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From the
We have cloned a protein tyrosine kinase, MATK, which is
expressed abundantly in megakaryocytes and the brain. We investigated
whether MATK participates in the c-Kit ligand/stem cell factor (KL/SCF)
signaling pathway in the megakaryocytic cell line CMK. After KL/SCF
stimulation, five major proteins of molecular masses of 145, 113, 92,
76, and 63 kDa were rapidly and transiently tyrosine-phosphorylated in
a time-dependent manner, peaking within 5 min, and returning to basal
levels within 60 min. To study the role of MATK in the KL/SCF signaling
pathway, glutathione S-transferase (GST) fusion proteins
containing SH2 and SH3 domains of MATK were cloned, expressed in
Escherichia coli, and purified. MATK-SH2, but not MATK-SH3,
precipitated the tyrosine-phosphorylated c-Kit (molecular mass of 145
kDa) in KL/SCF-stimulated CMK cells. Other GST fusion proteins
containing the SH2 domain of p85 of phosphatidylinositol 3-kinase,
phospholipase C
The human c-Kit ligand (KL),
The intracytoplasmic protein tyrosine
kinases play a critically important role in a diverse array of cellular
responses including proliferation, differentiation, and cell survival
(13) . After the binding of KL/SCF, the c-Kit tyrosine kinase
becomes activated and is autophosphorylated at a number of discrete
sites within its C-terminal cytoplasmic domain
(14) . Following
autophosphorylation of these sites, specific SH2 domain-containing
proteins such as the 85-kDa subunit (p85) of phosphatidylinositol
3-kinase (PI 3-kinase) and phospholipase C
We
have recently identified a novel cytosolic tyrosine kinase, termed
megakaryocyte-associated tyrosine kinase (MATK), which is abundantly
expressed in marrow megakaryocytes and the brain
(21) .
Concurrently, Ntk/Ctk, the respective murine and rat counterparts of
MATK, were cloned and observed to have 85% homology with the human MATK
cDNA
(22, 23) . Treatment of CMK cells or marrow
CD34
The Src
family of protein tyrosine kinases participates in cellular signaling
pathways, including growth factor signaling, integrin-mediated
signaling, T- and B-cell activation, and cellular transformation
(13, 25) . C-terminal phosphorylation of c-Src at
Tyr-527, identified in vivo, represses its kinase activity,
while kinase domain phosphorylation at Tyr-416 is stimulatory. Csk
(C-terminal Src kinase) was observed to negatively regulate the Src
kinase by phosphorylating Src at Tyr-527
(26, 27) . The
MATK has
MATK has a molecular mass of 60 kDa and is composed of an SH3
domain, an SH2 domain, and a kinase domain. Although the precise
functional role of MATK is currently unknown, the presence of these SH2
and SH3 domains suggests that MATK may play a role in the signal
transduction pathways in cells or tissues such as megakaryocytes and
the brain which express the MATK protein. In the present study, we
expressed the SH2 and SH3 domains of MATK into GST fusion proteins and
demonstrated the association of the activated c-Kit with MATK via its
SH2 domain. MATK appears to have a role in the KL/SCF signaling pathway
in human megakaryocytic cells.
To test the role of MATK in the
signaling pathway of KL/SCF in CMK cells, the SH2 domain of human MATK
(MATK-SH2) was prepared as described under ``Experimental
Procedures.'' Serum-starved CMK cells were stimulated with KL/SCF
up to 30 min, and the lysed cells were analyzed. The supernatants were
precipitated with the purified MATK-SH2 protein, analyzed on SDS-PAGE,
and immunoblotted with PY-20 (Fig. 1 B). A
tyrosine-phosphorylated protein with the molecular mass of 145 kDa
(p145) was precipitated with MATK-SH2 within 2 min of KL/SCF
stimulation. The association of the p145 with MATK-SH2 peaked at 2 min
after stimulation and gradually decreased.
To confirm the identity
of this band as a c-Kit receptor, the precipitates were immunoblotted
with polyclonal anti-c-Kit antibodies (Fig. 1 C).
Anti-c-Kit antibodies indeed recognize the 145-kDa protein band. This
result indicates that the MATK protein can associate with the
tyrosine-phosphorylated 145-kDa protein, and that this protein is the
c-Kit receptor. The role of the phosphotyrosine moiety in this
interaction is first emphasized by the elimination of the phosphatase
inhibitors (Na
In
summary, we show that MATK associates with c-Kit via its SH2 domain and
that p85 of PI 3-kinase, phospholipase C
We examined components of the signal transduction pathway of
KL/SCF in model CMK megakaryocytic cells. KL/SCF stimulation rapidly
and transiently induced tyrosine phosphorylation of a number of
proteins including the cognate c-Kit receptor. We and others recently
cloned a novel tyrosine kinase, MATK, which contains an SH2 domain, an
SH3 domain, and a tyrosine kinase domain, and exhibits Csk-like
tyrosine phosphorylation of Src
(21, 22, 23, 24, 31) . In this
study, we demonstrated that MATK associated with KL/SCF-stimulated
c-Kit. This association of MATK with c-Kit occurred within the same
time period in which tyrosine phosphorylation of c-Kit was detected
following KL/SCF stimulation. Our results also demonstrate that the
association of MATK with c-Kit involves the SH2 domain, not the SH3
domain. The analysis of the amino acid sequence involved in the
association of MATK with c-Kit was not addressed in the present study.
Cytokine specificity of the association of MATK and c- kit was addressed in this study. Neither GM-CSF nor IL-6 induced the
association of MATK-SH2 with c- kit, although treatment with
GM-CSF induced tyrosine phosphorylation of a number of proteins in a
time-dependent manner in a pattern that was similar to stimulation with
KL/SCF. These results indicate the specificity of KL/SCF to induce
association of MATK-SH2 with c- kit.
In addition, we have
observed the association of the p85 subunit of PI 3-kinase and
phospholipase C
Interestingly, divergent results from several laboratories have been
obtained regarding the association of ras-GAP with c-Kit
(14, 35, 36) . This discrepancy may originate
from differences in either distinct cell types expressing c-Kit or the
experimental design. To our knowledge, none of the above studies has
addressed the question of whether a fusion protein containing the SH2
domain of ras-GAP could associate with c-Kit. Using a GST
fusion protein containing the SH2-SH3-SH2 domain of ras-GAP,
we observed the association of ras-GAP with the activated
c-Kit at a comparable intensity to the GST fusion proteins containing
the SH2 domain of p85 or the SH2-SH3-SH2 domain of phospholipase
C
Because of the amino acid sequence homology between MATK and Csk,
MATK may be predicted to share certain functional properties with Csk.
Csk is a protein tyrosine kinase that can phosphorylate the C-terminal
tyrosine of Src and suppress its kinase activity
(26, 27) . Similarly, MATK was shown to phosphorylate
both purified Src protein in vitro and the C-terminal
conserved tyrosine of the Src family. Csk does not directly bind to
c-Src or v-Src, but was found to co-localize at podosomes with Src,
where the catalytic activity of Csk is not necessary but its SH2 and
SH3 domains are required
(36, 37) . Recently, the SH2
domain of Csk has been shown to bind to both tyrosine phosphorylated
pp125
In megakaryocytes, the association
of MATK with the KL/SCF- stimulated c-Kit suggests that MATK
participates in KL/SCF-induced cell proliferation. Exposure of
CD34
We would like to thank Dr. J. E. Groopman and Dr. S.
Avraham for their critical review of the manuscript and Patricia DeLapp
and Janet Delahanty for preparation of the manuscript. We also would
like to thank Dr. L. Bennett (Amgen) for providing recombinant KL/SCF
and anti-c-Kit antibodies.
Volume 270,
Number 16,
Issue of April 21, pp. 9661-9666, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-1, and ras-GAP also precipitated c-Kit.
The tyrosine-phosphorylated c-Kit was co-immunoprecipitated with
anti-MATK and anti-p85 antibodies in KL/SCF-stimulated CMK cells, but
not in granulocyte-macrophage colony stimulating factor or
interleukin-6-stimulated cells, suggesting receptor specificity. These
results indicate that MATK associates with the c-Kit receptor following
specific stimulation by KL/SCF via its SH2 domain and likely
participates in transduction of growth signals induced by this cytokine
in megakaryocytes.
()
also known
as stem cell factor (SCF), mast cell factor, or steel factor stimulates
the proliferation of mast cells and early hematopoietic progenitors
(1, 2) . The cell surface receptor for KL/SCF is the
product of the proto-oncogene c- kit, a transmembrane protein
tyrosine kinase belonging to the subfamily of the platelet-derived
growth factor (PDGF) receptor
(3, 4) . In the
hematopoietic system, c-Kit mutations affect the stem cell compartment,
erythroid precursors, tissue mast cells, and platelets
(5) . We
and others have previously reported that c-Kit was expressed on the
surface of human megakaryocytes and functions as a growth factor for
cells of this lineage
(6, 7, 8) . It has been
proposed that c-Kit and its ligand may play a role in the pathogenesis
of acute myeloid leukemia
(9) , and several studies provide
evidence that c-Kit and its ligand are involved in the clonogenic
growth of acute myeloid leukemia blasts
(10, 11, 12) . Permanent megakaryocytic leukemia
cell lines, including the CMK cell line, appear to highly express
c- kit mRNA and protein
(7, 8, 12) , but
less is known about the role of c-Kit and KL/SCF in human
megakaryocytic leukemia.
-1 are recruited from
the surrounding cytosol into complexes with the activated c-Kit. This
recruitment is mediated by a specific phosphotyrosine-SH2 domain
interaction with high-affinity binding and rapid dissociation and
exchange
(15, 16, 17) . SH2 domain-mediated
binding of proteins to the phosphotyrosine-containing sequences is not
a random event, but is a precise interaction between specific SH2
domains and a limited range of phosphorylated target sequences. Short
tyrosine-phosphorylated peptides of a specific target receptor can
competitively inhibit the binding of specific SH2 domain-containing
proteins to that receptor and can be used to purify the appropriate SH2
domain-containing protein
(18, 19, 20) .
cells, the progenitors of bone marrow
megakaryocytes, with antisense oligodeoxynucleotide to MATK inhibited
the growth and maturation of these cells in vitro (24) , suggesting that MATK may play an important role in
the signaling pathway regulating megakaryocytopoiesis.
50% amino acid homology with Csk and phosphorylates
purified Src protein
(21) as well as the C-terminal conserved
tyrosine of Src family members
(22, 23, 24) .
Materials
Recombinant KL/SCF and polyclonal
anti-c-Kit antibodies, were generously provided by Dr. L. Bennett,
Amgen, Inc. GM-CSF and IL-6 were purchased from R & D Systems. The
primers for polymerase chain reaction were synthesized by an automated
DNA synthesizer (Applied Biosystems, Model 394). Monoclonal
anti-phosphotyrosine antibody (PY-20) was obtained from ICN, and the
affinity-purified polyclonal anti-p85 antibodies were obtained from
Transduction Laboratories (Lexington, KY). GST-fusion proteins
containing the N-terminal SH2 domain (amino acids 333-430) of
human p85 of PI 3-kinase, the SH2-SH3-SH2 domain (amino acids
171-448) of human ras-GAP, and the SH2-SH2-SH3 domain
(amino acids 530-850) of human phospholipase C-1 were
obtained from Santa Cruz Biotechnology. Electrophoresis reagents were
obtained from Bio-Rad. All other reagents were purchased from Sigma.
Cell Line
The CMK cell line, provided by Dr. T.
Sato (Chiba University, Japan), was maintained in RPMI 1640 with 10%
fetal calf serum as described previously
(7) . The CMK cell line
was derived from a child with megakaryoblastic leukemia and has
properties of cells of the megakaryocytic lineage, including surface
expression of glycoproteins Ib and IIb/IIIa, synthesis of platelet
factor 4, PDGF and, von Willebrand factor and their change into
polyploid cells on induction with phorbol ester
(7, 28) .
Generation of SH2 and SH3 Domains of
MATK
Oligonucleotides flanking both SH2 and SH3 domains of MATK
(21) and containing appropriate restriction sites were
synthesized. The polymerase chain reaction was used with MATK cDNA as a
template to amplify the appropriate fragments. These fragments were
precleaved with BamHI and EcoRI and ligated to
pGEX-2T which had also been cleaved with BamHI and
EcoRI. Competent Escherichia coli JM109 were
transformed, and recombinant clones were screened by SDS-PAGE analysis
of overexpressed fusion proteins and restriction enzyme analysis.
GST-fusion proteins were produced by 10 m
M isopropyl
-thiogalactopyranoside induction and purified on a large scale by
affinity chromatography on glutathione-Sepharose beads (Pharmacia
Biotech Inc.). The proteins were eluted with 10 m
M glutathione
followed by concentration in a Centricon 30 filter (Amicon), and the
buffer was exchanged to a 5 m
M NaPO
and 100
m
M KCl, pH 7.4. The fusion proteins expressed the following
fragments of human MATK: SH2 domain (amino acids 110 to 221) and SH3
domain (amino acids 43 to 116).
Affinity Precipitation with GST Fusion Proteins
To
detect binding of proteins to GST fusion proteins, approximately
10
CMK cells (2 10
cells/ml) were
starved for 4 h at 37 °C in serum-free Dulbecco's modified
Eagle's medium and stimulated for 5 min at 37 °C with either
KL/SCF (500 ng/ml), GM-CSF (500 ng/ml), or IL-6 (50 ng/ml). The
stimulation was terminated with the addition of an ice-cold stopping
buffer (137 m
M NaCl, 1 m
M MgCl, 1 m
M
CaCl
, 100 µ
M Na
VO, 20
m
M Tris-HCl, pH 7.5), followed by centrifugation (1,500 rpm
for 5 min). The cells were lysed with lysis buffer (137 m
M
NaCl, 1 m
M MgCl, 1 m
M CaCl, 100
µ
M NaVO, 10% glycerol, 1% Nonidet
P-40, 2 m
M phenylmethylsulfonyl fluoride, aprotinin (10
µg/ml), leupeptin (10 µg/ml), 20 m
M Tris-HCl, pH 7.5)
for 30 min, and undissolved particles were removed by centrifugation
(14,000 rpm, 10 min). Phenylarsine oxide (10 µ
M) was added
in one experiment (Fig. 2). In some cases, cells were extracted
in the same buffer but in the absence of NaVOand phenylarsine oxide. The cell lysate was incubated for 90 min
at 4 °C with 10 µg of GST-fusion proteins coupled to
glutathione-Sepharose beads. The beads were washed three times with
lysis buffer, and proteins were separated by 7.5% SDS-PAGE. Bound
proteins were immunoblotted with anti-phosphotyrosine antibody (PY-20)
and polyclonal anti-c- kit antisense. The blots were developed
by enhanced chemiluminescence (ECL) (Amersham).
Figure 2:
Effect of phosphatase inhibitors on the
association of the SH2 domain of MATK and the c-Kit receptor in CMK
cells. CMK cells unstimulated ( lane 1) or stimulated with
KL/SCF (500 ng/ml) ( lanes 2-5) were extracted in the
presence ( lanes 1, 2, and 4) or absence
( lanes 3 and 5) of phosphatase inhibitors. Extracts
were precipitated directly as described in Fig. 1 ( lanes 1,
2, and 3) or preincubated for 30 min at room
temperature prior to precipitation ( lanes 4 and 5).
CMK lysates were analyzed by 7.5% SDS-PAGE followed by immunoblotting
with anti-phosphotyrosine (PY-20) antibody.
Immunoprecipitation
The cell lysates were obtained
as above and immunoprecipitated with polyclonal anti-MATK antibodies
(1:100 dilution)
(21) or polyclonal anti-p85 antibodies (5
µg/ml) for 16 h at 4 °C. The mixtures were incubated with
Protein A-coupled Sepharose beads for 30 min at 4 °C, and the beads
were washed three times with lysis buffer. Precipitates were analyzed
by 7.5% SDS-PAGE and immunoblotted with PY-20. Blots were developed by
ECL.
Characterization of the Association of MATK and
c-Kit
To address the role of MATK in signal transduction
pathways in megakaryocytes, experiments were performed using the model
CMK megakaryocytic cell line. CMK cells were starved in serum-free
medium for 4 h and stimulated with KL/SCF (500 ng/ml) for the indicated
times. Cells were lysed, analyzed on SDS-PAGE, and immunoblotted with
anti-phosphotyrosine antibody (PY-20) (Fig. 1 A). KL/SCF
stimulation induced the rapid appearance of five prominent
tyrosine-phosphorylated bands with molecular masses of 145, 113, 92,
76, and 63 kDa. Tyrosine phosphorylation of these proteins was rapid
and transient, observed within 2 min, peaked at 5 min, and returned to
basal levels within 60 min.
VOand phenylarsine oxide),
resulting in the complete disappearance of the c- kit band from
the blots (Fig. 2). In the presence of
NaVO, which inhibits tyrosine
dephosphorylation, the association of MATK-SH2 and c-Kit was greater
than in the absence of NaVO, particularly after
preincubation of the extract for 30 min at room temperature (Fig. 2).
Taken together, these results indicate that MATK-SH2/c- kit association is dependent on tyrosine phosphorylation of
c- kit. The association of MATK-SH2 with the c- kit was
saturated at a concentration of 5 µg of MATK-SH2 and was not
further increased up to 20 µg of MATK-SH2 (data not shown).
MATK-SH2 precipitated additional tyrosine-phosphorylated proteins;
however, these proteins were precipitated even in the basal cell lysate
and did not change dramatically over the time course tested.
Figure 1:
Time course of KL/SCF-induced tyrosine
phosphorylation and the association of the SH2 domain of MATK
(MATK-SH2) and the c-Kit receptor in CMK cells. Serum-starved CMK cells
(2 10
cells/ml) were stimulated with KL/SCF (500
ng/ml) for the indicated times at 37 °C. A, these CMK
lysates were analyzed by 7.5% SDS-PAGE followed by immunoblotting with
anti-phosphotyrosine (PY-20) antibody. B, lysates were
incubated with MATK-SH2 (10 µg) and glutathione-Sepharose 4B beads
for 90 min at 4 °C. After intensive washing, the
tyrosine-phosphorylated proteins associated with MATK-SH2 were
separated as mentioned above and immunoblotted with monoclonal
anti-phosphotyrosine antibody (PY-20). C, the association of
MATK-SH2 with the c-Kit receptor was detected by polyclonal anti-c-Kit
antibodies (1:500 dilution). Molecular size markers (in kilodaltons)
are denoted on the left. TCL is total cell
lysates.
Cytokine Specificity of the Association of MATK and
c-Kit
We then investigated whether this association of MATK and
c-Kit was a receptor-specific effect by comparing KL/SCF to two other
cytokines known to stimulate CMK cells. GM-CSF and IL-6 treatment
induce the growth and maturation of CMK cells and megakaryocytes
(7, 29) . We addressed whether MATK played a role in the
signaling pathways of these cytokines in CMK cells. Cell activation of
serum-starved CMK cells by GM-CSF (500 ng/ml) induced tyrosine
phosphorylation of a number of proteins in a time-dependent manner
(Fig. 3 A). No newly phosphorylated protein was observed in
IL-6-stimulated cells under these conditions (Fig. 3 A). Only
KL/SCF induced the association of MATK-SH2 with c-Kit
(Fig. 3 B). Both GM-CSF and IL-6 were used at
concentrations that induce the proliferation of CMK cells.
Figure 3:
Time course of GM-CSF or IL-6-induced
tyrosine phosphorylation and the association of MATK-SH2 with the c-Kit
receptor. Serum-starved CMK cells (2 10
cells/ml)
were stimulated with GM-CSF (500 ng/ml) or IL-6 (50 ng/ml) for the
indicated times at 37 °C and processed for immunoblotting.
A, lysates were analyzed by 7.5% SDS-PAGE followed by
immunoblotting with PY-20. B, cell lysates from a 5-min
stimulation with either KL/SCF, IL-6, or GM-CSF were incubated for 90
min at 4 °C with 10 µg of MATK-SH2. The associated proteins
were visualized PY-20.
The
association of activated c-Kit with other SH2 domain-containing
signaling molecules such as p85 of PI 3-kinase, phospholipase C-1,
and ras-GAP was also examined. This was done using the GST
fusion protein in the same manner in which we used the MATK-SH2
protein. KL/SCF-stimulated CMK cell lysates were incubated with the
N-terminal SH2 domain of p85 of PI 3-kinase (p85-SH2N) and then
analyzed with PY-20 (Fig. 4 A) and anti-c-Kit antibodies
(Fig. 4 B). Fig. 4shows that in this experiment
p85-SH2N coupled beads also precipitated tyrosine-phosphorylated c-Kit
after KL/SCF stimulation. We did not detect any association of any
tyrosine-phosphorylated protein with p85-SH2N at the basal level or
after GM-CSF or IL-6 stimulation. These results indicate that p85 of PI
3-kinase associates with activated c-Kit via its N-terminal SH2 domain.
Figure 4:
Association of SH2-containing p85 of PI
3-kinase, phospholipase C-1, and ras-GAP with the c-Kit
receptor in cytokine-stimulated CMK cells. The same group of
cytokine-stimulated cell lysates as in Fig. 3 B were incubated
with 10 µg of the GST fusion proteins containing either the SH2
domain of MATK ( MATK-SH2), the N-terminal SH2 domain of p85 of
PI 3-kinase ( p85-SH2N), the SH2-SH2-SH3 domain of
phospholipase C
-1 ( GST-PLC), or the SH2-SH3-SH2 domain of
ras-GAP ( GST-GAP) for 90 min at 4 °C. The
associated proteins were analyzed as in Fig. 1 with either PY-20
( A) or anti-c-Kit antibodies applied to the same membranes
after stripping ( B). The top left panel in this
figure was reproduced for Fig. 4 B for
comparison.
The association of c-Kit with ras-GAP and phospholipase
C-1 was also examined. A previous study
(30) showed that a
fusion protein containing SH2-SH3-SH2 of ras-GAP was bound to
the PDGF receptor after PDGF stimulation, while a fusion protein
containing only the SH2 domain did not associate with this PDGF
receptor. For this reason, we selected GST fusion proteins containing
the SH2-SH2-SH3 fragment of phospholipase C
-1 and the SH2-SH3-SH2
fragment of ras-GAP. As shown in Fig. 4, A and
B, the c-Kit receptor is a target for all four signaling
molecules in KL/SCF-activated CMK cells.
SH3 Is Not Involved in MATK and c-Kit
Interaction
The potential role of the SH3 domain of MATK was
examined using an experimental protocol similar to that described for
the SH2 domain. A GST fusion protein containing the SH3 domain of human
MATK (MATK-SH3) was cloned and purified using the same methods employed
for the MATK-SH2 fusion protein. KL/SCF-, GM-CSF-, and IL-6-stimulated
CMK cell lysates were incubated with MATK-SH2 and MATK-SH3 and
immunoblotted with PY-20, anti-c-Kit antibodies, and anti-GST
antibodies (Fig. 5). Unlike MATK-SH2, MATK-SH3 did not precipitate any
protein, suggesting that the putative target protein for this SH3
domain in activated CMK cells is either much lower in affinity or that
it is not a phosphotyrosine-containing protein. Alternatively, this
protein may reside in the detergent insoluble compartment and may have
been removed with the insoluble fraction following cell lysis. In
GM-CSF- or IL-6-stimulated CMK cells, MATK-SH3 also did not precipitate
any tyrosine-phosphorylated protein. Upon comparing the amino acid
sequence of the SH3 domain with that of other signaling proteins, we
found that MATK is lacking a characteristic SH3 motif, ALYDY, and also
the relatively conserved PSNYV motif found in the C-terminal region of
the SH3 domain of the Src family. Further work will be necessary to
investigate the role of MATK-SH3 in megakaryocyte signaling pathways.
Studies of the Association of Intact MATK with
c-Kit
To further confirm the association between c-Kit and MATK
in CMK cells, immunoprecipitation was performed with anti-MATK and
anti-p85 antibodies (as opposed to the direct SH2 domain mediated
precipitation). Cells were stimulated with KL/SCF, GM-CSF, or IL-6, and
the associated proteins were analyzed with PY-20 (Fig. 6). In
KL/SCF-stimulated CMK cells, the tyrosine-phosphorylated c-Kit was
immunoprecipitated with anti-MATK antibodies, while in GM-CSF- or
IL-6-stimulated cells, no precipitated phosphotyrosine protein was
detected. Taken together, these data suggest that the MATK protein
associated with the c-Kit via its SH2 domain, not its SH3 domain. Using
a similar method, anti-p85 antibodies immunoprecipitated c-Kit as well
as several additional tyrosine-phosphorylated proteins in the
KL/SCF-treated, but not in the GM-CSF- or IL-6-treated cells.
-1, and ras-GAP
also bind to the activated c-Kit receptor.
-1 with the activated c-Kit. A GST fusion protein
containing the SH2 domain of p85 and anti-p85 antibodies precipitated
the activated c-Kit. Similarly, a GST fusion protein containing the
SH2-SH2-SH3 domain of phospholipase C
-1 precipitated the activated
c-Kit as well. Consistent with our results, several laboratories have
reported that activated c-Kit associates with p85 in mast cells and
Cos-1 cells, phospholipase C
-1 in mast cells, and Tec kinase in
Mo7e cells, as well as protein tyrosine phosphatase 1C (PTP1C) in Mo7e
human cell lines
(14, 32, 33, 34) .
-1. Since currently available published data were obtained in a
variety of laboratories using diverse tools, experimental designs, and,
most importantly, cellular systems, it is premature to favor any
conclusion. Our results demonstrated that the SH2 domain of
ras-GAP has the ability to associate with the activated c-Kit.
and paxillin
(36) . These results suggest
that despite a high degree of sequence homology, MATK and Csk may
participate in different signaling pathways leading to cell
proliferation and Src regulation.
marrow cells, the progenitors of bone marrow
megakaryocytes, to MATK antisense oligodeoxynucleotides inhibited the
growth of megakaryocyte progenitors
(24) , suggesting that MATK
expression was involved in the signaling pathway for survival,
proliferation, and/or maturation of cells of this lineage. For that
reason, we explored whether the signals induced by cytokines like
KL/SCF known to modulate CD34
progenitors and
megakaryocytopoiesis might be transduced along pathways that involve
MATK. Our findings of an association of MATK with the c-Kit receptor
suggest that MATK likely plays an important role in this specific
pathway leading to the proliferation of human megakaryocytes.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. Hirao, X.-L. Huang, T. Suda, and N. Yamaguchi Overexpression of C-terminal Src Kinase Homologous Kinase Suppresses Activation of Lyn Tyrosine Kinase Required for VLA5-mediated Dami Cell Spreading J. Biol. Chem., April 17, 1998; 273(16): 10004 - 10010. [Abstract] [Full Text] [PDF]
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 M. Kozlowski, L. Larose, F. Lee, D. M. Le, R. Rottapel, and K. A. Siminovitch SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain Mol. Cell. Biol., April 1, 1998; 18(4): 2089 - 2099. [Abstract] [Full Text]
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S. Zrihan-Licht, B. Deng, Y. Yarden, G. McShan, I. Keydar, and H. Avraham Csk Homologous Kinase, a Novel Signaling Molecule, Directly Associates with the Activated ErbB-2 Receptor in Breast Cancer Cells and Inhibits Their Proliferation J. Biol. Chem., February 13, 1998; 273(7): 4065 - 4072. [Abstract] [Full Text] [PDF]
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D. J. Price, B. Rivnay, Y. Fu, S. Jiang, S. Avraham, and H. Avraham Direct Association of Csk Homologous Kinase (CHK) with the Diphosphorylated Site Tyr568/570 of the Activated c-KIT in Megakaryocytes J. Biol. Chem., February 28, 1997; 272(9): 5915 - 5920. [Abstract] [Full Text] [PDF]
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S. Zrihan-Licht, J. Lim, I. Keydar, M. X. Sliwkowski, J. E. Groopman, and H. Avraham Association of Csk-homologous Kinase (CHK) (formerly MATK) with HER-2/ErbB-2 in Breast Cancer Cells J. Biol. Chem., January 17, 1997; 272(3): 1856 - 1863. [Abstract] [Full Text] [PDF]
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C. Bougeret, S. Jiang, I. Keydar, and H. Avraham Functional Analysis of Csk and CHK Kinases in Breast Cancer Cells J. Biol. Chem., August 31, 2001; 276(36): 33711 - 33720. [Abstract] [Full Text] [PDF]
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