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INTRODUCTION |
The pyrazolo-pyrimidine compound
PP11 was identified as a high
potency inhibitor of Src tyrosine kinase family members that acts as a
competitive inhibitor of ATP binding (1). PP1 has been used in a number
of studies to evaluate the role of Src tyrosine kinases in cellular
function (2-7). PP1 does not affect the activity of other non-receptor
tyrosine kinases, such as Jak-2 and Zap-70 (1). Src kinases have been
reported to be critically involved in signaling via the stem cell
factor (SCF) receptor c-Kit; inhibition of Src kinases has been shown
to abrogate SCF induced proliferation of hemopoietic (3) and small cell
lung cancer cells (8) and to block internalization of c-Kit after
ligand binding (4). These findings have relied largely on the use of
PP1 as a Src-selective inhibitor. c-Kit is expressed on hemopoietic
progenitors, mast cells, and germ cells (for review, see Refs. 9 and
10). Loss of either c-Kit or its ligand in mice results in impaired
hemopoiesis (in particular anemia), abnormalities of mast cell
development, and impaired melanogenesis and gametogenesis (11). c-Kit
has been implicated in tumor cell development, including mast cell disorders, (12) acute myeloid leukemia, gastrointestinal stromal tumors
(13), Ewing's sarcoma, peripheral neuro-ectodermal tumors, malignant
melanoma (11) and cancers of lung, ovary, and breast (14-16). In some
instances, this is because of activating mutations in c-Kit
(e.g. in mastocytosis, acute myeloid leukemia, and
gastrointestinal stromal tumors) (17); in others, the receptor is wild
type but there is associated autocrine production of SCF (16).
The development of inhibitors of c-Kit-mediated signals would be useful
to investigate the role of c-Kit in maintaining these tumors and could
provide the basis for a potential therapeutic agent. In this study, we
have examined the effects of PP1 on signaling via c-Kit. We find that
PP1 and the related compound PP2 block SCF-induced proliferation and
activation of the ERK mitogen-activated protein kinase and Akt. This is
associated with an inhibition of c-Kit autophosphorylation both in
intact cells and in in vitro kinase assays. PP1 decreases
the activity of constitutively active c-Kit and triggers the apoptosis
of mast cell leukemia cells expressing this mutant. PP1 also inhibits
Bcr-Abl kinase activity and triggers apoptosis in
Bcr-Abl-dependent cells. These results have implications for the use of PP1 and related compounds in the experimental
investigation of cell signaling pathways and suggest that this family
of molecules may be useful in the development of treatment for diseases
with abnormal c-Kit signaling.
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MATERIALS AND METHODS |
Cell Culture--
The RBL-2H3 cell line (18) (kindly supplied by
S. Cockcroft, Dept. of Physiology, University College, London, UK) was
maintained in Earle's minimal essential medium supplemented
with 10% (v/v) heat-inactivated fetal calf serum (FCS). M07e cells
(originally described by Avanzi et al. (19)) were maintained
in RPMI 1640 medium with 10% (v/v) FCS, supplemented with 20 ng/ml
recombinant human GM-CSF and 20 ng/ml human IL-3 (Sandoz,
Frimley Park, UK). FDCP1 cells expressing p210 Bcr-Abl were generated
as described previously (20) and were maintained in Dulbecco's
modified Eagle's medium with 10% FCS without additional growth
factors. PP1 was from Affiniti (Exeter, UK), whereas PP2 and the
inactive PP3 were from Calbiochem-Novabiochem Biosciences. Human SCF
and thrombopoietin (Tpo) were from PeproTech EC Ltd. (London, UK), and
Escherichia coli-derived recombinant human GM-CSF was from
Behringwerke-Hoechst (Marburg, Germany).
Cell Proliferation Assay--
After overnight growth factor
withdrawal in RPMI 1640 medium with 10% FCS, M07e cells were plated at
3 × 105/ml in 12-well plates, and inhibitors were
added. After 60 min of incubation, growth factors were added. Cells
were then incubated at 37 °C, 5% CO2 for up to 72 h. Cell proliferation was measured using an MTS assay (CellTiter 96;
Promega). The assay was performed according to manufacturer's
instructions. Briefly, at specified time points, 100 µl of sample was
added to 20 µl of MTS reagent in a 96-well plate and incubated at
37 °C for 2 h. Absorbance at 490 nm was measured using a
96-well plate reader. All measurements were performed in triplicate.
Annexin-V Binding/Propidium Iodide Uptake--
RBL-2H3 cells
were plated at 1 × 105/ml in 12-well plates in
Earle's minimal essential medium with 10% FCS and allowed to adhere before addition of inhibitors. Subsequently, cells were removed from
wells at 24-h intervals using trypsin/EDTA (5 min at 37 °C, 5%
CO2) and pelleted by centrifugation. Fluorescein
isothiocyanate-conjugated Annexin-V (Roche Molecular Biochemicals) was
used according to the manufacturer's instructions. Briefly, cells were
incubated at room temperature for 5 min in Annexin-V binding buffer
(140 mM NaCl2 10 mM HEPES, pH 7.4, 5 mM CaCl2) with 40 µg/ml propidium iodide
(Sigma-Aldrich) and 10 µl/ml Annexin-V-fluorescein isothiocyanate. Samples were then placed on ice and immediately analyzed for Annexin-V binding and propidium iodide uptake by flow cytometry (Epics Elite; Beckman Coulter).
Plasmid Generation and 293T Cell Transfection--
Murine c-Kit
cDNA in pUC19 was cloned into the pCDNA3.1 vector and
site-directed mutagenesis (Stratagene QuikChange kit) was used to
generate D814V and D814Y mutants. Human embryonic kidney 293T cells
were transfected by the calcium phosphate method using a Promega kit
according to the manufacturer's instructions. Cells were transfected
on 10-cm dishes with the respective constructs and replated into 6-well
plates 24 h later. Incubation with PP1 at the indicated
concentrations for 2 h was carried out 48 h after initial
transfection, and cells were lysed as detailed below. Immunoprecipitation with goat anti-c-Kit antibody (Santa Cruz) was for
4 h; the last hour included protein G-Sepharose (Sigma).
SDS-PAGE and Immunoblotting--
Cells were incubated overnight
in the absence of growth factor before resuspension at 4 × 106/ml in PBS with 5 mM glucose. The indicated
concentrations of inhibitor were added, and cells were incubated for 60 min at 37 °C. M07e were then stimulated with 20 ng/ml human SCF for
10 min at 37 °C. Cells were pelleted by centrifugation and lysed in
lysis buffer (50 mM HEPES, pH 7.5, 100 mM NaCl,
1% Triton X-100, 2 mM EDTA, 20 mM NaF, 1 mM NaVO4, 10 µg/ml aprotinin, 10 µg/ml
pepstatin, 10 µg/ml leupeptin, 1 mM Pefabloc, and 5 µM microcystin) for 10 min at 4 °C. Lysates were
cleared of nuclear debris (centrifuged at 14,000 rpm for 5 min at
4 °C), added to sample buffer, and boiled for 5 min. Equal volumes
of sample were resolved using SDS-PAGE and transferred to
nitrocellulose membranes (Hybond-C extra; Amersham Biosciences).
Activated proteins were detected using phosphospecific primary
antibodies. The monoclonal anti-phosphotyrosine antibody 4G10 was from
Upstate Biotechnology, and polyclonal antibodies raised against
phosphorylated (active) ERK1&2 (T202/Y204), Akt (S473), and STAT5
(Y694) were from New England Biolabs, as was the Akt antibody.
Polyclonal ERK2, c-Kit, and monoclonal Abl antibodies were from Santa
Cruz Biotechnology. Peroxidase conjugated secondary antibodies were
from Amersham Biosciences. Phosphorylation was quantified using NIH
Image software.
c-Kit and Bcr-Abl Kinase Assay--
M07e cells taken from
culture were lysed at 40 × 106/ml as described above,
and c-Kit was immunoprecipitated by incubation overnight at 4 °C
with 2 µg/ml polyclonal antibody. c-Kit receptor-antibody complexes
were captured by protein-G Agarose (incubated at 4 °C for 2 h),
and immunoprecipitates were washed at 4 °C three times in
phosphate-buffered saline, 0.1% Triton X-100, and 2 mM
EDTA and twice in kinase buffer (20 mM HEPES, 20 mM PIPES) pH 7.4, and 10 mM MnCl2
before resuspension in 25 µl of kinase buffer containing varying
concentrations of PP1 or other inhibitors. After a 30-min incubation at
37 °C, a further 25-µl kinase buffer (in which PP1 concentrations
were maintained) containing 100 µM ATP and 10 µCi of
[
-32P]ATP was added, and samples were incubated for 10 min at 30 °C. Reactions were terminated by the addition of 1 ml of
kinase buffer at 4 °C and washed rapidly. c-Kit was eluted with the
addition of sample buffer, and samples were resolved by SDS-PAGE.
Bcr-Abl was immunoprecipitated from either 293T cells expressing
MSCV-p210Bcr-Abl (kind gift of R. Van Etten, Boston, MA) or
Bcr-Abl-FDCP1 cells and processed as for c-Kit kinase assays. Abl
kinase assay was carried out in kinase buffer (20 mM HEPES,
20 mM PIPES), pH 7.4, 10 mM MnCl2,,
10 mM MgCl2 and 10 µCi of
[
-32P]ATP supplemented with 4 µg GST-Crk as
substrate. Phosphorylation of GST-Crk and c-Kit was detected using
autoradiography and quantified using NIH Image software.
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RESULTS |
The Src Selective Inhibitor PP1 Abolishes SCF-stimulated
Proliferation--
SCF and GM-CSF, Tpo, and IL-3 stimulate
proliferation of the human myeloid factor-dependent cell
line M07e. It has previously been shown that of the Src family of
tyrosine kinases, Lyn is present in M07e cells and that activation of
this kinase can be detected after SCF stimulation (3, 21). To
investigate the potential role of Src family kinases in the SCF-induced
proliferation of M07e cells, we used PP1, a selective inhibitor of Src kinases.
M07e cells became quiescent without undergoing apoptosis after 24 h of factor withdrawal and remained so for 48-72 h in growth factor-free conditions (data not shown). Proliferation was measured using the colorimetric MTS assay over a 72-h period after factor re-addition. Fig. 1A shows
that PP1 inhibited SCF-induced proliferation in a
dose-dependent manner (IC50, 0.5-1
µM), whereas 2.5 µM PP1 completely
prevented SCF-induced proliferation. PP2 had a similar effect
(IC50, ~1 µM), whereas the inactive
analogue PP3 had no significant effect on proliferation (Fig.
1B).

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Fig. 1.
PP1 blocks SCF- but not Tpo-induced
proliferation. M07e cells were incubated with the indicated
concentrations of either PP1 (A) or PP2 or PP3
(B) for 60 min before stimulation with 20 ng/ml SCF. Both
PP1 and PP2 blocked SCF-induced proliferation (IC50 <1
µM), but the inactive analogue PP3 had no effect.
C, M07e cells were incubated with PP1 as before, and
stimulated with 45 ng/ml Tpo. The small reduction in Tpo-induced
proliferation with the addition of PP1 indicates that the effects seen
on SCF stimulation are not caused by nonspecific inhibition.
Proliferation was measured using an MTS assay. Results shown are a mean
of three independent experiments.
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In contrast, PP1 had only a minor effect on thrombopoietin-induced
proliferation of M07e cells (Fig. 1C), indicating that the
effects of PP1 on SCF-stimulated proliferation were not caused by
nonspecific toxicity.
PP1 Inhibits SCF-induced Autophosphorylation of c-Kit in Intact
Cells and c-Kit Tyrosine Kinase Activity in Vitro--
These effects
of PP1 suggested a very significant role for Src family kinases in
SCF-induced biological responses. However, these results could
potentially come about from the direct inhibition of c-Kit by PP1 and
not exclusively from inhibition of Src kinases. To investigate this
possibility, M07e were incubated with or without PP1, stimulated with
SCF, and cell extracts were assessed by immunoblotting with an
anti-phosphotyrosine antibody. Fig.
2A shows that c-Kit autophosphorylation was inhibited in a dose-dependent
manner by PP1 and completely blocked at a concentration of 1 µM PP1.

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Fig. 2.
PP1 inhibits c-Kit autophosphorylation in
intact cells and in vitro kinase assays. A,
growth factor-deprived M07e cells were incubated with PP1 for 60 min
before stimulation with 20 ng/ml SCF for 10 min. Lysates were
immunoblotted with an anti-phosphotyrosine antibody, and for total
c-Kit. Phosphorylated c-Kit is indicated. 1 µM PP1
completely abrogated c-Kit autophosphorylation. B, c-Kit was
immunoprecipitated from unstimulated M07e lysates, in vitro
kinase assay performed in the presence of varying PP1 concentrations,
and c-Kit phosphorylation quantified after SDS-PAGE/autoradiography.
PP1 directly inhibited in vitro c-Kit activity
(IC50, ~75 nM). Results shown are mean ± S.E. of three independent experiments. C, PP1, PP2, and
STI571 but not SU6656 inhibit c-Kit activity. C-kit in vitro
kinase assays were carried out in the presence of the indicated
inhibitor concentrations and analyzed by SDS-PAGE
autoradiography.
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These results suggested that PP1 could be inhibiting c-Kit kinase
activity. To examine this further, unstimulated M07e were lysed and
c-Kit immunoprecipitated, and in vitro autophosphorylation kinase assays were performed in the absence or presence of different PP1 concentrations. Fig. 2B shows that PP1 (and PP2)
directly inhibit c-Kit kinase activity in vitro in a
dose-dependent manner (PP1 IC50, ~75
nM).
PP1 but Not the Unrelated Src Inhibitor SU6656 Inhibits c-Kit
Autophosphorylation and Activation of Downstream Signaling
Pathways--
To further exclude the possibility that the effects of
PP1 on c-Kit autophosphorylation were dependent on Src kinases, we used
the unrelated Src inhibitor SU6656 (22, 23). This inhibits Src family
kinases with IC50 values in the nanomolar range, with an
IC50 for lyn kinase of 130 nM. Inhibition by
SU6656 of lyn kinase activity was confirmed in an in vitro
kinase assay using immunoprecipitated lyn from MO7e cells with complete
inhibition of autophosphorylation at 1 µM inhibitor (data
not shown). SU6656 failed to inhibit c-Kit in an in vitro
kinase assay, whereas imatinib mesylate/STI571 (24, 25) was able to
reduce activity (Fig. 2C). Fig.
3A shows that SU6656 is unable
to inhibit c-Kit autophosphorylation in whole cells even at
concentrations of up to 10 µM. Preincubation with PP1 and
STI571, but not SU6656, was also able to inhibit the activation of Akt
and ERK1/2 in response to SCF (Fig. 3B). Inhibition of Akt
and extracellular signal-regulated kinase activation was nearly
complete at concentrations between 1 and 2.5 µM PP1. In
contrast, PP1 at 20 µM did not affect the tyrosine
phosphorylation of STAT5 in response to GM-CSF (Fig. 3C), in
keeping with its reported lack of activity against Jak family
kinases.

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Fig. 3.
Effects of PP1, SU6656, and STI571 on c-Kit
activation. MO7e cells were incubated with the indicated
concentrations of kinase inhibitors for 60 min and then stimulated with
SCF 20 ng/ml for 10 min. Cell lysates were probed with an
anti-phosphotyrosine antibody (A) and antibodies to
phosphorylated active Akt (S473) and ERK mitogen-activated protein
kinase (B). C, MO7e cells were incubated with 20 µM PP1 for 60 min and stimulated with 20 ng/ml GM-CSF for
the indicated times. Lysates were probed for tyrosine-phosphorylated
activated STAT5.
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PP1 Inhibits the Kinase Activity of Constitutively Active Mutants
kit D814V and D814Y--
Several kinase inhibitors, including STI571
and SU9529, have been reported to potently inhibit the activity of
wild-type c-Kit but to have low or no activity against enzymatic site
point mutants (D814V and D814Y in murine or D816V and D816Y in human
numbering) (26, 27). We investigated the effects of PP1 on wild-type and activated kit mutants by transfection of 293 cells. Incubation of
cells expressing wild-type kit with PP1 confirmed the inhibition of
SCF-induced kinase activity (Fig.
4A) with complete block at 0.2 µM PP1. PP1 potently inhibited the basal kit
autophosphorylation in whole-cell assays of the D814V mutant
(IC50, ~1 µM) and the D814Y mutant
(IC50, ~0.2 µM) and markedly reduced the
phosphorylation of multiple intracellular substrates at these
concentrations (Fig. 4B). These results indicate that PP1
has significant activity against both wild-type c-Kit and enzymatic
site point mutants found in mast cell disorders.

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Fig. 4.
Effects of PP1 on the phosphorylation and
activity of wild-type and constitutively active mutant forms of c-Kit.
A, 293 cells were transiently transfected with wild-type
c-Kit, incubated with the indicated concentration of PP1 for 60 min,
stimulated with 20 ng/ml murine SCF for 10 min, and lysates were
immunoprecipitated with anti-c-Kit. Samples were probed with an
anti-phosphotyrosine antibody. B, 293 cells were transiently
transfected with constitutively active kit mutants, incubated with the
indicated concentration of PP1 for 120 min, lysed, and part of the
extract was immunoprecipitated with anti-c-Kit. Total lysates and
immunoprecipitated kit were probed with an anti-phosphotyrosine
antibody. C, RBL-2H3 basophil leukemia cells expressing
constitutively active c-Kit were incubated with PP1 and apoptosis
measured by analysis of annexin-V binding after 24 and 48 h (mean
of three experiments).
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PP1 Induces Apoptosis in RBL-2H3 Basophilic Leukemia Cells
That Express a Constitutively Active Mutant c-Kit--
RBL-2H3 cells
are a factor independent, rat basophilic leukemia cell line with a
point mutation in c-Kit (D817Y), which renders it constitutively active
(28). Cells in complete medium were incubated with increasing
concentrations of PP1 and apoptosis was measured by
annexin-V-fluorescein isothiocyanate binding using flow cytometry (Fig.
4C). Incubation with PP1 triggered apoptosis in these cells.
PP2 had similar effects, whereas the inactive analogue PP3 had no
effect on cell viability (data not shown).
PP1 Also Inhibits Bcr-Abl Tyrosine Kinase Activity and Triggers
Apoptosis in Bcr-Abl-transformed Cells--
The kinase inhibitor
imatinib mesylate/STI571, currently being employed in the treatment of
chronic myelogenous leukemia, blocks the activity of Abl, Bcr-Abl, and
c-Kit tyrosine kinases (25). Having detected the unexpected effects of
PP1 on c-Kit, we investigated whether it could also inhibit Bcr-Abl
kinase activity. FDCP1 cells were transfected with p210 Bcr-Abl and
clonal stable cell lines isolated by standard techniques. Parental
FDCP1 cells are normally dependent on IL-3, but the p210 Bcr-Abl/FDCP1
cells are factor-independent for survival and proliferation and can be
cultured without IL-3 (20). Fig. 5 shows
that incubation of p210 Bcr-Abl/FDCP1 with PP1 led to a
dose-dependent reduction in Bcr-Abl autophosphorylation
(Fig. 5A) and inhibited the activation of STAT5 and ERK1&2
(Fig. 5B). PP1 and PP2 directly inhibited Bcr-Abl kinase
activity in an in vitro assay (Fig. 5C) with an IC50 for PP1 of ~1 µM. A decrease in the
number of viable cells as measured by an MTS assay (IC50,
~3 µM) (Fig. 5D) was also observed upon
incubation of p210 Bcr-Abl/FDCP1 cells with PP1. Assessment of cell
survival by flow cytometric assay of annexin-V binding showed that this
was caused by rapid apoptosis (Fig. 5E). In
contrast, survival of parental cells incubated with PP1 (in the
presence of IL-3) was unaffected (data not shown).

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Fig. 5.
PP1 blocks constitutive activation of Bcr-Abl
and causes rapid apoptosis in Bcr-Abl/FDCP1 cells. A and
B, P210 Bcr-Abl/FDCP1 cells were incubated with PP1 for 60 min, and whole-cell lysates were immunoblotted with
anti-phosphotyrosine to detect activated Bcr-Abl (A) and for
phosphorylated forms of STAT5 and ERK1&2 (B). Parental
FDCP-1 cells are shown to compare activation of STAT5 and ERK1&2 in
response to IL-3 stimulation. C, immunoprecipitated Bcr-Abl
was subjected to an in vitro kinase assay using GST-Crk as a
substrate in the presence of the indicated concentrations of PP1 or
PP2. Samples were analyzed by SDS-PAGE/autoradiography. D,
p210 Bcr-Abl/FDCP-1 cells were incubated with PP1 for 18 h and
analyzed for the number of viable cells using an MTS assay. A reduction
in total cell number (IC50, 3 µM) (mean of
two experiments) was caused by increased apoptosis, as measured by
analyzing annexin-V binding (E). Results shown are the mean
and S.E. of three experiments.
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DISCUSSION |
PP1 was originally described as a selective, ATP-competitive
inhibitor of Src family kinases (1) and has been widely used to
investigate the contribution of Src kinases to a number of biological
functions. In vitro kinase assays carried out by Hanke et al. (1) showed that Src family kinases were inhibited at low nanomolar concentrations and that Zap-70 and Jak-2 were not inhibited at concentrations up to 50 µM.
Biological effects on intact T-cell function were seen with an
IC50 around 0.5 µM
this higher concentration
requirement may be caused by high intracellular levels of ATP compared
with the low concentrations used in in vitro kinase assays.
Since then, Waltenberger and colleagues (29) have shown that PP1 can
also directly inhibit the platelet-derived growth factor-
receptor
tyrosine kinase with an in vitro IC50 of 0.1 µM and an IC50 in intact cells of 0.6 µM; more recently, Carlomagno et al. have
shown inhibition of Ret tyrosine kinase by PP1 (30). In this study, we
investigated the effect of PP1 on signaling via the SCF receptor c-Kit.
We found that PP1 abrogated the proliferative effects of SCF on M07e
cells and that this was associated with blockade of downstream
signaling pathways, including mitogen-activated protein kinase and Akt,
and of receptor autophosphorylation. c-Kit autophosphorylation in
intact cells was blocked with an IC50 of ~100
nM. In vitro kinase assays done with
immunoprecipitated c-Kit and incubated with PP1 gave an
IC50 of ~75 nM. This indicates that PP1 is a
direct inhibitor of c-Kit. Bondzi and colleagues (31) have also shown
that PP1, at a single concentration of 10 µM, could
inhibit the in vitro kinase activity of c-Kit. We found that
the closely related inhibitor PP2 is also an inhibitor of c-Kit. Src
kinases have been postulated to play a significant role in signaling
downstream from c-Kit; these conclusions have partly relied on the
effects of PP1 and PP2 (3, 4, 8, 32) Our data suggest that, as with
most inhibitor studies, corroborating evidence using other experimental
techniques should be obtained where possible.
c-Kit plays a significant role in the pathophysiology of a number of
human malignancies. Mutations have been identified in mast cell
disorders, gastrointestinal stromal tumors, and acute myeloid leukemia,
among others (13, 17, 33). These mutations take a number of forms; the
majority have been shown to confer constitutive activation. The best
characterized mutation is in the kinase domain at aspartate 816 (human
numbering) (34, 35); in a recent series, this mutation was identified
in all cases of adult sporadic cutaneous mastocytosis and in a
significant proportion of pediatric cases (36). In addition, many tumor types, including ovarian, lung, and breast malignancies, express both
c-Kit and SCF, indicating the possibility of autocrine growth stimulation (16). Therefore, there has been interest in developing kinase inhibitors that target c-Kit to increase the treatment options
for patients with c-Kit abnormalities.
Investigation of a number of indolinone compounds showed that several
could inhibit wild type c-Kit but were generally inefficient at
inhibiting the kinase activity of c-Kit with a mutation in the kinase
domain (814 murine, 816 human, 817 rat); only SU6577 was effective at
the high concentration of 40 µM (26). The Abl-selective tyrosine kinase inhibitor STI571 can inhibit wild-type c-Kit with an
IC50 around 0.1 µM (24) but has been reported
not to inhibit the activity of 814/816 mutant c-Kit at concentrations
up to 10 µM (27). We found that PP1 could potently
inhibit the activation of D816V and D816Y mutant forms of c-Kit. PP1
triggered apoptosis in rat basophilic leukemia cells with the
homologous D817Y mutation. These results indicate that further
investigation of the effects of PP1 and related compounds on primary
mastocytosis cells expressing the human counterpart of this mutation
are required. The inhibitory effects of PP1 on additional kinases, such
as Src, Abl, and the platelet-derived growth factor receptor have the
potential to increase the toxic effects of PP1 on normal cells. Our
data show a relative lack of effect of PP1 on thrombopoietin-induced
proliferation, but this area requires further study with other growth
factors and cell types. Using PP1 as a starting point, it may be
possible to refine the development of an inhibitor that is more
selective for c-Kit and its mutants.
We have also identified PP1 (and PP2) as direct inhibitors of the
Bcr-Abl kinase. Inhibition of Bcr-Abl autophosphorylation was seen in
intact cells with an IC50 of ~1 µM, and
apoptosis was induced in Bcr-Abl-transformed cells with an
IC50 of ~2 µM. This compares with
biological effects of STI571 on Bcr-Abl-transformed cell lines that are
seen at concentrations between 0.1 and 0.5 µM. STI571 is
being investigated widely for its effects on Bcr-Abl in chronic
myelogenous leukemia and acute lymphoblastic leukemia, its effects on
c-Kit in a number of disorders, including mast cell neoplasms, and its
effects on the platelet-derived growth factor receptor in malignant
glioblastomas (37). It is interesting that PP1 is also able to inhibit
each of these kinases. Resistance to STI571 may result from a number of
factors, such as overexpression of Bcr-Abl (38, 39) tumor-specific
selection of point mutations that are resistant to STI571 (40, 41), and
binding to plasma proteins such as
1 acidic glycoprotein (42) (tumor
nonspecific). It will be of interest to determine whether there is
cross-resistance to PP1 in these different cases.
The data presented in this article extend the range of tyrosine kinases
that are inhibited by PP1 from Src-family members (1), platelet-derived
growth factor receptor-
(29), and Ret (30) to include c-Kit (and its
mutants) and Bcr-Abl. Hanke et al. (29) showed no inhibition
of Jak-2 and Zap-70 by PP1, and Waltenberger and colleagues (1, 29)
found no effect of PP1 on the kinase activity of KDR/VEGFR-2, FGFR-1,
and IGF-1R. There is conflicting evidence of the effects of PP1 on EGFR
activity (1, 29). This indicates that although PP1 shows some
selectivity, it can inhibit a significant number of tyrosine kinases.
In our study we found that PP2 also inhibits c-Kit and Bcr-Abl; it is likely that this compound has an inhibitory profile similar to that of
PP1.
Recent articles have shed some light on the structural basis of PP1
inhibition of Src kinases. A single residue, corresponding to threonine
338 in c-Src, largely controls the ability of PP1 to inhibit protein
kinases with high potency (43). Mutation of this residue to, or the
natural occurrence of, a larger amino acid (e.g. methionine
or phenylalanine) is sufficient to confer resistance to PP1. The
corresponding residue in the PP1-sensitive kinases platelet-derived
growth factor receptor, c-Kit, and c-Abl is a threonine but in both
Jak2 and Zap-70 is a methionine, which have both been shown to be
resistant to PP1.
In conclusion, we have shown that, at concentrations routinely used by
investigators to study the role of Src kinases in cell function, the
Src-selective inhibitors PP1 and PP2 are also potent inhibitors of the
c-Kit and the Bcr-Abl tyrosine kinases. In addition, PP1 is able to
inhibit the activity of mutant forms of kit commonly found in mast cell
disorders. These results have implications for the use of PP1 and PP2
for the investigation of intracellular signaling pathways and
biological function and also indicate a potential role for PP1 or
related compounds in the treatment of human disease.