the insufficient transport of
mutant-type KIT from cytoplasm to cell surface membrane, the present
results revealed that the highly activated KIT
and
KIT
as well as modestly activated KIT
and KIT
were continuously degraded in the
absence of rmSCF, whereas KIT
required ligand
stimulation to undergo rapid degradation. These results are consistent
with previous findings that activating mutants of CSF-1 receptor show
rapid internalization and degradation in the absence of ligand (19) and suggest that the constitutive low surface expression
of mutant-type KIT is attributable, at least in part, to the
ligand-independent degradation. Furthermore, these findings raise the
possibility that the transforming activity of mutant-type KIT does not
result from a failure of the receptor to down-regulate, as described in
an internalization-defective mutant of epidermal growth factor receptor (20, 21) .
Despite the dramatic effects of the
activating mutations at the Asp residue on the function
and expression of KIT, the precise mechanisms underlying the
constitutive activation and degradation of mutant-type KIT remain to be
determined. A number of previous studies have suggested that activating
mutations of growth factor receptors tend to involve changes that mimic
ligand-stimulated activation, such as receptor dimerization and
down-regulation(19, 22, 23) . In the case of
Val to Gly
mutation in the juxtamembrane of KIT, we found
that KIT
was organized at the plasma membrane in a
dimerized form in the absence of rmSCF(17) , suggesting that
the Gly
mutation may yield receptor dimerization with
resulting enzymatic activation. However, a dimeric form of
KIT
was barely detectable without the addition of
rmSCF, although it was detectable after stimulation with
rmSCF(17) . It is therefore possible that the c-kit mutations at the Asp
residue may be unique in
causing constitutive activation and degradation independently of
receptor dimerization, although it is still possible that the c-kit mutations at the Asp
residue may induce
conformational changes that lead to association of constitutively
activating KIT in the cytoplasmic domain.
Interestingly, our
preliminary experiments indicate that KIT is capable
of inducing neoplastic transformation of normal hematopoietic stem
cells more efficiently than KIT
. (
)
Furthermore, recent surveys on human leukemias
suggested the presence of Asp to Val mutation,
corresponding to the mouse Val
mutation, of c-kit in a fraction of human hematological malignancies(24) .
Molecular identification of the downstream targets of the
constitutively activated KIT will provide important insights not only
into fundamental mechanisms regulating enzymatic activity and
expression of KIT but also into novel signaling events associated with
tumorigenesis.