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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-01-0177.
NEOPLASIA
From the Department of Pathology and the Department of
Hematology and Oncology, Osaka University Graduate School of Medicine,
Suita; and the Department of Pathology, Hyogo College of Medicine,
Nishinomiya, Japan.
Substitution of valine (Val) for aspartic acid (Asp)
at codon 814 constitutively activates murine c-kit receptor
tyrosine kinase (KIT), and Asp816Val mutation, corresponding to murine Asp814Val mutation, is found in patients with mastocytosis and acute
myelocytic leukemia. However, the signal transduction pathways responsible for oncogenesis by the Asp814Val mutant
(KITVal814) are not fully understood. To examine the
oncogenic signal transduction of KITVal814, we converted 20 tyrosine (Tyr) residues to phenylalanine (Phe) in the cytoplasmic
domain of KITVal814 or deleted the C-terminal region
containing 2 other tyrosine residues (Del). Among various
KITVal814- derived mutants,
KITVal814-Tyr719Phe and KITVal814-Del
severely impaired receptor tyrosine phosphorylation and association with the p85 subunit of phosphatidylinositol 3'-kinase (p85
PI3-K). Moreover, KITVal814-Tyr719Phe
and KITVal814-Del failed to induce ligand-independent
growth in Ba/F3 cells, indicating that Tyr719, the binding site for
p85PI3-K, and the C-terminal region are indispensable for
factor-independent growth by KITVal814. Although the
C-terminal region was also required for ligand-dependent growth by
wild-type KIT (KITWT), the Tyr719Phe substitution
had negligible effects on ligand-dependent growth by
KITWT. Furthermore, dominant-negative PI3-K significantly
inhibited ligand-independent growth by KITVal814. These
results demonstrate that Tyr719 is crucial for constitutive activation
of KITVal814, but not for the ligand-induced activation of
KITWT, and that the downstream signaling of PI3-K plays an
important role in ligand-independent growth and tumorigenicity by
KITVal814, thereby suggesting that KITVal814 is
a unique activating mutation that leads to a distinguishable function
from the effects of KITWT.
(Blood. 2003;101:1094-1102) The proto-oncogene c-kit encodes a
receptor tyrosine kinase (RTK) that is a member of the same RTK
subfamily (type 3 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 structurally characterized by the presence of 5 immunoglobulinlike motifs in the extracellular domain and a cytoplasmic kinase domain interrupted by a hydrophilic kinase insert sequence that divides the
kinase domain into an adenosine triphosphate (ATP) binding region and a
phosphotransferase region.1-6 The ligand for
c-kit receptor tyrosine kinase (KIT) is stem cell factor
(SCF), also known as kit ligand, mast cell growth
factor, or steel factor.7-10 Binding of SCF to KIT
promotes dimerization and autophosphorylation of the receptor at the
specific tyrosine residues. Tyrosine-phosphorylated KIT then serves as
a docking site for the assembly of multisubunit complexes that are
further activated and that transmit a series of biochemical signals
leading to a variety of cellular responses. Several pathways have been
implicated in SCF/KIT-mediated signal transduction, including
phosphatidylinositol 3'-kinase (PI-3K), Ras-Raf-MAP (mitogen-activated
protein) kinase cascade, Src family kinase, and Janus family
kinase/signal transducer and activator of transcription (JAK/STAT)
pathways.4,11-15
Although the enzymatic activity of KIT is tightly regulated by SCF
binding, we have found that KIT is constitutively activated by
mutations of c-kit in neoplastic mast cell lines. The human mast cell leukemia cell line (HMC-1) carried 2 types of activating mutations of c-kit SCF-stimulated receptor dimerization in the extracellular domain is
known to be a key event in the activation of intrinsic protein kinase
activity of KIT. Activating mutations within the juxtamembrane domain
of c-kit, such as the murine Val559Gly (Val560Gly in human)
mutation, led to constitutive dimerization of KIT in the extracellular
domain without SCF stimulation. In contrast, the activation mutation of
murine Asp814Val (Asp816Val in human) did not yield dimerization in
chemical cross-linking analysis but was found to cause receptor
self-association in the cytoplasmic region without SCF
stimulation.20,22 Moreover, the Asp814Val mutant has been
shown to have altered sites of receptor autophosphorylation and altered
specificity for peptide substrates.26,27 These results
suggest that the Asp814Val mutant has undergone certain changes in
receptor binding or catalytic properties that selectively activate
signal transduction pathways leading to phenotypic changes that are
distinguishable from the effects of the wild-type KIT. However, the
molecular mechanisms of constitutive activation and subsequent
signaling responsible for the oncogenesis mediated by D814V mutation
are not fully understood. In this study, we converted a series of
tyrosine residues to phenylalanines in the cytoplasmic domain by
site-directed mutagenesis or deleted the C-terminal region containing 2 tyrosine residues of the Asp814Val mutant, and we examined how the
Asp814Val mutation yields oncogenic signal transduction.
Reagents
Cell lines
Construction of transgene and transfection Murine KIT contains 22 tyrosine residues in the cytoplasmic domain. To generate various mutants of KITVal814 and KITWT in which each cytoplasmic tyrosine residue was replaced by phenylalanine, site-directed mutagenesis was carried out on the expression vector pEF-BOS carrying c-kitVal814 or c-kitWT cDNA.26,32 In brief, point mutations were generated by overlap extension polymerase chain reaction (PCR) with synthetic oligonucleotides encoding the desired amino acid substitutions using c-kitVal814 or c-kitWT as a template. Digested fragments of amplified products were exchanged by the corresponding fragment of c-kitVal814 or c-kitWT cDNA in pEF-BOS. Because it was difficult to create Tyr Phe mutants of Tyr 898 and 934, deletion
mutants of KITVal814 and KITWT, in which the
C-terminal region (70 amino acids) containing these 2 tyrosine residues
were excluded, were created by introducing a stop codon into the
nucleotide position 2675 of c-kitVal814 and
c-kitWT cDNA. The resultant plasmids were
sequenced to confirm the mutations and were named as shown in Figure
1. The expression vector pEF-BOS carrying
various types of c-kit cDNA (10 µg) was transfected into 293T cells by the calcium-phosphate method,33 and the
cells were used for further analysis 2 days after transfection. For gene transfer into Ba/F3 cells, the linearized expression vector pEF-BOS carrying various types of c-kit cDNA (30 µg) and
pSTneoB carrying the neomycin-resistant gene (1 µg) were added to the cell suspension (1 × 107) in 0.7 mL phosphate-buffered
saline (PBS), and electroporation (975 µF, 350 V) was performed by
Gene Pulser II (Bio-Rad Laboratories, Hercules, CA). Two days after
electroporation, 1000 µg/mL G418 sulfate was added to the complete
culture medium to select neomycin-resistant cells. Cells expressing
various types of KIT were selected by limiting-dilution assay, and 3 different clones for each KIT construct were used for further
study.
Flow cytometry Cells were incubated with ACK2 mAb at 4°C for 30 minutes and were stained with fluorescein isothiocyanate-conjugated rabbit antirat immunoglobulin antibody (DAKO A/S, Glostrup, Denmark). After washing, cells were analyzed using FACScan (Becton Dickinson, Los Angeles, CA).Immunoblotting Cell lysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting were performed according to methods described previously.34 Briefly, after the depletion of serum and factors, cells were treated with rmSCF (100 ng/mL) at 37°C for 15 minutes. Cells were then washed with cold PBS and lysed in lysis buffer (20 mM Tris HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, and protease and phosphatase inhibitors). After the removal of insoluble materials by centrifugation, cell lysates were incubated with ACK2 mAb or a p85PI3-K antibody and protein G-Sepharose beads (Pharmacia AB, Uppsala, Sweden). 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, Bedford, MA). Immunoblotting was performed with -KIT,
-P-Tyr, and -p85PI3-K antibodies.
Immune complex kinase assay The immune complex kinase assay was performed according to the method described previously.16-19,35 Briefly, cell lysates were incubated with ACK2 mAb and Protein-G Sepharose beads to collect the antigen-antibody complexes. After washing, the immune complexes were incubated in kinase buffer (10 mM MnCl2, 20 mM Tris-HCl, pH 7.4) containing 1 mL -[32P]-ATP
(Dupont/NEN Research Products, Boston, MA; 10 mCi/mL [370 MBq]) for 20 minutes at 25°C and separated by SDS-PAGE with
5% to 20% gradient polyacrylamide. The gel was dried, and radioactive proteins were detected by autoradiography.
Cell proliferation assay Proliferation of cells was quantified by [3H]thymidine incorporation as previously described.36 Triplicate aliquots of cells (2 × 104) suspended in 100 µL Cosmedium-001 (Cosmo Bio, Tokyo, Japan) were cultured in 96-well microtiter plates at 37°C with various concentrations of rmSCF and rmIL-3. At 72 hours after initiation of the culture, 0.5 µCi (18.5 KBq) [3H]thymidine (specific activity, 5 Ci/mmol [185 GBq/mmol]; Amersham, Arlington Heights, IL) was added to each well. Four hours after the addition of [3H]thymidine, the cells were harvested with a semiautomatic cell harvester (model 1295; Pharmacia LKB Biotechnology, Piscataway, NJ), and the incorporation of [3H]thymidine was measured with a liquid scintillation counter.Induction of dn-p85PI3-K expression The Lac Switch II inducible expression system (Stratagene, La Jolla, CA) was used to examine the effect of dn-p85PI3-K on the ligand-independent growth by KITVal814. In this expression system, the expression of target genes is ordinarily suppressed by Lac-repressor (Lac-R) through the lactose operon because the expression of genes, subcloned into pOPRSVI, is regulated by RSV promoter linked to the Escherichia coli lactose operon. After treatment with IPTG, Lac-R is released from lactose operon, and the transcription of target genes is then initiated. To obtain Ba/F3 cells expressing Lac-R, pCMV-LacI was first transfected into Ba/F3 cells by electroporation.37 The Ba/F3 cells expressing Lac-R (Lac-R+-Ba/F3 cells) were then transfected with pOPRSVI carrying a neomycin-resistant gene and dn p85PI3-K cDNA or were cotransfected with pOPRSVI carrying a neomycin-resistant gene and dn-p85PI3-K cDNA and pEF-BOS carrying c-kitVal814 or c-kitWT cDNA. The Lac-R+-Ba/F3 transfectants were screened by cultivating with G418 sulfate at a concentration of 1.5 mg/mL, and neomycin-resistant cells were cloned. The induction of dn-p85PI3-K by treatment with 1 mmol/L IPTG was examined by Northern blot and Western blot analyses, and the KIT expression on the cell surface was examined by flow cytometry using ACK2 mAb.Northern blot analysis RNA was extracted by using the TRIZOL reagent (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. RNA (20 µg) was fractionated by agarose formaldehyde gel electrophoresis, transferred to a Hybond N+ nylon membrane (Amersham International, Buckinghamshire, United Kingdom), and hybridized with the 32P-labeled p85PI3-K and -actin cDNA. After
washing, the blot was subjected to autoradiography.38
PI-3K assay Cells were pretreated with 1 mmol/L IPTG for 24 hours at 37°C and then stimulated with rmSCF (100 ng/mL) for 15 minutes at 37°C. Cells were lysed in lysis buffer (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.5, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 10% glycerol, 1% Nonidet P-40, and protease and phosphatase inhibitors). After removal of insoluble materials by centrifugation, cell lysates were incubated with-phosphotyrosine mAb and protein G-Sepharose preconjugated with
rabbit antimouse immunoglobulin G (IgG) antibody. Immunoprecipitates
were resuspended in 30 µL of 10 mM phenylphosphate. Elutes containing
immunocomplexes were incubated with 0.2 mg/mL L--phosphatidylinositol, 40 mM ATP, 30 mM MgCl2, and 20 mCi (740 MBq) of -[32P]-ATP for 10 minutes at 37°C.
Reactions were stopped by the addition of 200 µL of 1 N HCl, and
lipids were extracted with 200 µL chloroform/methanol (1:1). After
washing with methanol/1 N HCl (1:1), phosphorylated lipids were
extracted and resolved by thin-layer chromatography using
chloroform/methanol/H2O/NH4OH (43:38:7:5) as
solvent. Radioactive spots of phosphatidylinositol 3'-phosphate (PI-3P)
were detected by autoradiography.
Effects of substitution of phenylalanine for tyrosine and removal of the C-terminal region on the constitutive activation of KITVal814 KIT is constitutively activated by the substitution of Val for Asp at codon 814 (Val814 mutation) in the phosphotransferase domain, and this Val814 mutation confers a factor-independent and tumorigenic phenotype on IL-3-dependent hematopoietic cell lines. To examine how KITVal814 yields oncogenic signal transduction, we constructed the expression vector pEF-BOS carrying various c-kit mutants that encode the TyrPhe-substituted
KITVal814 or the C-terminal-deleted KITVal814
(Figure 1). After the substituted or deleted KITVal814 were
expressed in 293T cells by transfection with various c-kit mutants, KITs were immunoprecipitated with ACK2 mAbs from cell lysates
that were prepared without stimulation by rmSCF. Immunoprecipitated KITs were then subjected to immunoblotting with a KIT antibody and the
immune complex kinase assay. As shown in Figure
2, KIT proteins were apparently expressed
in 293T cells. All TyrPhe-substituted KITVal814 were
composed of 145-kDa (mature) and 125-kDa (immature) forms and
KITVal814. As for the C-terminal-deleted
KITVal814, its molecular size was smaller than that of
KITVal814, as expected. When immune complex kinase assay
was performed, KITVal814 showed striking kinase activity
regardless of rmSCF stimulation. The constitutive kinase activity of
KITVal814 was not impaired, even if tyrosine residues were
changed to phenylalanines at codons 544, 552, 569, 645, 671, 674, 702, 728, 745, 772, and 853. On the other hand, the substitution of
phenylalanine for tyrosine at codons 546, 567, 577, 608, 719, 821, 844, 868, and 878 and the deletion of C-terminal region impaired the
constitutive kinase activity of KITVal814. Among these
mutations, the Tyr719Phe mutation and C-terminal deletion led to the
abolishment of constitutive kinase activity (Figure 2). These results
obtained from 293T cells suggest that tyrosine residues at codons 546, 567, 577, 608, 719, 821, 844, 868, and 878 and the C-terminal region
may be required for the constitutive activation of
KITVal814.
Effects of substitution of phenylalanine for tyrosine and removal of the C-terminal region on factor-independent growth by KITVal814 To examine the effect of TyrPhe substitution at codons 546, 567, 577, 608, 719, 821, 844, 868, and 878 and of C-terminal deletion
on the factor-independent growth by KITVal814, the
expression vector pEF-BOS carrying c-kit mutants that encode the TyrPhe-substituted KITVal814 or the
C-terminal-deleted KITVal814 and pST2neo were
cotransfected into the IL-3-dependent Ba/F3 cell line by
electroporation. As controls, Ba/F3 cells were also transfected with
the expression vector pEF-BOS carrying c-kitWT
or c-kitVal814. After selection in a
medium containing G418 and rmIL-3 for 3 weeks, Ba/F clones expressing
KITWT, KITVal814,
KITVal814-Tyr546Phe, KITVal814-Tyr567Phe,
KITVal814-Tyr577Phe,
KITVal814-Tyr608Phe,
KITVal814-Tyr719Phe, KITVal814-Tyr821Phe,
KITVal814-Tyr844Phe,
KITVal814-Tyr868Phe, KITVal814-Tyr878Phe,
or KITVal814-Del were isolated by
limiting-dilution assays and were designated as
Ba/FWT, Ba/FVal814,
Ba/FVal814-Tyr546Phe,
Ba/FVal814-Tyr567Phe,
Ba/FVal814-Tyr577Phe,
Ba/FVal814-Tyr608Phe,
Ba/FVal814-Tyr719Phe,
Ba/FVal814-Tyr821Phe,
Ba/FVal814-Tyr844Phe, Ba/FVal814-Tyr868Phe,
Ba/FVal814-Tyr878Phe, or
Ba/FVal814-Del cells, respectively. Flow cytometry with
ACK2 mAb showed the cell surface expression of KIT proteins on all
transfectants, except Ba/FVector cells that were
transfected with vector alone (data not shown).
The various Ba/F transfectants were cultured with rmIL-3 (0 to 10 ng/mL) or rmSCF (0 to 100 ng/mL) for 72 hours at 37°C, and their
proliferative potential was measured by means of
[3H]-thymidine incorporation assay. Cultivation with
rmIL-3 induced the dose-dependent proliferation of
Ba/FVector cells, and rmSCF had no effect. In addition
to rmIL-3, Ba/FWT cells dose dependently proliferated
in response to rmSCF. Ba/FVal814 cells proliferated in a
factor-independent manner, as previously reported.
Ba/FVal814-Tyr608Phe,
Ba/FVal814-Tyr844Phe, Ba/FVal814-Tyr868Phe, and
Ba/FVal814-Tyr878Phe cells showed
factor-independent growth at a level almost similar to that for
Ba/FVal814 cells. Ba/FVal814-Tyr546Phe,
Ba/FVal814-Tyr567Phe,
Ba/FVal814-Tyr577Phe, and Ba/FVal814-Tyr821Phe
cells also proliferated in a factor-independent manner, but their magnitude of proliferation was lower than that for
Ba/FVal814 cells (Figure 3).
On the other hand, Ba/FVal814-Tyr719Phe and
Ba/FVal814-Del cells failed to autonomously proliferate and
resulted in cell death as determined morphologically (data not
shown). Moreover, rmSCF had no effects on the proliferation of
Ba/FVal814-Tyr719Phe and Ba/FVal814-Del cells,
whereas Ba/FVal814-Tyr719Phe and Ba/FV814-Del
cells dose dependently proliferated in response to rmIL-3 (Figure 3).
These findings indicate that tyrosine at codon 719 and the C-terminal
region are indispensable for the factor-independent growth induced by
KITVal814.
Tyr719Phe substitution and C-terminal deletion inhibit tyrosine phosphorylation of KITVal814 and association between KITVal814 and PI3-K in Ba/F3 cells To examine the state of KIT-tyrosyl phosphorylation of TyrPhe-mutated or C-terminal-deleted Val814 in Ba/F3 cells, these mutants expressing Ba/F3 cells, together with Ba/FVector,
Ba/FWT, and Ba/FVal814 cells, were deprived of
serum and rmIL-3 for 12 hours and were stimulated with or without rmSCF
(100 ng/mL) for 15 minutes at 37°C, and KIT was then
immunoprecipitated with ACK2 mAb from the cell lysates. Based on
-KIT immunoblot data as shown in the lower portion of Figure
4A, approximately equivalent amounts of
KIT proteins were immunoprecipitated before and after stimulation with
rmSCF in Ba/FWT, Ba/FVal814, and all
KITVal814-derived mutant cells, but not in
Ba/FVector cells. Immunoblotting with -phosphotyrosine
mAb showed increased phosphotyrosine of KITWT after
treatment with rmSCF and the abundant phosphotyrosine of KITVal814, regardless of rmSCF stimulation. Among
KITVal814 mutants, only KITVal814-Tyr719Phe and
KITVal814-Del showed significantly suppressed tyrosine
phosphorylation, whereas other TyrPhe mutants showed comparable
tyrosine phosphorylation with KITVal814 (Figure 4A). These
results clearly correspond to the previous results that only
Ba/FVal814-Tyr719Phe and Ba/FVal814-Del cells
failed to autonomously proliferate.
Because tyrosine at codon 719 of KIT was reported to be the binding site for the 85-kDa subunit of PI3-K (p85PI3-K),12 we evaluated the association of p85PI3-K with KITVal814-derived mutants. First, the cell lysates were immunoprecipitated with anti-p85PI3-K Ab, and the immunoprecipitates were subjected to immunoblotting with anti-p85PI3-K Ab. As shown in the lower portion of Figure 4B, approximately equivalent amounts of p85PI3-K were expressed in Ba/FVector, Ba/FWT, Ba/FVal814, and all KITVal814-derived mutants. Next, the cell lysates were immunoprecipitated with ACK2 mAb, and the immunoprecipitates were subjected to immunoblotting with anti-p85PI3-KAb. The p85PI3-K was coimmunoprecipitated with KITWT after stimulation with rmSCF and with KITVal814, even before stimulation with rmSCF. Among KITVal814-derived mutants, p85PI3-K was not coimmunoprecipitated with KITVal814-Tyr719Phe or KITVal814-Del before or after stimulation with rmSCF, whereas other mutants showed coimmunoprecipitation with p85PI3-K (Figure 4B, upper portion). These results indicate that KITVal814-Tyr719Phe and KITVal814-Del lost the association with p85PI3-K in Ba/F3 cells, consistent with both mutants showing significantly suppressed receptor autophosphorylation. These results suggest that PI3-K-mediated signal transduction plays a crucial role in factor-independent growth by KITVal814. Roles of tyrosine719 and C-terminal portion in ligand-induced activation of KITWT To determine whether tyrosine at codon 719 and the C-terminal region are required for ligand-dependent activation of KITWT, the expression vector pEF-BOS carrying c-kitWT-Tyr719Phe or c-kitWT-Del and pST2neo were cotransfected into Ba/F3 cells by electroporation. After selection in a medium containing G418 and rmIL-3 for 3 weeks, Ba/F clones expressing KITWT-Tyr719Phe and KITWT-Del were isolated by limiting-dilution assays and were designated as Ba/FWT-Tyr719Phe and Ba/FWT-Del cells, respectively. Flow cytometry with ACK2 mAb showed the cell surface expression of KIT on both cells (data not shown). Ba/FWT-Tyr719Phe and Ba/FWT-Del cells, together with Ba/FVector and Ba/FWT cells, were cultured with rmIL-3 (0 to 10 ng/mL) or rmSCF (0 to 100 ng/mL) for 72 hours at 37°C, and their proliferative potential was measured by means of [3H]thymidine incorporation assay. Cultivation with rmIL-3 induced the dose-dependent proliferation of Ba/FWT-Del cells, and rmSCF had no effect, as was the same with Ba/FVal814-Del cells. By contrast, Ba/FWT-Tyr719Phe cells could dose dependently proliferate in response to rmSCF (Figure 5A).
We next examined the state of tyrosine phosphorylation of KIT in
Ba/FVector, Ba/FWT,
Ba/FWT-Tyr719Phe, and Ba/FWT-Del cells. These
cells were deprived of serum and rmIL-3 for 12 hours and were
stimulated with or without rmSCF (100 ng/mL) for 15 minutes at 37°C,
and KIT was then immunoprecipitated with ACK2 mAb from the cell
lysates. Immunoblotting with the Effects of dominant-negative p85PI-3K on the proliferation of Ba/FVal814 cells To examine whether PI3-K-mediated signal transduction participates in factor-independent growth by KITVal814, we chose the Lac Switch II inducible expression system, in which the expression of target genes is initiated after releasing Lac-R from lactose operon by treatment with ITPG. For setting up the Lac Switch II inducible expression system, we transfected pCMV-LacI into Ba/F3 cells by electroporation, and the obtained Lac-R+-Ba/F3 cells were then cotransfected with dn-p85PI3-K cDNA and c-kitVal814 or c-kitWT cDNA. Flow cytometry using ACK2 mAbs showed that the expression of KIT on the surfaces of Lac-R+-Ba/F3 cells cotransfected with dn-p85PI3-K cDNA and c-kitWT or with dn-p85PI3-K cDNA and c-kitVal814 cDNA, but not on the surfaces of Lac-R+-Ba/F3 cells transfected with dn-p85PI3-K cDNA alone (data not shown). The induction of dn-p85PI3-K by treatment with IPTG was then examined by Northern blot and Western blot analyses. Northern blot analysis showed that treatment with IPTG could induce dn-p85PI-3K transcripts in the Lac-R+-Ba/F3 cells that were transfected with dn-p85PI3-K cDNA alone or that were cotransfected with dn-p85PI3-K cDNA and c-kitVal814 or with dn-p85PI-3K cDNA and c-kitWT cDNA (Figure 6A). Moreover, immunoblotting with anti-p85PI3-K Ab in Lac-R+-Ba/F3 cells transfected with dn-p85PI3-K cDNA showed that dn-p85PI3-K proteins were detectable 4 hours after treatment with IPTG and continued up to 96 hours, indicating that dn-p85PI3-K proteins suppress the PI3-K-mediated signal transduction at least for 96 hours (Figure 6B).
Lac-R+-Ba/F3 cells expressing KITWT and dn-p85PI3-K were preincubated with or without IPTG for 24 hours and were stimulated with rmSCF. Lysates were then immunoprecipitated with antiphosphotyrosine Ab and underwent PI3-K assay. Although PI3-P, a product of PI3-K, was detected in the immunoprecipitates from Lac-R+-Ba/F3 cells expressing KITWT and dn-p85PI3-K after stimulation with rmSCF, it was not detected in the immunoprecipitates from Lac-R+-Ba/F3 cells expressing KITWT and dn-p85PI3-K preincubated with IPTG, even after stimulation with rmSCF. As for Lac-R+-Ba/F3 cells expressing KITVal814 and dn-p85PI3-K, PI3-P was detected in their immunoprecipitates even in the absence of rmSCF. When Lac-R+-Ba/F3 cells expressing KITVal814 and dn-p85PI3-K were preincubated with IPTG, PI3-P was not detected in their immunoprecipitates (Figure 6C). To confirm that PI-3K-mediated signal transduction is crucial for the factor-independent proliferation by KITVal814, [3H]-thymidine incorporation assay was performed in Lac-R+-Ba/F3 cells expressing KITVal814 and dn-p85PI3-K. The proliferation of Lac-R+-Ba/F3 cells expressing dn-p85PI3-K and of Lac-R+-Ba/F3 cells expressing KITWT and dn-p85PI3-K was also measured by means of [3H]thymidine incorporation assay. Induction of dn-p85PI3-K by IPTG did not impair the proliferation of Lac-R+-Ba/F3 cells expressing dn-p85PI3-K by rmIL-3 or the proliferation of Lac-R+-Ba/F3 cells expressing KITWT and dn-p85PI3-K by rmSCF. On the other hand, dn-p85PI3-K led to a marked reduction in the factor-independent growth activity of Ba/F3Val814 cells (Figure 6D).
Oncogenic activations of KIT are provoked by 2 types of mutation; one involves the juxtamembrane domain, and the other involves the phosphotransferase domain.16-22 Phosphotransferase domain mutations are the point mutations at the Asp814 codon in murine c-kit or the Asp816 in human c-kit, and they have been detected in various types of hematologic disorders of stem cell or mast cell origin.23-25 We have previously reported that the Asp814 mutant (KITVal814) induced a more aggressive neoplastic phenotype in murine hematopoietic cells than did the juxtamembrane domain mutant.21 By deleting the extracellular domain in KITVal814, we have found that the constitutive activation of KITVal814 was induced by causing receptor self-association in the cytoplasmic domain, not by dimerization in the extracellular domain.22 It has also been suggested that the KITVal814 mutant may alter its catalytic properties quantitatively and qualitatively, thereby generating a more aggressive oncoprotein.26,27 Recently, others39 and we40 have reported that the activation of the juxtamembrane domain KIT mutant was effectively suppressed by tyrosine kinase inhibitors such as imatinib (STI571), an excellent inhibitor of Bcr-Abl, and AG1296, an inhibitor of tyrphostin class.39,40 However, in sharp contrast, neither inhibitor has an effect on the activation of KITVal814.39,40 Hence, the catalytic domain KIT mutant is characterized by a unique activation mechanism that resists the tyrosine kinase inhibitors and by a more aggressive oncogenic potential than the juxtamembrane domain mutant has. Therefore, to clarify, the activation mechanisms and downstream effectors of the catalytic domain mutants are necessary for the development of novel therapeutic interventions. To identify the critical signaling pathways of KITVal814,
we generated a series of Tyr Consistent with the report that Tyr719 is identified as the binding site of p85PI3-K,12 we found that p85PI3-K was constitutively associated with KITVal814, and this association was completely abrogated by Tyr719F. These results suggest that p85PI3-K binding is involved in KITVal814-mediated constitutive activation and subsequent signal transduction. Furthermore, the proliferation of KITVal814 was significantly suppressed by inducible expression of dominant-negative p85PI3-K, whereas the proliferation of ligand-stimulated KITWT was not affected. These results indicated that KITVal814-mediated oncogenic signaling involves PI3-K activation. In agreement with our results, it was recently reported that PI3-K activation, mediated by Tyr721 of human KIT (corresponding to Tyr719 in murine KIT), was important for the proliferation of Val816 human c-kit mutant (corresponding to murine KITVal814).41 Inconsistent with our results, however, they reported that KITVal814-Tyr721Phe significantly decreased, but did not abolish, the proliferation and colony formation of murine fetal liver cells partially transformed by Myb. We consider that the different effect of Tyr719Phe (Tyr721Phe) mutation on the KITVal814-mediated proliferation may reflect the difference of the cellular background. They also reported that the KITVal814-Tyr721Phe mutant could not induce tumor formation in mice, whereas all mice injected with KITVal814 cells rapidly developed tumors, as did 1 of 6 mice injected with KITWT cells. These results indicate that Tyr721Phe repressed the transforming activity of KITVal816, and they coincide with our results that Tyr719 was a critical residue for the kinase activity of KITVal814. Recently, it was reported that the KITWT-Tyr719Phe knock-in mouse shows impaired spermatogenesis and oogenesis but retains steady state hematopoiesis.42,43 This report indicated that Tyr719-associated PI3-K activation is dispensable for KITWT-mediated proliferation and survival of hematopoietic cells. Again, this is in sharp contrast to the critical role of Tyr719/PI3-K in KITVal814. The role of PI3-K in oncogenic signaling has also been demonstrated in Bcr-Abl and in constitutively active forms of receptor tyrosine kinases such as platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and RET proto-oncogene.44-47 Among them, constitutively active RET, through multiple endocrine neoplasia 2 (MEN2) germline mutations, induced the strong activation of PI3-K, and the abolishment of PI3-K activation by the dominant-negative molecule significantly suppressed transformation activity, suggesting a major role of PI3-K in constitutively active RET, similar to that of KITVal814.46 MEN2 mutations of RET are divided into an extracellular domain mutation, MEN2A, and a catalytic domain mutation, MEN2B. MEN2B is a more potent activator of PI3-K than MEN2A, which is thought to be associated with the aggressive clinical features of MEN2B.45 These results suggest the important role of PI3-K in the catalytic domain mutations of receptor tyrosine kinase, such as KITVal814 and RET/MEN2B mutation. In our study, in spite of the complete suppression of PI3-K activation,
dominant-negative p85PI3-K did not totally abolish the
proliferation, nor cause the apoptosis, of Ba/F3Val814
(data not shown). These results suggested that the activation of PI3-K
was the major, but not the sole, pathway for
KITVal814-mediated proliferation and survival. Ning et
al48 reported that KITVal814 constitutively
activated STAT3 and STAT1 in addition to PI3-K, which resulted in the
up-regulation of STAT3 target genes Bcl-xL and
Myc. They reported that the suppression of STAT3 by a
dominant-negative molecule could suppress the transforming activity of
Val814 but not completely abolish it, suggesting that the cooperative
activation of PI3-K and STAT3 is necessary for the full oncogenic
activity of Val814. On the other hand, Tyr719Phe completely suppressed kinase activity itself, suggesting that Tyr719 may be necessary for the
tertiary structure of KITVal814 mutant, which was critical
for the tyrosine kinase activity of KITVal814, not of
KITWT. The structural change of KITVal814 might
be further supported by the findings that KITVal814
resisted several tyrosine kinase inhibitors, including STI571, that are
effective against KITWT and KIT juxtamembrane
mutant.39,49 Recently, several mutations in Bcr-Abl have
been reported to confer resistance to STI571 in patients with
Bcr-Abl-positive leukemia. Although most of these mutants affected the
ATP-binding site, where STI571 directly interacts, one mutation in the
activation loop In addition to the Tyr719Phe substitution, factor-independent growth of
Ba/F3 cells by KITVal814 was found to be suppressed by
Tyr
We thank Dr S. I. Nishikawa of Kyoto University for ACK2 and c-kit cDNA, Dr S. Nagata of Osaka University for pEF-BOS, Dr M. Kasuga of Kobe University for dn-p85PI3-K cDNA, and Kirin Brewery Company Ltd for rmSCF and rmIL-3.
Submitted January 22, 2002; accepted September 2, 2002.
Prepublished online as Blood First Edition Paper, September 12, 2002; DOI 10.1182/blood-2002-01-0177.
Supported by grants from the Japanese Ministry of Education, Science and Culture and the Japanese Ministry of Health and Welfare and by the Medical Research Award from the Japan Medical Association.
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: Yuzuru Kanakura, 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.
1.
Qiu FH, Ray P, Brown K, et al.
Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family 2. Yarden Y, Kuang WJ, Yang-Feng T, et al. Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J. 1987;6:3341-3351[Medline] [Order article via Infotrieve]. 3. Fantl WJ, Johnson DE, Williams LT. Signalling by receptor tyrosine kinases. Annu Rev Biochem. 1993;62:453-481[Medline] [Order article via Infotrieve]. 4. Reith AD, Ellis C, Lyman SD, et al. Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase. EMBO J. 1991;10:2451-2459[Medline] [Order article via Infotrieve]. 5. Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990;61:203-212[CrossRef][Medline] [Order article via Infotrieve]. 6. Yarden Y, Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443-478[CrossRef][Medline] [Order article via Infotrieve]. 7. Flanagan JG, Leder P. The kit ligand: a cell surface molecule altered in steel mutant fibroblasts. Cell. 1990;63:185-194[CrossRef][Medline] [Order article via Infotrieve]. 8. Huang E, Nocka K, Beier DR, et al. The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell. 1990;63:225-233[CrossRef][Medline] [Order article via Infotrieve]. 9. Williams DE, Eisenman J, Baird A, et al. Identification of a ligand for the c-kit proto-oncogene. Cell. 1990;63:167-174[CrossRef][Medline] [Order article via Infotrieve]. 10. Zsebo KM, Williams DA, Geissler EN, et al. Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell. 1990;63:213-224[CrossRef][Medline] [Order article via Infotrieve].
11.
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
12.
Serve H, Hsu YC, Besmer P.
Tyrosine residue 719 of the c-kit receptor is essential for binding of the P85 subunit of phosphatidylinositol (PI) 3-kinase and for c-kit-associated PI 3-kinase activity in COS-1 cells.
J Biol Chem.
1994;269:6026-6030
13.
Yi T, Ihle JN.
Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand.
Mol Cell Biol.
1993;13:3350-3358
14.
Lev S, Givol D, Yarden Y.
Interkinase domain of kit contains the binding site for phosphatidylinositol 3' kinase.
Proc Natl Acad Sci U S A.
1992;89:678-682
15.
Brizzi MF, Dentelli P, Rosso A, et al.
STAT protein recruitment and activation in c-Kit deletion mutants.
J Biol Chem.
1999;274:16965-16972 16. Furitsu T, Tsujimura T, Tono T, et al. Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product. J Clin Invest. 1993;92:1736-1744[Medline] [Order article via Infotrieve]. 17. Tsujimura T, Furitsu T, Morimoto M, et al. Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3. Int Arch Allergy Immunol. 1995;106:377-385[Medline] [Order article via Infotrieve].
18.
Tsujimura T, Furitsu T, Morimoto M, et al.
Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation.
Blood.
1994;83:2619-2626
19.
Tsujimura T, Morimoto M, Hashimoto K, et al.
Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain.
Blood.
1996;87:273-283
20.
Kitayama H, Kanakura Y, Furitsu T, et al.
Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines.
Blood.
1995;85:790-798
21.
Kitayama H, Tsujimura T, Matsumura I, et al.
Neoplastic transformation of normal hematopoietic cells by constitutively activating mutations of c-kit receptor tyrosine kinase.
Blood.
1996;88:995-1004
22.
Tsujimura T, Hashimoto K, Kitayama H, et al.
Activating mutation in the catalytic domain of c-kit elicits hematopoietic transformation by receptor self-association not at the ligand-induced dimerization site.
Blood.
1999;93:1319-1329
23.
Longley BJ Jr, Metcalfe DD, Tharp M, et al.
Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.
Proc Natl Acad Sci U S A.
1999;96:1609-1614
24.
Ferrao P, Gonda TJ, Ashman LK.
Expression of constitutively activated human c-Kit in Myb-transformed early myeloid cells leads to factor independence, histiocytic differentiation, and tumorigenicity.
Blood.
1997;90:4539-4552
25.
Nagata H, Worobec AS, Oh CK, et al.
Identification of a point mutation in the catalytic domain of the proto-oncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.
Proc Natl Acad Sci U S A.
1995;92:10560-10564
26.
Moriyama Y, Tsujimura T, Hashimoto K, et al.
Role of aspartic acid 814 in the function and expression of c-kit receptor tyrosine kinase.
J Biol Chem.
1996;271:3347-3350
27.
Piao X, Paulson R, van der Geer P, et al.
Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1.
Proc Natl Acad Sci U S A.
1996;93:14665-14669
28.
Ogawa M, Matsuzaki Y, Nishikawa S, et al.
Expression and function of c-kit in hemopoietic progenitor cells.
J Exp Med.
1991;174:63-71
29.
Mizushima S, Nagata S.
pEF-BOS, a powerful mammalian expression vector.
Nucleic Acids Res.
1990;18:5322 30. Kotani K, Yonezawa K, Hara K, et al. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling. EMBO J. 1994;13:2313-2321[Medline] [Order article via Infotrieve].
31.
Graham FL, Smiley J, Russell WC, et al.
Characteristics of a human cell line transformed by DNA from human adenovirus type 5.
J Gen Virol.
1977;36:59-74
32.
Higuchi R, Krummel B, Saiki RK.
A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.
Nucleic Acids Res.
1988;16:7351-7367
33.
Wigler M, Pellicer A, Silverstein S, et al.
DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells.
Proc Natl Acad Sci U S A.
1979;76:1373-1376
34.
Kanakura Y, Druker B, Cannistra SA, et al.
Signal transduction of the human granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins.
Blood.
1990;76:706-715
35.
Majumder S, Brown K, Qiu FH, et al.
c-kit protein, a transmembrane kinase: identification in tissues and characterization.
Mol Cell Biol.
1988;8:4896-4903
36.
Kuriu A, Ikeda H, Kanakura Y, et al.
Proliferation of human myeloid leukemia cell line associated with the tyrosine-phosphorylation and activation of the proto-oncogene c-kit product.
Blood.
1991;78:2834-2840
37.
Matsumura I, Nakajima K, Wakao H, et al.
Involvement of prolonged ras activation in thrombopoietin-induced megakaryocytic differentiation of a human factor-dependent hematopoietic cell line.
Mol Cell Biol.
1998;18:4282-4290
38.
Matsumura I, Kanakura Y, Kato T, et al.
Growth response of acute myeloblastic leukemia cells to recombinant human thrombopoietin.
Blood.
1995;86:703-709
39.
Ma Y, Zeng S, Metcalfe DD, et al.
The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors: kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations.
Blood.
2002;99:1741-1744 40. Ueda S, Ikeda H, Mizuki M, et al. Constitutive activation of c-kit by the juxtamembrane but not the catalytic domain mutations is inhibited selectively by tyrosine kinase inhibitors STI571 and AG1296. Int J Hematol. In press.
41.
Chian R, Young S, Danilkovitch-Miagkova A, et al.
Phosphatidylinositol 3 kinase contributes to the transformation of hematopoietic cells by the D816V c-Kit mutant.
Blood.
2001;98:1365-1373 42. Blume-Jensen P, Jiang G, Hyman R, et al. Kit/stem cell factor receptor-induced activation of phosphatidylinositol 3'-kinase is essential for male fertility. Nat Genet. 2000;24:157-162[CrossRef][Medline] [Order article via Infotrieve]. 43. Kissel H, Timokhina I, Hardy MP, et al. Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses. EMBO J. 2000;19:1312-1326[CrossRef][Medline] [Order article via Infotrieve].
44.
Dierov J, Xu Q, Dierova R, et al.
TEL/platelet-derived growth factor receptor beta activates phosphatidylinositol 3 (PI3) kinase and requires PI3 kinase to regulate the cell cycle.
Blood.
2002;99:1758-1765 45. Murakami H, Iwashita T, Asai N, et al. Enhanced phosphatidylinositol 3-kinase activity and high phosphorylation state of its downstream signaling molecules mediated by ret with the MEN 2B mutation. Biochem Biophys Res Commun. 1999;262:68-75[CrossRef][Medline] [Order article via Infotrieve].
46.
Segouffin-Cariou C, Billaud M.
Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/AKT signaling pathway.
J Biol Chem.
2000;275:3568-3576 47. Skorski T, Bellacosa A, Nieborowska-Skorska M, et al. Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt- dependent pathway. EMBO J. 1997;16:6151-6161[CrossRef][Medline] [Order article via Infotrieve].
48.
Ning ZQ, Li J, Arceci RJ.
Signal transducer and activator of transcription 3 activation is required for Asp(816) mutant c-Kit-mediated cytokine-independent survival and proliferation in human leukemia cells.
Blood.
2001;97:3559-3567 49. Ikeda H, Ueda S, Kitayama H, et al. Preferential inhibition of constitutive activating juxtamembrane mutant rather than kinase domain mutant and wild-type c-Kit receptor tyrosine kinase by tyrophostin [abstract]. Blood. 2000;96:99. 50. von Bubnoff N, Schneller F, Peschel C, et al. BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukaemia to STI571: a prospective study. Lancet. 2002;359:487-491[CrossRef][Medline] [Order article via Infotrieve].
51.
Kang H, Schneider H, Rudd CE.
Phosphatidylinositol 3-kinase p85 adapter function in T-cells: co-stimulation and regulation of cytokine transcription independent of associated p110.
J Biol Chem.
2002;277:912-921 52. Serve H, Yee NS, Stella G, et al. Differential roles of PI3-kinase and Kit tyrosine 821 in Kit receptor-mediated proliferation, survival and cell adhesion in mast cells. EMBO J. 1995;14:473-483[Medline] [Order article via Infotrieve]. 53. Timokhina I, Kissel H, Stella G, et al. Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation. EMBO J. 1998;17:6250-6262[CrossRef][Medline] [Order article via Infotrieve].
54.
Yamamoto Y, Kiyoi H, Nakano Y, et al.
Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies.
Blood.
2001;97:2434-2439
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  J. Sun, M. Pedersen, and L. Ronnstrand The D816V Mutation of c-Kit Circumvents a Requirement for Src Family Kinases in c-Kit Signal Transduction J. Biol. Chem., April 24, 2009; 284(17): 11039 - 11047. [Abstract] [Full Text] [PDF]
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 V. Munugalavadla, E. C. Sims, J. Borneo, R. J. Chan, and R. Kapur Genetic and pharmacologic evidence implicating the p85{alpha}, but not p85{beta}, regulatory subunit of PI3K and Rac2 GTPase in regulating oncogenic KIT-induced transformation in acute myeloid leukemia and systemic mastocytosis Blood, September 1, 2007; 110(5): 1612 - 1620. [Abstract] [Full Text] [PDF]
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A. Tefferi Mutant Kit: thwarting the message down below Blood, March 15, 2005; 105(6): 2240 - 2241. [Full Text] [PDF]
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G. Lefevre, A.-L. Glotin, A. Calipel, F. Mouriaux, T. Tran, Z. Kherrouche, C.-A. Maurage, C. Auclair, and F. Mascarelli Roles of Stem Cell Factor/c-Kit and Effects of Glivec(R)/STI571 in Human Uveal Melanoma Cell Tumorigenesis J. Biol. Chem., July 23, 2004; 279(30): 31769 - 31779. [Abstract] [Full Text] [PDF]
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 R. Shivakrupa, A. Bernstein, N. Watring, and D. Linnekin Phosphatidylinositol 3'-Kinase Is Required for Growth of Mast Cells Expressing the Kit Catalytic Domain Mutant Cancer Res., August 1, 2003; 63(15): 4412 - 4419. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
the Val560Gly mutation in the
juxtamembrane domain and the Asp816Val mutation in the
phosphotransferase domain.
Thr573-His579) in the juxtamembrane domain.
