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(Received for publication, July 31, 1995; and in revised form, December 12, 1995) From the
Human leukemic cell line K562 is induced to differentiate into
the megakaryocytic lineage by stimulation with
12-O-tetradecanoylphorbol-13-acetate (TPA). We demonstrate
here that TPA stimulation increases tyrosine phosphorylation of an
80-kDa protein at an early stage of megakaryocytic differentiation and
that this 80-kDa protein is identical with cortactin. Since tyrosine
kinase Syk was activated by TPA stimulation, we examined the
possibility that cortactin is a potential substrate of Syk in K562
cells. TPA-induced tyrosine phosphorylation of cortactin was decreased
profoundly by overexpression of dominant-negative Syk. Furthermore,
cortactin was associated with Syk even before TPA stimulation. Since
cortactin was previously referred as an 80/85-kilodalton
pp60
The generation of functional cells of the hematopoietic system
is a complex process requiring both the constant production of large
numbers of differentiated cells and the maintenance of primitive
precursor cells. As model systems to investigate the mechanisms of
hematopoietic differentiation, several hematopoietic cell lines that
can be induced to various cell lineages have been used. In these cell
lines, K562 cells, established from a patient with chronic myeloid
leukemia in blast crisis(1) , have a potential to differentiate
into a variety of hematopoietic cell
lineages(2, 3, 4, 5, 6, 7, 8, 9) .
Several lines of evidence indicate that this cell line can be
differentiated into megakaryocytic lineage by stimulation with
12-O-tetradecanoylphorbol-13-acetate
(TPA)( Evidence has been accumulating
that protein tyrosine phosphorylation and dephosphorylation play
important roles in a variety of processes, leading to cell growth and
differentiation in hematopoietic cells. Indeed, K562 cells transfected
with c-fes, one of the non-receptor-type protein-tyrosine
kinases, undergo myeloid differentiation(18) . In contrast,
erythroid differentiation of these cells can be induced by herbimycin
A, an inhibitor of tyrosine kinase (19) . A non-receptor-type
protein-tyrosine kinase Syk is expressed in almost all the
hematopoietic cells. Although the functions of Syk in mast cell, B
cell, and platelet activation have been elucidated
extensively(20) , its roles in hematopoietic cell
differentiation remain to be addressed. We found here that an
80-kilodalton (kDa) protein, cortactin, is tyrosine-phosphorylated in
K562 cells upon TPA treatment, and cortactin associates physically and
functionally with Syk. These findings suggest that Syk mediates
tyrosine phosphorylation of cortactin at an early stage of
megakaryocytic differentiation in K562 cells.
To examine the
association of Syk with cortactin, digitonin lysis buffer (1%
digitonin, 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 100 µM vanadate) supplemented with 2 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin was used
instead of Nonidet P-40 lysis buffer. After washing, immunoprecipitates
were boiled with Laemmli SDS sample buffer and subjected to immunoblot
analysis.
Tyrosine phosphorylation in K562 cells following TPA
stimulation was assessed by immunoblot analysis of whole cell lysates
with anti-phosphotyrosine mAb. Following TPA stimulation, cells were
harvested at the indicated times, and whole cell lysates were resolved
by SDS-PAGE and subjected to immunoblot analysis with
anti-phosphotyrosine mAb. As shown in Fig. 1, a protein with
molecular mass of 80 kDa was tyrosine-phosphorylated, reaching a
maximum within 10 min. Two other phosphotyrosine-containing proteins,
approximately 160 and 110 kDa, were dephosphorylated with different
time courses after TPA stimulation.
Figure 1:
Tyrosine
phosphorylation following TPA stimulation in K562 cells. K562 cells
were stimulated by 10 nM TPA and solubilized at the indicated
times. Whole cell lysates were separated on 12% SDS-PAGE gel and
subjected to immunoblot analysis probed with anti-phosphotyrosine mAb.
The positions of the molecular markers are shown to the left in kDa. Arrowheads indicate the positions of the 160-,
110-, and 80-kDa proteins, respectively.
To identify this 80-kDa protein,
we performed immunoblot analysis with several Abs to known
phosphoproteins of similar size. Among various Abs tested, we found
that this 80-kDa protein migrates to the same mobility as cortactin. Fig. 2A shows immunoblotting of whole cell lysates with
anti-phosphotyrosine mAb or anti-cortactin mAb. By long time
development, the lower extra band was visualized. To confirm that this
80-kDa protein is cortactin, immunoblotting of anti-cortactin
immunoprecipitates with anti-phosphotyrosine mAb was carried out. As
shown in Fig. 2B, tyrosine phosphorylation of cortactin was
induced by TPA stimulation. The amount of precipitated cortactin did
not differ in both samples judging from the immunoblot analysis with
anti-cortactin mAb. We next performed depletion experiments with
anti-cortactin mAb. TPA-induced tyrosine phosphorylation of the 80-kDa
protein was not detected following depletion with anti-cortactin Ab,
whereas it was not affected by the depletion with same
isotype-irrelevant mAb, anti-Rb mAb (Fig. 2C). These
results demonstrate that this 80-kDa protein is identical with
cortactin.
Figure 2:
Tyrosine phosphorylation of cortactin in
K562 cells. A, whole cell lysates were separated on 12%
SDS-PAGE gel and blotted on membrane. Blotted membrane was cut into two
pieces, and one membrane was probed with anti-phosphotyrosine mAb and
the other membrane was probed with anti-cortactin mAb. The positions of
molecular markers are shown to the left in kDa. The arrowhead indicates the position of the 80-kDa protein. B, tyrosine phosphorylation of cortactin in K562 cells. a, cells were stimulated by 10 nM TPA for 20 min.
Stimulated and unstimulated cells were solubilized and
immunoprecipitated with anti-cortactin mAb. Immunoprecipitates were
subjected to immunoblot analysis probed with anti-phosphotyrosine mAb. b, each immunoprecipitated sample was subjected to immunoblot
analysis with anti-cortactin mAb. The positions of cortactin and
immunoglobulin heavy chain are indicated. C, depletion of
cortactin from TPA-stimulated cells. Cells were stimulated as above,
and cell lysates were obtained before (pre IP) and after
immunoprecipitation with anti-cortactin mAb or the same
isotype-irrelevant mAb, anti-Rb mAb. Lysates were separated on 12%
SDS-PAGE gel and subjected to immunoblot analysis with
anti-phosphotyrosine mAb. The positions of molecular markers are shown
to the left in kDa. The arrowhead indicates the
position of 80 kDa.
A report that cortactin is tyrosine-phosphorylated after
stimulation of platelets with thrombin(23) , together with the
observation that this stimulant activates Syk(21) , prompted us
to examine the involvement of Syk in TPA-induced tyrosine
phosphorylation in K562 cells. We first examined whether TPA activates
Syk in this cell line or not. After TPA stimulation, cells were
solubilized with Nonidet P-40 lysis buffer at the indicated times, and
Syk was immunoprecipitated by anti-human Syk mAb. Then, an in vitro kinase assay was performed with an exogenous substrate H2B
histone. Syk kinase activity was increased within 1 min and reached to
a maximum at 5 min after TPA stimulation (Fig. 3A). The
amount of precipitated Syk did not change throughout the time course
judging from the immunoblot analysis with anti-human Syk mAb (Fig. 3B).
Figure 3:
Activation of Syk kinase in response to
addition of TPA. A, K562 cells were stimulated by 10 nM TPA and solubilized at the indicated times. Syk was
immunoprecipitated with anti-human Syk mAb and subjected to an in
vitro kinase assay using H2B histone as an exogenous substrate. B, each immunoprecipitated sample was subjected to immunoblot
analysis with anti-human Syk mAb. The positions of Syk and H2B histone
are indicated.
To address the relation between Syk
activation and phosphorylation of cortactin in TPA-treated K562 cells,
cell lines expressing a dominant-negative mutant form of Syk were
established. A point mutation was created in the ATP binding site of
porcine syk cDNA, leading to loss of its kinase activity,
Syk(K
Figure 4:
Expression of kinase-negative Syk in K562
cells. K562 cells were transfected with kinase-negative porcine syk cDNA in which a point mutation was created in the ATP binding
site. Transfected cells were selected in the presence of puromycin and
cloned by limiting dilution. A, expression of transfected
cDNA. Whole cell lysates of wild type K562 (Wt) and 2 clones
of Syk(K
To get insights of whether Syk
directly or indirectly phosphorylates cortactin upon TPA treatment,
co-immunoprecipitation experiments were performed. K562 cells were
stimulated and Syk was immunoprecipitated as described in Fig. 3with a modification that digitonin lysis buffer was used
to solubilize cells instead of Nonidet P-40 lysis buffer.
Immunoprecipitates were subjected to immunoblot analysis with
anti-cortactin mAb. As shown in Fig. 5, cortactin was
co-immunoprecipitated with Syk both before and after stimulation. The
amount of cortactin was not different throughout the time course.
Although the isotype of anti-human Syk mAb has not been determined, we
conveniently used anti-human Rb mAb for negative control
immunoprecipitation to exclude the possibility that cortactin binds
nonspecifically to protein A. Co-precipitation of cortactin with Syk
was also found when Nonidet P-40 lysis buffer was used to solubilize
cell lysates (data not shown), demonstrating that this association is
stable. The amount of precipitated Syk did not differ throughout the
time course (data not shown). Syk could not be detected in the
precipitates with anti-cortactin mAb. One possibility is that as
anti-human Syk mAb is not so suitable for immunoblot analysis, we could
not detect a small amount of co-precipitated Syk. Another possibility
is that the amount of cortactin might be greater than that of Syk in
this cell, and cortactin that does not associate with Syk might be
present. So, a detectable amount of Syk could not be precipitated with
cortactin.
Figure 5:
Association of cortactin with Syk. K562
cells were stimulated and Syk was immunoprecipitated as described in Fig. 3with a modification that digitonin lysis buffer was used
instead of Nonidet P-40 lysis buffer. Immunoprecipitates were subjected
to immunoblot analysis with anti-cortactin mAb. For irrelevant Ab
negative control, anti-Rb mAb was used. The positions of cortactin and
immunoglobulin heavy chain are indicated.
Since cortactin has been reported to be a potential
substrate of Src, we assessed the kinase activity of Src following TPA
stimulation in wild type cells and Syk(K
Figure 6:
Activation of Src kinase in response to
addition of TPA. A, K562 and Syk(K
In this study, we demonstrate that cortactin is
tyrosine-phosphorylated following TPA stimulation in K562 cells by
using immunoprecipitation experiments. TPA is well known to be an
activator of protein kinase C, suggesting that the protein kinase C
activates tyrosine kinase(s), leading to the induction of tyrosine
phosphorylation of cortactin. TPA-induced tyrosine phosphorylation was
reported(24, 25, 26, 27, 28, 29, 30, 31, 32) ,
whereas it was not necessarily elucidated whether this process is
mediated by protein kinase C. It was reported that the biological
effects by TPA cannot be explained completely by protein kinase C
activation in K562
cells(33, 34, 35, 36) . It is also
possible that TPA-induced tyrosine phosphorylation of cortactin is
independent of protein kinase C activation. Cortactin was initially
described as an 80/85-kilodalton pp60 An interaction between Syk and Src family
protein-tyrosine kinases has been proposed(44) . Coexpression
of Src family protein-tyrosine kinases and Syk leads to a remarkable
increase in net tyrosine phosphorylation, whereas Src family
protein-tyrosine kinases or Syk alone induce only marginal
phosphorylation in COS cells. Our finding that TPA-induced Src
activation is not affected by overexpression of dominant-negative Syk
may exclude the possibility that Syk activates Src kinase activity,
resulting in tyrosine phosphorylation of cortactin. It would be less
possible that Syk and Src are activated independently, considering the
close relationship between Syk and Src or Src and cortactin. Src might
be upstream to Syk. As mentioned, TPA treatment of K562 cells
induces the increase of nuclear DNA ploidy and cell volume. Cortactin
is concentrated in a cytoskeleton-associated structure that is rich in
focal adhesion proteins(37) . Since cortactin is able to
associate with F-actin through its tandem helix-turn-helix
domain(41) , it may be conceivable that cortactin is involved
in this TPA-mediated morphological change. Cortactin is related to a
putative transcriptional factor HS1(45) . This protein is
specifically expressed in hematopoietic cells and functions as a major
substrate of protein-tyrosine kinase(s) involved in B-cell antigen
receptor-mediated signaling. Tyrosine-phosphorylated HS1 is
demonstrated to be localized mainly in nucleus, proposing the idea that
HS1 is translocated from the cytoskeleton to the nucleus through its
phosphorylation(46, 47) . Similarly, cortactin may be
transported to the nucleus through its tyrosine phosphorylation where
it may be involved in nuclear events such as transcriptional
regulation, leading to differentiation into megakaryocytic lineage.
Although these notions await further investigation, the present study
demonstrates that cortactin may be the substrate of Syk rather than
Src.
Volume 271,
Number 12,
Issue of March 22, 1996 pp. 6631-6635
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
substrate, we examined the association
between Src and cortactin, whereas its association could not be
detected. These data suggest that Syk phosphorylates cortactin in K562
cells upon TPA treatment.
)(6, 10) . The expression of platelet
glycoprotein IIIa (GPIIIa) and thromboxane A
receptor,
which have been used as a marker of megakaryocytic differentiation, is
strongly enhanced on the surface of K562 cells upon treatment with TPA (11, 12) . Furthermore, during TPA induction, nuclear
DNA ploidy of these cells is increased to 4-16n simultaneously
with an increase in cell volume(13, 14) . K562 cells
can also be induced to undergo erythroid differentiation by various
compounds, including hemin(2) . When K562 cells are treated by
hemin, the transcription of -,
-,
-, and
-globin
mRNA is increased (15, 16) and hemoglobin is
accumulated(2, 17) .
Cell Culture
K562 cells were cultured in RPMI
1640 supplemented with 10% heat-inactivated fetal bovine serum, 100
IU/ml penicillin, and 100 µg/ml streptomycin. Passages were
performed every 3 days when the cells were in exponential phase of
growth. The cells were induced to differentiation by adding 10 nM TPA and harvested at the indicated times. The differentiation of
stimulated cells was confirmed by Giemsa staining.Antibodies
Antibody (Ab) against porcine Syk was
generated by immunizing rabbit with synthesized peptides as
described(21) . The sequence of synthesized peptides to
generate anti-porcine Syk Ab is not conserved in human Syk, so this Ab
reacts specifically with porcine Syk. Monoclonal Ab (mAb) against human
Syk (101) was obtained from Wako Chemicals, Tokyo, Japan. Anti-human
retinoblastoma protein (Rb) mAb (G99-2005) was purchased from
Pharmingen. Anti-phosphotyrosine mAb (4G10), anti-cortactin mAb
(anti-p80/85 pp60 substrate, 4F11), and anti-Src mAb
(GD11) were purchased from Upstate Biotechnology, Inc.
Generation of Dominant-negative Mutant
A point
mutation (Lys
Arg) in the ATP binding site of
porcine syk cDNA was created by polymerase chain reaction as
described(22) , and this mutated cDNA was inserted into the EcoRI site of pApuro vector, harboring the chicken actin
promoter and puromycin-resistant gene. This plasmid was linearized and
transfected into human hematopoietic cell line K562 cells by
electroporation using Electroporator II (Invitrogen) at 300 V, 1000
microfarads, and selected in the presence of 1.0 µg/ml puromycin.
Cells were cloned by limiting dilution, and the expression of
transfected cDNA was confirmed by immunoblot analysis using
anti-porcine Syk Ab.
Immunoprecipitation and in Vitro Kinase
Assay
Cells were sedimented by centrifugation and the pellets
were solubilized in Nonidet P-40 lysis buffer (1% Nonidet P-40, 20
mM Tris, pH 7.5, 150 mM NaCl, 10 mM EDTA,
100 mM NaF, 1 mM vanadate) supplemented with 2 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin. The
lysates were incubated with Ab-conjugated protein A-Sepharose for 1 h
at 4 °C. The immunoprecipitates were washed three times with lysis
buffer and twice with 10 mM HEPES, pH 8.0. Each sample was
incubated in 60 µl of reaction mixture (45 mM HEPES, pH
8.0, 150 mM NaCl, 50 mM MgCl, 10
µM vanadate, 1 µM ATP) containing 5 µCi
of [-
P]ATP with 0.2 mg/ml H2B histone or
0.04 mg/ml of acid-treated enolase. After 10 min at 30 °C, the
reaction was terminated by adding Laemmli SDS sample buffer and boiling
for 3 min. The samples were separated on SDS-PAGE gels and analyzed
using a Fujix imaging analyzer (BAS 2000).
Immunoblot Analysis
Whole cell lysates were
prepared from nonstimulated or stimulated cells by boiling with Laemmli
SDS sample buffer for 3 min. Whole cell lysates or immunoprecipitates
were separated on SDS-PAGE gels and transferred onto polyvinylidene
difluoride membrane. The blots were blocked with 5% milk, 0.05% Tween
20 in phosphate-buffered saline and incubated with primary Ab for 1 h
at room temperature. After washing with 0.05% Tween 20 in
phosphate-buffered saline, filters were developed with goat anti-mouse
or donkey anti-rabbit secondary Ab conjugated to horseradish
peroxidase.
)(22) . This mutated cDNA was
transfected into K562 cells, and stable transformants were isolated in
the presence of puromycin. Expression of mutated porcine Syk was
examined by immunoblot analysis with anti-Syk Ab which recognizes only
porcine species, and 4 stable transformants were cloned. Fig. 4A shows the expression of mutated porcine Syk in 2
clones of Syk(K
) expressing cells. To examine whether
overexpression of Syk(K
) affects tyrosine
phosphorylation in K562 cells, whole cell lysates of stimulated and
unstimulated cells were subjected to immunoblot analysis with
anti-phosphotyrosine mAb. In parental K562 cells, an 80-kDa protein was
tyrosine-phosphorylated by TPA stimulation, whereas in 4 transformants
expressing Syk(K
), this induction was abolished. Fig. 4B shows tyrosine phosphorylation of the 80-kDa
protein in parental K562 cells and 2 clones of Syk(K
)
expressing cells. To confirm that this 80-kDa protein is cortactin,
cell lysates from parental K562 cells and transformant expressing
Syk(K
) were immunoprecipitated by anti-cortactin Ab
and probed with anti-phosphotyrosine mAb (Fig. 4C).
Consistent with a whole cell lysate data, TPA-induced tyrosine
phosphorylation of cortactin was not observed in transformant
expressing Syk(K
). These data demonstrate a strong
correlation between Syk activity and TPA-induced tyrosine
phosphorylation of cortactin.
) transfected cells were analyzed by
immunoblot analysis with anti-porcine Syk Ab. B, tyrosine
phosphorylation of wild type K562 (Wt) and 2 clones of
Syk(K
) transfected cells following TPA stimulation.
Whole cell lysates of nonstimulated cells and TPA-stimulated cells were
subjected to immunoblot analysis with anti-phosphotyrosine mAb.
Stimulation was performed by adding 10 nM TPA for 20 min. The
positions of the molecular markers are shown to the left in
kDa. The arrowhead indicates the position of 80 kDa. C, tyrosine phosphorylation of cortactin in wild type and
Syk(K
) expressing cells. a, cortactin before
and after stimulation in these cells was immunoprecipitated, followed
by immunoblot analysis with anti-phosphotyrosine mAb. b, each
immunoprecipitated sample was subjected to immunoblot analysis with
anti-cortactin mAb. The positions of cortactin and immunoglobulin heavy
chain are indicated.
) expressing
cells. As shown in Fig. 6A, both in K562 cells and
transformant expressing Syk(K
), autophosphorylation
of Src and phosphorylation of enolase were increased after addition of
TPA, suggesting that Syk activity does not affect TPA-induced
activation of Src. The amount of precipitated Src did not differ in
each sample (Fig. 6B). In the same condition for
detecting the association of cortactin with Syk, its association with
Src could not be detected (data not shown).
)
expressing cells were stimulated by 10 nM TPA for the
indicated times. Src was immunoprecipitated by anti-Src mAb, and in
vitro kinase assay was performed with enolase as an exogenous
substrate. B, each immunoprecipitated sample was subjected to
immunoblot analysis with anti-Src mAb. The positions of Src, enolase,
and immunoglobulin heavy chain are
indicated.
substrate. It
became phosphorylated on tyrosine residues in v-Src-transformed chicken
embryo (CE) cells(37) . In normal cells, cortactin is known to
be tyrosine-phosphorylated by various stimulants including growth
factors and
thrombin(23, 38, 39, 40) . Since Src
is recruited to growth factor receptors possessing tyrosine kinase in
those cytoplasmic domains by SH2 domain/phosphotyrosine interactions
and consequently activated, it was proposed that cortactin is
phosphorylated by this activated Src(40) . However, it has been
noted previously that cortactin could not be detected in Src
immunoprecipitates, and involvement of other tyrosine kinase(s) in
tyrosine phosphorylation of cortactin has been
suggested(37, 41, 42, 43) . In
platelets, tyrosine phosphorylation of cortactin and activation of Syk
were induced by thrombin treatment, although a direct correlation
between these events was not clarified. We show here a possibility that
cortactin is a substrate of Syk upon TPA stimulation by the following
evidence. 1) Syk is activated by TPA stimulation, and, following the
activation of Syk, cortactin is tyrosine-phosphorylated. 2) Expression
of dominant-negative Syk abolishes the TPA-induced tyrosine
phosphorylation of cortactin. 3) Cortactin is associated with Syk even
before TPA stimulation. Since Src is also activated by TPA stimulation,
it is possible that cortactin is a direct substrate of Src in
TPA-treated K562 cells. However, in contrast to the association of
cortactin with Syk, we could not detect the association of cortactin
with Src, suggesting that it is unlikely that Src phosphorylates
cortactin directly.
)), kinase-negative Syk;
SH2, Src homology 2.
We thank Dr. M. Asahi for 4G10. We also thank Dr. T.
Nogochi for helpful advice and K. Sakai for her skillful secretarial
assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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