From the
Aurin tricarboxylic acid (ATA), a general nuclease inhibitor,
was reported to prevent PC12 cells from cell death caused by serum
starvation(1) . In our study, ATA also protected PC12 cells, but
not NIH3T3 cells, from serum-starved cell death. When we investigated
the mechanism of action of ATA on these cells, ATA was found to
increase tyrosine phosphorylation in PC12 cells, but not in NIH3T3
cells. Further investigation on tyrosine-phosphorylated proteins
revealed that ATA, similar to nerve growth factor and epidermal growth
factor, induced tyrosine phosphorylation of mitogen-activated protein
kinases. Since the tyrosine phosphorylation of mitogen-activated
protein kinases is thought to play an important role in growth
factor-dependent signal pathways, this finding suggests that the action
of ATA on PC12 cells is mediated by tyrosine phosphorylation cascade,
similar to growth factor signaling. In addition, we found that Shc
proteins, phosphatidylinositol 3-kinase, and phospholipase C-
PC12 cells, derived from rat pheochromocytoma, are widely used
as a model system to study the effects of neurotrophic factors,
especially those of NGF.
The PC12 cell death under serum-free condition is accompanied
by one of the apoptotic properties, DNA
fragmentation(1, 2) . The protective effect of NGF is
mediated by the activation of p140
ATA can
prevent the PC12 cell death caused by serum and NGF starvation (1).
ATA, which is a general endonuclease inhibitor, has been thought to
inhibit the internucleosomal DNA cleavage during the
apoptosis(1) . ATA can also protect cultured sympathetic neurons
from cell death caused by the absence of trophic supports(2) . In vivo, ATA protects hippocampal neurons from NMDA- and
ischemia-induced death(3) . In addition, ATA has been reported
to regulate the expression of
ATA is also known to inhibit macromolecule
synthesis(5, 6, 7, 8) , nucleic
acid-protein interaction(9) , and activities of other enzymes
involved in cellular metabolism, for example glucose-6-phosphate
dehydrogenase (5) and phosphofructokinase(10) . However, these
inhibitory activities of ATA were defined in vitro. It is not
clear whether ATA is able to penetrate the plasma membrane to exert
these activities.
In this study, investigating the possibility that
ATA affected the growth factor signaling pathway, we found that ATA
could stimulate most of major signal transduction cascades which are
stimulated by NGF and EGF in PC12 cells.
Initially, we
examined whether ATA induces tyrosine phosphorylation of MAPKs/ERKs,
which proteins are known to be phosphorylated at their tyrosine and
threonine residues in response to a variety of growth factors,
including NGF and
EGF(11, 12, 13, 14, 15) . The
phosphorylated MAPKs were detected by anti-MAPK immunoblotting in
anti-phosphotyrosine immunoprecipitates of cell lysate. The
phosphorylated MAPKs, both of MAPK1 (44 kDa) and MAPK2 (42 kDa), were
clearly increased in the immunoprecipitates prepared from the cells
treated with ATA as well as NGF or EGF, as compared with those from
non-treated cells (Fig. 4). This result shows that ATA induced
the phosphorylation of tyrosine residues on MAPKs in PC12 cells.
We investigated whether Shc
proteins were also phosphorylated in the cells stimulated by ATA.
Lysates from PC12 cells treated with ATA, NGF, and EGF, for 5 min, were
immunoprecipitated with anti-Shc antibody. The immunoprecipitates were
immunoblotted with anti-phosphotyrosine or anti-Shc antibody. The
anti-Shc antibody immunoprecipitated three Shc proteins,
p46
We
examined whether ATA induced the phosphorylation of PI 3-kinase.
Lysates from cells treated with ATA, EGF, or NGF, was
immunoprecipitated with anti-PI 3-kinase antibody and the
immunoprecipitates were immunoblotted with anti-phosphotyrosine
antibody. In cells treated with ATA for 1 min, similar to those treated
with NGF or EGF, two tyrosine-phosphorylated proteins, whose molecular
masses were 100 kDa and 110 kDa, were detected. In the cells treated
with ATA for 5 min, these two proteins were more phosphorylated than in
the 1-min treated cells (Fig. 6A, upper panel).
It has been reported that the p100 and p110 were co-precipitated with
p85 in anti-PI 3-kinase immunoprecipitates of NGF- or EGF- treated PC12
cells(26) . The p100 and p110 are thought to be p85-binding
proteins and mediate the activation of PI 3-kinase by treatment with
NGF and EGF(26) . In cells treated with ATA for 5 min, and in
cells treated with NGF, or EGF for 1 min, two phosphorylated proteins
were detected around 50-60 kDa. These may be
pp52
As already reported by Batistatou and Greene(1) , ATA
promotes survival of serum-starved PC12 cells and inhibits the DNA
fragmentation which occurs during serum-starved cell death. ATA has
been thought to suppress the internucleosomal cleavage occurring in
apoptosis by inhibiting endonuclease activity(2, 32) .
In NIH3T3 cells, however, ATA did not prevent either the
internucleosomal cleavage or the cell death caused by serum starvation.
These facts suggest that the action of ATA have a selectivity between
these two types of cells. Batistatou and Greene have reported that ATA
rescued sympathetic neurons as well as PC12 cells from serum-starved
cell death(1) . This suggested to us that the survival action of
ATA may be selective to PC12 cells or neuronal cells. However, to
conclude the neuronal specificity of the survival action of ATA, it
will be necessary to investigate the effect on other varieties of cell
types.
The correlation between the induction of tyrosine
phosphorylation and the survival action of ATA in PC12 and NIH3T3 cells
indicates that tyrosine phosphorylation is the signal mediating the
survival action of ATA. This is further supported by the fact that the
survival action of neurotrophic factors is dependent on the activation
of a member of Trk family. However, we found that ATA did not
stimulate NGF or EGF receptor autophosphorylation in PC12 cells (data
not shown). In addition, the profile of tyrosine-phosphorylated
proteins in PC12 cells stimulated by ATA was different from that by NGF
or EGF (Fig. 3B). Furthermore, ATA was not able to
stimulate differentiation or proliferation of PC12 cells (data not
shown). These three observations indicate that the survival action of
ATA may share some parts of signals related to the survival with the
pathway that is also stimulated by growth factors. However, ATA-induced
survival is not directly mediated by the stimulation of receptors for
NGF or EGF.
MAPKs are important components of the signal
transduction pathway in the differentiation, proliferation, and trophic
response of cells and are known to be phosphorylated on tyrosine
residues in PC12 cells in response to NGF or EGF. The observation that
ATA also stimulates the tyrosine phosphorylation of MAPKs in PC12 cells
indicates the presence of a common pathway between ATA and growth
factors and suggests that the pathway may be responsible for the
survival activity of ATA.
In signal transduction stimulated by
growth factors, the activation of p21
ATA protects cultured sympathetic neurons
from cell death induced by the deprivation of serum and NGF(1) .
The mechanisms of this action of ATA on neuronal cells are still
unclear; however, there is a possibility that the receptor that can be
activated by ATA in PC12 cells is expressed on sympathetic neurons.
It has been reported that ATA protects hippocampal neurons from
NMDA- and ischemia-induced toxicity in vivo(3) . The
NMDA antagonistic activity of ATA may account for the protective effect
of ATA on this ischemia-induced toxicity(40) . However, growth
factors, such as brain-derived neurotrophic factor, are also reported
to protect neurons from NMDA- or ischemia-induced cell
death(43) . Therefore, the effect of ATA in vivo may
also explained by stimulation of tyrosine kinase pathways. Further
experiments are necessary to correlate the tyrosine kinase activation
and neuroprotection by ATA in vivo.
ATA can form
heterogeneous polymers, and some of the effects of ATA, such as
anti-human immunodeficiency virus activity (44) and inhibition
of protein-nucleic acid interaction(45) , are reported to
increase with the molecular mass. It remains to be determined which
molecular mass species of ATA can act on PC12 cells.
Here, we showed
that the MAPKs, Shc proteins, PI 3-kinase, and PLC-
We are grateful to Dr. B. Goldsmith for reading this
manuscript. We also thank K. Bessho for excellent technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
were
also phosphorylated in ATA-treated PC12 cells. These key proteins in
signal transduction pathways are known to associate with
ligand-activated growth factor receptors and are phosphorylated on
tyrosine. Thus, the phosphorylation of these three proteins by ATA
stimulation supports the speculation that ATA activates a certain
receptor tyrosine kinase.
(
)Recent studies
revealed that PC12 cells provide a good model system to study not only
neuronal differentiation but also neuroprotection from cell death by
NGF. Without trophic supports, PC12 cells will undergo apoptotic cell
death.
, a NGF
receptor having protein tyrosine kinase activity, and the subsequent
stimulation of cellular signal transduction pathways.
-amyloid precursor protein in
neuroblastoma(4) . Although a variety of mechanisms are proposed
for the neuroprotective effect of ATA, all of this evidence indicates
that ATA is a very interesting compound in terms of neuroprotective
activity.
Materials
The MTT cell growth assay kit was
purchased from Chemicon International Inc. (Temecula, CA). Monoclonal
anti-MAPK (ERK1+2) antibody was from Zymed (San Francisco, SF),
polyclonal anti-SHC antibody was from Transduction Laboratories
(Lexington, KY), and monoclonal anti-phosphotyrosine antibody (4G10),
polyclonal anti-PI 3-kinase antibody, and monoclonal anti-PLC-
antibody were from Upstate Biotechnology, Inc. (Lake Placid, NY).
Anti-mouse Ig and anti-rabbit Ig horseradish peroxidase-linked F(ab`)2
fragments, and ECL Western blotting detection kit were from Amersham
(Buckinghamshire, United Kingdom). Protein G-Sepharose beads were from
Pharmacia LKB (Uppsala, Sweden). PC12 cells were generously provided by
Dr. Gordon Guroff, National Institutes of Health. NIH3T3 cells were
obtained from ATCC (Rockville, MD). ATA was from Sigma, EGF and bFGF
were from Upstate Biotechnology, Inc. Recombinant human NGF was
prepared with baculovirus expression system in insect cells and
purified before use. DMEM and supplements were obtained from Life
Technologies, Inc.
Cell Culture
PC12 cells were grown in 100- or
60-mm dishes in DMEM with 10% horse serum and 5% fetal bovine serum.
NIH3T3 cells were grown in 60-mm dishes in DMEM with 10% fetal bovine
serum. The cells were maintained at 37 °C in a humidified
atmosphere with 5% CO.
Analysis of DNA Fragmentation
PC12 cells
(approximately 5 10
cells) or NIH3T3 cells
(approximately 1
10
cells) were incubated at 37
°C for indicated time under the experimental conditions. After the
treatment, cells were harvested mechanically with a rubber policeman
and collected by centrifugation at 650
g for 10 min at
4 °C. Cells were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5% Triton X-100) and kept
on ice for 10 min. After centrifugation at 27,000
g for 10 min, the soluble DNA was isolated and extracted with
TE-saturated phenol and phenol/chloroform (1:1), followed by
ethanol-precipitation. The DNA was dissolved in TE buffer and incubated
with RNase A at 37 °C overnight. All recovered soluble DNA per
condition were subjected to 1.5% agarose gel electrophoresis and
visualized by a UV transilluminator.
MTT Assay
The MTT assay was performed according to
supplied procedures with some modification. The cells (approximately 5
10
cells/well) were cultured in 96-well culture
plates to subconfluence in 50 µl of medium/well. After treatment of
cells under experimental conditions, 10 µl of MTT solution was
added to each well, and the plates were incubated at 37 °C for
another hour in a CO
incubator. Next, 100 µl of 0.04 N HCl/isopropanol was added to each well and the plates were
shaken to dissolve the crystals of the tetrazolium salt. The results
were quantified by measuring the absorbance at 570 nm with 650 nm as a
reference.
Anti-phosphotyrosine Immunoblot of Whole Cell
Lysates
Cells were grown in 60-mm dishes, and the medium was
replaced with serum-free medium overnight. Unless otherwise stated,
ATA, NGF, EGF, or bFGF was added directly to the medium and incubated
for 5 min, to a final concentration of 100 µM, 100 ng/ml,
20 ng/ml, or 10 ng/ml, respectively. The medium was then removed, and
cells were lysed with 200 µl of SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS w/v, 10% glycerol) containing 1 mM orthovanadate, a tyrosine phosphatase inhibitor. The lysates were
sonicated for about 10 s to degrade the DNA. After determination of
protein concentration in each preparation with BCA protein assay
reagent (Pierce) and boiling with 5% of -mercaptoethanol, 25
µg of protein was applied on 8% SDS-PAGE. After transferring to a
nitrocellulose membrane, blots were blocked with 5% bovine serum
albumin, then probed with 0.5 µg/ml anti-phosphotyrosine antibody
(4G10) for 1 h. After repeating the 5-min wash four times with washing
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2%
Nonidet P-40), blots were incubated with peroxidase-labeled anti-mouse
Ig antibody at 1:2000 dilution for 1 h, followed by washing as
described above. The resultants were visualized using ECL Western
blotting detection kit by exposure to x-ray films (Kodak).
Immunoprecipitation
Cells grown in 100-mm dishes
were replaced with serum-free medium overnight. Unless otherwise
indicated, ATA, NGF, or EGF was directly added to the medium and
incubated for 5 min, to a final concentration of 100 µM,
100 ng/ml, or 20 ng/ml, respectively. After removing the medium, cells
were lysed with 500 µl of ice-cold TNE buffer (10 mM
Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, 10 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, 1 mM orthovanadate) on ice
for 10 min and the lysates centrifuged at 12,000 g for
10 min. For immunoprecipitation with anti-phosphotyrosine antibody, the
protein content in each supernatant was normalized in each experiment.
The supernatants were rotated at 4 °C for 2 h with 20 µl of 50%
(v/v) protein G-Sepharose beads. After the proteins nonspecifically
bound to protein G were removed by centrifugation (1500
g, for 3 min), 5 µg of a primary antibody was added to
supernatants and rotated at 4 °C for 2 h. Then 20 µl of protein
G-Sepharose beads were added and incubated for 2 h at 4 °C while
rotating. The immunocomplexes bound to the beads were washed 5 times
with 500 µl of TNE buffer and collected by centrifugation. For
immunoprecipitation with anti-phosphotyrosine antibody, the
tyrosine-phosphorylated proteins bound to the antibody were eluted by
rotating for 2 h at 4 °C with 20 µl of 100 µM phenylphosphate in TNE buffer, and 5 µl of 5
SDS-PAGE
sample buffer containing 25%
-mercaptoethanol (v/v) was added to
the eluted solution. For immunoprecipitation with other antibodies, the
proteins were solubilized in 20 µl of SDS-PAGE sample buffer. The
samples were boiled after addition of
-mercaptoethanol, to a final
concentration of 5% (v/v), and loaded to SDS-polyacrylamide gel. The
immunoblotting with the indicated antibody was performed as described
as above. For reprobing the blots, the membrane was incubated for 30
min in 62.5 mM Tris-HCl buffer, pH 6.7, containing 2% of SDS
and 10 mM
-mercaptoethanol, blocked, and reprobed with
the indicated antibody.
ATA Supports the Survival of PC12 Cells under Serum
Starvation, but Not That of NIH3T3 Cells
ATA has been reported
to prevent PC12 cells from death caused by serum
deprivation(1) . We have confirmed the result by the MTT assay.
Under serum-free conditions for 72 h, PC12 cells cultured in the
presence of 100 ng/ml ATA showed a comparable survival rate to that in
the presence of 100 ng/ml NGF (Fig. 1A). In the presence
of 100 µM NGF, no sign of cell death was found by
microscopic observation of cells under serum-free condition. The higher
formation of MTT in the cells with serum than that with NGF or ATA is
due to the serum-dependent proliferation of PC12 cells and not due to
the partial cell death in the presence of NGF or ATA. Internucleosomal
DNA fragmentation, which is a characteristic of apoptosis, was shown in
the cell death of PC12 cells caused by serum starvation(2) .
This DNA fragmentation was clearly blocked by ATA as well as NGF after
12 h incubation (Fig. 1B). On the other hand, ATA did
not show any survival effect on mouse fibroblast NIH3T3 cells cultured
in serum-free medium for 24 h (Fig. 2A). In addition,
ATA did not block the DNA fragmentation in NIH3T3 cells which was also
observed under serum-free conditions, whereas bFGF completely blocked
it (Fig. 2B). These results indicated that the survival
action of ATA has cell selectivity.
Figure 1:
Effect of ATA on the cell death caused
by serum starvation in PC12 cells. A, after cultured in
indicated conditions for 72 h, the cell viabilities were measured by
MTT assay as described under ``Experimental Procedures.'' The
numbers of cell viability are expressed relative to those cultured in
serum-supplemented medium (designated as 100%). Experimental data are
mean ± S.D. (n = 6). B, PC12 cells were
cultured in serum-supplemented medium (+serum), or
serum-free medium (-) with 100 ng/ml NGF
(+NGF) or 100 µM ATA (+ATA)
for 12 h. The soluble DNA was isolated and analyzed as described under
``Experimental Procedures.'' Molecular size markers (in
kilobase pairs) are indicated on the left.
Figure 2:
Effect of ATA on the cell death caused by
serum starvation in NIH3T3 cells. A, after cultured in
indicated conditions for 24 h, the cell viabilities were measured by
MTT assay as described under ``Experimental Procedures.'' The
numbers of cell viability are expressed relative to those cultured in
serum-supplemented medium (designated as 100%). Experimental data are
means ± S.D. (n = 6). B, cells were
cultured in serum-supplemented medium (+serum), or in
serum-free medium (none) with 10 ng/ml bFGF
(+bFGF), or 100 or 200 µM ATA
(+ATA) for 12 h. The soluble DNA was isolated and
analyzed as described under ``Experimental Procedures.''
Molecular size markers (in kilobase pairs) are indicated on the left.
ATA Increases Tyrosine Phosphorylation in PC12
Cells
It is known that tyrosine phosphorylation cascade plays an
important role in the action of growth factors and trophic factors.
Therefore, to determine whether ATA affects tyrosine phosphorylation of
any protein in PC12 cells, we examined the profile of
tyrosine-phosphorylated proteins in the lysate of PC12 cells treated
with 100 µM ATA. After 5 min of treatment of cells, ATA
clearly increased the tyrosine phosphorylation of several proteins,
mainly 180-, 130-, 85-, and 60-kDa proteins, and the phosphorylation of
each protein declined in 60 min (Fig. 3A). The profile
of tyrosine-phosphorylated proteins of ATA-treated cells were different
from that of NGF- or EGF-treated cells (Fig. 3B). In
NIH3T3 cells, which ATA does not prevent from serum-starved cell death,
bFGF increased the tyrosine phosphorylation of several proteins.
However, ATA treatment did not increase tyrosine phosphorylation (Fig. 3C). The correlation between the survival response
and the stimulation of tyrosine phosphorylation in PC12 cells and
NIH3T3 cells suggests that the survival action of ATA on PC12 cells is
mediated by the stimulation of tyrosine phosphorylation.
Figure 3:
Effects of ATA on tyrosine phosphorylation
in PC12 cells (A and B) and in NIH3T3 cells (C). Whole cell lysates were subjected to 8% SDS-PAGE and
immunoblot with an anti-phosphotyrosine antibody as described under
``Experimental Procedures.'' A, PC12 cells treated
with 100 µM ATA for indicated times; B, PC12
cells treated with 20 ng/ml EGF, 100 ng/ml NGF or ATA for 5 min; C, NIH3T3 cells treated with ATA or 10 ng/ml bFGF for 5 min.
The arrowheads on the right indicate the major
phosphorylated proteins by ATA (A) and by bFGF (C).
Molecular mass markers (in kDa) are indicated on the left.
Tyrosine Phosphorylation of MAPK
If the increased
tyrosine phosphorylation mediates the survival action of ATA on PC12
cells, it is quite possible that ATA stimulates the phosphorylation of
proteins phosphorylated following treatment with other growth factors
such as NGF or EGF. Therefore, we further investigated the
phosphorylation of proteins that are involved in the signal
transduction pathway of peptide growth factors.
Figure 4:
Tyrosine phosphorylation of MAPKs in PC12
cells treated with ATA, EGF, and NGF. The cells were treated with 100
µM ATA, 20 ng/ml EGF, and 100 ng/ml NGF, for 5 min. Then
the cell lysates after normalized protein content were
immunoprecipitated with an anti-phosphotyrosine antibody. The
immunocomplexes precipitated with protein G-Sepharose beads were
subjected to 8% SDS-PAGE, followed by immunoblot with an anti-MAPK
antibody as described under ``Experimental Procedures.'' The
positions of MAPKs are indicated by arrowheads on the right. Molecular mass marker is indicated on the left.
Shc Proteins Are Phosphorylated by ATA as Well as by NGF
and EGF
The activation of MAPKs by growth factors is considered
to be mediated through p21-GAP activation.
Recently, Shc proteins have been found to play a role upstream of
p21
activation by NGF/EGF
stimulation(16, 17, 18) . Shc proteins associate
with the tyrosine-autophosphorylated receptors and are phosphorylated
on their tyrosine residues(19) .
, p52
, and
p66
(Fig. 5, rightpanel), and the Shc proteins were phosphorylated on
tyrosine in response to each of ATA, NGF, and EGF (Fig. 5, leftpanel). The p66
was
phosphorylated most by EGF treatment, and least by ATA treatment. The
finding that the phosphorylation of Shc proteins is induced by ATA
suggests that the phosphorylation of MAPKs in PC12 cells stimulated by
ATA is mediated by a pathway similar to that activated by NGF or EGF.
Figure 5:
Tyrosine phosphorylation of Shc proteins
in PC12 cells treated with ATA, EGF, and NGF. The cells were treated
with 100 µM ATA, 20 ng/ml EGF, and 100 ng/ml NGF, for 5
min. All lysates were immunoprecipitated with an anti-Shc antibody and
analyzed by 8% SDS-PAGE, followed by immunoblot with an
anti-phosphotyrosine antibody (leftpanel). The
membrane after ECL detection was stripped of bound antibodies and
blotted with an anti-Shc antibody (rightpanel). All
procedures were performed as described under ``Experimental
Procedures.'' The positions of Shc proteins are indicated with arrowheads.
ATA Stimulates the PI 3-Kinase Pathway
The PI
3-kinase is phosphorylated on tyrosine by a number of receptor tyrosine
kinases (20, 21). Recent studies indicate that the 85-kDa subunit (p85)
of PI 3-kinase, similar to Shc proteins, forms a stable association
with the growth factor receptors and is phosphorylated on
tyrosine(22, 23, 24, 25) .
and pp58
, which are
thought to be associate with p85 via receptors(19) . The
increase in the amount of tyrosine-phosphorylated p85 was directly
detected in PC12 cells treated with ATA by anti-phosphotyrosine
immunoprecipitation followed by anti-PI 3-kinase immunoblotting (Fig. 6B). As shown in Fig. 3B, a major
tyrosine-phosphorylated protein was observed at a molecular mass
position similar to that for p85 of PI 3-kinase when the cells had been
stimulated by ATA. However, it was thought to be a different protein
from p85 of PI 3-kinase, because the dominant phosphorylation of the
85-kDa protein was detected in only ATA-treated cells, but neither in
NGF- nor in EGF-treated cells.
Figure 6:
Tyrosine phosphorylation of p85 of PI
3-kinase and p85-binding proteins in PC12 cells treated with ATA, EGF,
and NGF. A, the cells were treated with 100 µM ATA, 20 ng/ml EGF, and 100 ng/ml NGF, for indicated times. The
cell lysates were immunoprecipitated with an anti-PI 3-kinase antibody.
The immunocomplexes were analyzed by 8% SDS-PAGE followed by immunoblot
with an anti-phosphotyrosine antibody (upperpanel).
After stripping, the membrane was reblotted with an anti-PI 3-kinase
antibody (lowerpanel). B, cells treated
with 100 µM ATA or non-treated were lysed. After
normalization of protein content, the cell lysates were
immunoprecipitated with an anti-phosphotyrosine antibody. The
immunocomplexes were subjected to 8% SDS-PAGE, followed by
immunoblotting with anti-PI 3-kinase antibody (lowerpanel). After stripping the membrane was reblotted with
an anti-phosphotyrosine antibody (upperpanel). All
procedures were performed as described under ``Experimental
Procedures.'' The position of p85 of PI 3-kinase is indicated by arrowheads in lower panels. Molecular mass markers are
indicated on the right and left.
PLC-
PLC- Was Also Tyrosine-phosphorylated by
ATA
can also stably associate with receptors in a
growth factor-dependent fashion via the SH2 domain and is
phosphorylated on tyrosine by a number of receptor tyrosine kinases
(27-31). We also investigated tyrosine phosphorylation of
PLC-
in response to ATA. The tyrosine phosphorylation of PLC-
(148 kDa) in ATA-treated PC12 cells was detected by
anti-phosphotyrosine immunoprecipitation of cell lysates followed by
anti-PLC-
immunoblotting (Fig. 7). The tyrosine
phosphorylation of PLC-
was increased by ATA treatment, though the
phosphorylation was less than that induced by NGF and EGF treatment.
Figure 7:
Tyrosine phosphorylation of PLC- in
PC12 cells treated with ATA, EGF, and NGF. The cells were treated with
100 µM ATA, 20 ng/ml EGF, and 100 ng/ml NGF, for 5 min.
The cell lysates after normalized protein content were
immunoprecipitated with an anti-phosphotyrosine antibody. The
immunocomplexes were analyzed by 8% SDS-PAGE followed by immunoblot
with an anti-PLC-
antibody as described under ``Experimental
Procedures.'' The position of PLC-
is indicated by an arrowhead on the right. Molecular mass markers are
indicated on the left.
takes
place between the activations of receptor and MAPKs. The Shc proteins
reside in the signal pathway right after the receptor tyrosine kinase
and upstream of the activation p21
(33). Shc
proteins associate with the tyrosine-phosphorylated receptors via their
SH2 domains and are themselves phosphorylated on tyrosine residues. The
Shc proteins can also associate with growth factor receptor-bound
protein 2 (Grb2), which contains SH2 and SH3 domains (34). This
association of Shc proteins with Grb2 mediates the activation of
p21
in response to growth factors via Son of
Sevenless (Sos), the Ras nucleotide exchange
factor(19, 35, 36, 37, 38, 39) .
The observation that tyrosine phosphorylation of Shc proteins is
induced by ATA, as well as NGF and EGF, indicates that the same pathway
is stimulated by both ATA and the growth factors and results in the
tyrosine phosphorylation of MAPKs. This also suggests that ATA
activates a protein tyrosine kinase that behaves like a receptor for
growth factors. In addition, the increase in the tyrosine
phosphorylation of p85 subunit of PI 3-kinase and PLC-
by ATA
treatment further supports the existence of such a tyrosine kinase,
because both of them are known to associate with activated growth
factor receptors through their SH2 domains. Since it is unclear whether
ATA penetrates the plasma membrane or not, we cannot assert whether the
activation of the signal cascade is triggered by the binding of ATA to
a receptor or by action of intracellular ATA on a tyrosine kinase.
However, the other actions of ATA on cells, which are antagonizing both
NMDA binding on NMDA receptor (40) and human immunodeficiency
virus binding on CD4(41, 42) , suggest that the site of
the action of ATA may be at the surface of cells rather than on
endonuclease in nucleus.
were
phosphorylated on tyrosine by ATA treatment, each of which was also
phosphorylated by growth factor stimulation. However, the major
proteins phosphorylated by ATA treatment (180, 130, 85, and 60 kDa)
have not been identified yet. This suggests that the survival action of
ATA on PC12 cells is mediated by tyrosine phosphorylation cascade,
similar to the action of growth factors. Our results are not clear
enough to demonstrate that the survival action of ATA on PC12 cells
depends on the ATA-induced tyrosine phosphorylation that is shown here.
However, it supports the idea that ATA activates a certain growth
factor receptor tyrosine kinase and triggers the same signal pathway.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.