From the Division of Neuroscience, Institute of
Biomedical Sciences, Academia Sinica,
Taipei 11529, Taiwan, Republic of China, the
§ Department of Neurology, University of California,
San Francisco, California 94608, and the Ernest Gallo Clinic
and Research Center, Emeryville, California 94608
Received for publication, September 20, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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We found in the present study that stimulation
of A2A adenosine receptors (A2A-R)
prevents apoptosis in PC12 cells. This A2A-protective effect was blocked by protein kinase A (PKA) inhibitors and was not
observed in a PKA-deficient PC12 variant. Stimulation of PKA also
prevented apoptosis, suggesting that PKA is required for the protective
effect of A2A-R. A general PKC inhibitor, but not down-regulation of conventional and novel PKCs, readily blocked the
protective effect of A2A-R stimulation and PKA activation, suggesting that atypical PKCs (aPKCs) serve a critical role downstream of PKA. Consistent with this hypothesis, stimulation of
A2A-R or PKA enhanced nuclear aPKC activity. In addition,
the A2A-protective effect was blocked by a specific
inhibitor of one aPKC, PKC Adenosine, which is released from metabolically active cells by
facilitated diffusion or is generated extracellularly by degradation of
released ATP, is a potent biological mediator (1). It is well known
that adenosine modulates the activity of numerous cell types including
various neuronal populations, platelets, neutrophils, and smooth muscle
cells (1). To date, four adenosine receptors (A1,
A2A, A2B, and A3) have been
identified. These receptors all contain seven transmembrane domains and
belong to the G protein-coupled receptor
(GPCR)1 family (2). We
previously cloned the cDNA and the gene for the rat A2A
adenosine receptor (A2A-R; see Refs. 3 and 4). In the
central nervous system, the rat A2A-R gene is heavily
expressed by striatal neurons and colocalizes with the D2
dopamine receptor in GABAergic striopallidal neurons (5). Low
level A2A-R expression is also observed in the cortex,
hippocampus, cerebellum, and other areas of the brain (6). Importantly,
A2A-R has been regarded as a potential therapeutic target
in protecting against neurodegeneration (e.g. Parkinson's
disease and Huntington's disease; see Refs. 7 and 8) and neuronal
trauma (e.g. hypoxia/ischemia; see Ref. 9). Moreover,
stimulation of A2A-R delays apoptosis in human neutrophils
(10) and protects the hippocampus from excitotoxicity in a model of
kainate-induced neuronal cell death (11). The molecular mechanisms
underlying the protective effect of adenosine acting at
A2A-R remain largely uncharacterized in neuronal cells.
The rat pheochromocytoma cell line PC12 displays phenotypic traits
associated with both adrenal chromaffin cells and sympathetic neurons
and is a useful model for studying the actions of neurotrophic factors
and neurotransmitters. In the past decade, this cell line has also
served as a popular model system for studying the functions of various
survival factors, including nerve growth factor (NGF; see Ref. 12). We
previously demonstrated that stimulation of A2A-R increases
intracellular cAMP formation and activates novel protein kinase C
isozymes in PC12 cells (13, 14). In the present study, we found that
activation of A2A-R prevents apoptosis in serum-deprived
PC12 cells. This protective mechanism involves transient enhancement of
PKA activity and subsequent activation of atypical PKCs (aPKCs) and
requires the activity of a serine-threonine protein phosphatase
(PPase).
Reagents--
All reagents were purchased from Sigma except
where specified. Forskolin,
2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine (CGS 21680; CGS), 8-(3-chlorostyryl)caffeine (CSC), and okadaic acid
(OKA) were purchased from Research Biochemical, Inc. (Natick, MA).
DMEM, fetal bovine serum, and horse serum were purchased from Life
Technologies, Inc. Bisindolylmaleimide I-HCl (BiM), calyculin A (Caly
A), phorbol-12,13-didecanoate (PDD), and LY 294002 were purchased from
Calbiochem-Novabiochem. H-89 and KT-5720 were from Biomol (Plymouth
Meeting, PA). [ Cell Culture--
PC12 cells were maintained in DMEM
supplemented with 10% v/v horse serum and 5% v/v fetal bovine serum.
A123, a cAMP-dependent protein kinase (PKA)-deficient
variant of PC12 cells (15), was kindly provided by Dr. J. A. Wagner (Cornell University Medical College, New York). A123 cells were
maintained in DMEM supplemented with 5% v/v horse serum and 10% v/v
fetal bovine serum. Novel PKC-dominant-negative PC12 variants (16) were
maintained in DMEM supplemented with 10% v/v horse serum, 5% v/v
fetal bovine serum, and G418 (50 µg/ml).
DNA Fragmentation--
Cells were plated at the density of
3 × 106 cells per 100-mm plate. After 24 h,
cells were treated with the indicated reagent(s) for another 24 h
and harvested by centrifugation, resuspended in 100 µl of lysis
buffer (10 mM EDTA, 50 mM Tris-HCl, 0.5%
Sarkosyl, and 0.5 mg/ml proteinase K), and incubated at 50 °C for
3 h. RNase (2 mg/ml) was added to the lysate for another 15 h. The lysate was extracted with 200 µl of phenol/chloroform and then
centrifuged again for 5 min. DNA fragments present in the supernatant
were separated using a 2% agarose gel.
MTT Assay--
Cells grown on 150-mm plates were washed twice
with phosphate-buffered saline (PBS) and resuspended in DMEM. The
resuspended cells were plated on 96-well plates (1.5 × 104 cells/well) and treated with the indicated reagent(s)
for 24 h. MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was
then added to the medium (1 mg/ml), and cells were incubated at
37 °C for 3 h. Me2SO (100 µl) was then
applied to the medium to dissolve the formazan crystal derived from
mitochondrial cleavage of the tetrazolium ring of MTT. The absorbency
at 570 nm in each well was measured on a micro-enzyme-linked
immunosorbent assay plate reader. None of the reagents used in this
study interfered with the MTT values.
PKC Activity Assay--
PKC activity was measured as described
previously (14) with slight modifications. To measure atypical PKC
activity, PC12 cells were first treated with PDD (1 µM)
for 20 h to down-regulate conventional and novel PKCs. Cells were
then washed with twice DMEM and incubated with the indicated reagents
for the desired period of time. Different fractions of cells were then
collected as described below. PKC activity was measured in a 40-µl
reaction containing 136 mM NaCl, 5.4 mM KCl,
0.3 mM Na2HPO4, 0.3 mM
KH2PO4, 10 mM
Mg2SO4, 25 mM Isolation of Membrane, Cytosol, and Nuclear
Fractions--
Membrane, cytosol, and nuclear fractions were isolated
as described by Zhou et al. (18). Briefly, PC12 cells were
collected by centrifugation (1000 × g, 2 min),
resuspended, and incubated in 1 ml of PKC sonication buffer (2 mM Tris, pH 7.6, 50 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 100 µM leupeptin, 10 µM aprotinin, and 1 mM NaF) at room temperature for 2 min, and chilled on ice for 5 min. Nonidet P-40 was added to 1% (v/v) final concentration. Samples were forced once through a 20-gauge needle and then
MgCl2 was added to 5 mM. The samples were
centrifuged at 600 × g for 5 min to collect the
nuclear fractions in the pellets. The supernatants were collected as
the non-nuclear fraction or were further centrifuged at 100,000 × g for 45 min to separate the cytosol and membrane fractions.
The pellets (i.e. the membrane fractions) were resuspended in 300 µl of PKC sonication buffer containing 0.1% Triton X-100. The
nuclear fractions were resuspended in 150 µl of buffer C (20 mM HEPES, pH 8, 425 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 80 µg/ml PMSF, 1 mM NaVO4, 20 mM NaF, 100 nM okadaic acid, and 25% glycerol)
and incubated for 30 min on ice. The nuclear samples were centrifuged
at 3,000 rpm for 10 min at 4 °C to collect nuclear extracts in the
supernatants. Protein concentrations were measured using the Bio-Rad
Protein Assay Dye Reagent.
Western Blot Analysis--
PC12 cells were rinsed with ice-cold
PBS and lysed in ice-cold lysis buffer (20 mM HEPES, 1 mM dithiothreitol, 20 mM EGTA, 10% glycerol,
50 mM Transfection and Cell Viability Determinations--
All plasmids
used in transient transfection experiments were prepared by CsCl
purification. Cells were transfected using TfxTM (Promega),
following the manufacturer's protocol, and then harvested between 48 and 72 h post-transfection. Transfection efficiency was typically
between 10 and 15%. For survival analyses, cells were transiently
transfected with a control vector or with vectors encoding the gene of
interest along with one-seventh of the molar amount of an expression
construct (pEGFP, CLONTECH; Palo Alto, CA) encoding
green fluorescent protein (GFP), as indicated. Two days
post-transfection, cells were subjected to serum deprivation for
24 h. Transfected cells were identified by GFP expression. Survival was determined as percentage of GFP-expressing cells by
counting GFP-expressing cells in photomicrographs taken using a
fluorescent microscope and normalized to the total number of cells
counted from the corresponding photomicrographs of cells examined by
phase contrast. An average of 1500 to 4000 total cells was counted for
each experimental condition. Alternatively, GFP-expressing cells were
quantified by flow cytometry as indicated below. These two methods
produced similar results for the percentage of GFP-expressing cells.
The survival index of 100% is designated as the percentage of
GFP-expressing cells transfected with a control vector under the
indicated treatment conditions.
Flow Cytometry--
PC12 cells were transiently transfected with
the indicated plasmid construct plus one-seventh the amount of GFP
vector. Forty eight hours post-transfection, serum was withdrawn for
24 h, and cells were analyzed for the expression of GFP by gently
removing the cells from plates with PBS containing trypsin (0.15%) and EDTA (0.53 mM) and then analyzing cell samples by flow
cytometry with a Becton Dickinson FACScan. The transfected cells were
identified by the expression of GFP that was detected using the FL-1
channel (excitation, 488 nm; emission, 530/30 nm). For each transfected plasmid, 30,000 cells were analyzed.
Statistics--
Unless indicated otherwise, results were
analyzed by one-way analyses of variance. Differences between means
were assessed by the Student-Newman-Keuls method and were considered
significant where p < 0.05.
Effects of an A2A-R-selective Agonist, CGS 21680, on
Serum-deprived Apoptosis--
Serum deprivation for 24 h resulted
in significant DNA fragmentation in PC12 cells (Fig.
1A). Serum deprivation also
decreased cell survival, as measured by the MTT assay (Fig.
1C). Addition of an A2A-R-selective agonist,
CGS21680 (CGS, 0.1 µM), reversed the DNA fragmentation
and cell death induced by serum deprivation. Addition of CGS (0.1 µM) also significantly reduced phosphorylation of the
stress-activated kinases JNK1 and JNK2 (Fig. 1B), which are
implicated in apoptosis (12). Such protection by A2A-R
required new protein synthesis, because a protein synthesis inhibitor
blocked the prevention of apoptosis by activation of A2A-Rs
in a dose-dependent manner (Fig. 1C).
The Role of PKA in the A2A-protective Effect in PC12
Cells--
Since activation of A2A-R led to a transient
increase in cAMP in PC12 cells (13), we first examined whether PKA
plays an important role in preventing apoptosis due to serum
deprivation. As shown in Fig.
2A, two PKA inhibitors (H-89
and KT-5720) reduced the protective effect of CGS and forskolin (FK) in
serum-deprived apoptosis. In addition, CGS and FK exerted no effect on
serum-deprived apoptosis in a PKA-deficient PC12 variant (A123, Fig.
2B), further supporting our hypothesis that PKA is critical
for the protective effect of A2A-R against apoptosis.
Furthermore, the effect of CGS was markedly reduced by an
A2A-R-selective antagonist, 8-(3-chlorostyryl)caffeine (CSC) (Fig. 2A). Thus, the effect of CGS is mediated by
A2A-Rs.
Phosphatases and PKC Mediate A2A-R-evoked
Protection--
Because stimulation of A2A-R activates a
serine/threonine PPase in neutrophils (19), we examined whether a PPase
was involved in prevention of apoptosis by A2A-R
activation. As shown in Fig. 3, two
serine/threonine PPase inhibitors, okadaic acid (OKA) and calyculin A
(Caly A), blocked the protective effect of A2A-R activation in a dose-dependent manner. Maximal inhibition of the
A2A-protective effect by Caly A and OKA occurred at 1 and
10 nM, respectively. Caly A and OKA at these concentrations
also markedly reduced the protective effect of FK (Fig. 3). Caly A (1 nM) or OKA (10 nM) alone did not markedly
affect the survival of PC12 cells in the absence or presence of serum.
Thus, a serine-threonine PPase appears to act downstream of PKA to
facilitate survival of serum-starved cells upon A2A-R
stimulation.
Activation of A2A-R has been shown to stimulate the
ERK/MAPK pathway in several cell types, including PC12 cells (20).
Therefore we examined whether this pathway is involved in the
protection against apoptosis by A2A-R activation. As shown
in Fig. 4A, treatment with CGS
or FK increased phosphorylation of ERK1/ERK2 without altering protein
levels. A MAPK kinase inhibitor (PD98059) blocked the FK- and
CGS-mediated activation of ERK. However, PD98059 did not prevent the
protective effect of CGS in serum-deprived cells (Fig. 4B).
Therefore, activation of ERK is not required for
A2A-R-mediated protection against apoptosis. This finding
is consistent with the observation that ERK is not important for cAMP-
or NGF-mediated survival of primary sympathetic neurons (21).
We previously showed that stimulation of A2A-Rs activates
novel PKCs (14). Therefore, we used a PKC inhibitor bisindolylmaleimide I-HCl (BiM) to examine whether PKC is involved in the protective effect
of A2A-R. As shown in Table
I, BiM markedly reduced the protective
effect of CGS and FK. PKC therefore might be involved in
A2A-R-mediated protection and exert its effect downstream
of PKA.
Atypical PKCs Mediate A2A-R Prevention of
Serum-deprived Apoptosis--
PKC is a family of serine/threonine
protein kinases that is composed of three subfamilies as follows:
conventional, novel, and atypical. We previously demonstrated that two
novel PKC isozymes (
We next considered whether aPKCs are important for the
A2-R-mediated protection by measuring aPKC activity after
A2A-R stimulation in PC12 cells treated with PDD for
20 h. In these cells, CGS enhanced aPKC activity in both nuclear
and non-nuclear fractions (Fig. 6,
A and B). The increase in aPKC activity was much
greater in the nuclear fraction as compared with the non-nuclear
fraction. Western blot analysis showed that PKC
PKC has been implicated in survival following serum deprivation in PC12
cells (22). Therefore, we used a cell-permeable PKC
To examine whether stimulation of A2A-Rs regulates PKC The PI3K/Akt Pathway Is Not Involved in the
A2A-R-mediated Protection against Apoptosis in
Serum-deprived PC12 Cells--
Data from the above experiments suggest
that PKC
We next examined whether A2A-R stimulation activates Akt,
one of the downstream targets of PI3K implicated in cell survival. We
examined the phosphorylation levels of the two activating residues (26), threonine 308 and serine 473 of Akt, by using Western blot
analysis (Fig. 9, B and C). Our data show that
NGF markedly increases Akt phosphorylation, whereas stimulation of
A2A-R does not. We next employed a kinase-dead Akt (dnAkt)
that contains a point mutation in its catalytic domain (K179M; see Ref.
27) to determine whether Akt is involved in the
A2A-R-protective effect. This K179M-Akt mutant is unable to
transmit a signal downstream and effectively decreases upstream signals
mediated by 3'-phosphorylated phosphoinositides and PDK1, which
activate Akt (28). Overexpression of dnAkt reduced NGF-mediated cell
survival during serum deprivation but did not alter CGS-mediated
survival (Fig. 9D). Collectively, these results indicate
that although the PI3K/Akt pathway plays a role in NGF-mediated
survival, it is not activated by A2A-R stimulation nor is
it required for the protective effect of A2A-R activation
in serum-starved PC12 cells.
In the present study, we found that A2A-R activation
protects PC12 cells from apoptosis induced by serum deprivation. This protective effect requires PKA activation, since it is blocked by two
different PKA inhibitors (H-89 and KT 5720), and is absent in
PKA-deficient PC12 cells. In contrast to NGF-mediated survival, A2A-mediated protection does not require activation of PI3K
or Akt. Although MAPK was activated by stimulation of
A2A-R, blocking the MAPK pathway did not alter
A2A-R-mediated protection against apoptosis. Compared with
the well characterized mechanisms involving ERK/MAPKs and PI3K
underlying NGF-mediated survival in PC12 cells, the results of the
present study demonstrate that A2A-R utilizes a distinct
set of signaling pathways to activate key downstream mediators
(e.g. PKC In PC12 cells, cyclic AMP has been implicated in mitosis, apoptosis,
and differentiation. Long lasting elevation of cellular cAMP, either by
treatment with cAMP analogs or FK, rescues PC12 cells from cell death
(29). Treatment of PC12 with cAMP analogs also eventually leads to
neuronal differentiation, but this requires exposure to these agents
for several days (30). Although activation of A2A-R
increases cAMP, this response is transient (13) and does not stimulate
neural differentiation of PC12
cells.2 Results in the
present study suggest that transient activation of the cAMP/PKA pathway
is sufficient to protect against cell death due to serum deprivation.
Our data also suggest that downstream of PKA, aPKCs mediate the
protective effect of A2A-R against apoptosis in PC12 cells. This conclusion is based on the following evidence. First, the protective effect of A2A-R can be reversed by a general PKC
inhibitor (BiM, Table I) and by a PKC The second line of evidence supporting a role for aPKCs in
A2AR-mediated survival comes from our observation that
stimulation of A2A-R increased nuclear aPKC activity and
the amount of nuclear PKC This protective effect of aPKCs is consistent with previous studies
suggesting that aPKCs are potent anti-apoptotic kinases (36). For
example, low concentrations of ceramide transiently activate PKC The involvement of the PI3K/Akt pathway in preventing apoptosis has
been well established (25). Various GPCRs activate Akt in either a
PI3K-dependent or -independent manner (39, 40). In
NGF-differentiated PC12 cells, PI3K plays a critical role in cell
survival (41). We considered whether PI3K is important for
A2A-mediated survival, since PKC, whereas overexpression of a
dominant-positive PKC
enhanced survival. In contrast, inhibitors of
MAP kinase and phosphatidylinositol 3-kinase did not modulate
the A2A-protective effect. Dominant-negative Akt also did
not alter the A2A-protective effect, whereas it
significantly reduced the protective action of nerve growth factor.
Collectively, these data suggest that aPKCs can function downstream of
PKA to mediate the A2A-R-promoted survival of PC12 cells.
Furthermore, the results indicate that different extracellular stimuli
can employ distinct signaling pathways to protect against apoptosis induced by the same insult.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was obtained from PerkinElmer
Life Sciences. Anti-active c-Jun N-terminal kinase (JNK) and MAPK
antibodies and TfxTM were purchased from Promega Co.
(Madison, WI). Anti-phospho-Akt (Thr-308) and (Ser-473) antibodies were
obtained from Upstate Biotechnology, Inc. (Lake Placid, NY), and Cell
Signaling (Beverly, MA), respectively. The Akt antibody that recognizes
the total Akt protein was from Cell Signaling (Beverly, MA). The
cell-permeable myristoylated PKC
pseudosubstrate (myr-SIYRRGARRWRKL)
was obtained from Quality Controlled Biochemicals (Hopkinton, MA). NGF
was obtained from Alomone (Jerusalem, Israel).
-glycerophosphate,
5 mM EGTA, 2.5 mM CaCl2, 1 mM glucose, 0.5% Triton X-100, and 25 mM
HEPES, pH 7.2. Reactions were started by adding 150 µM of
substrate (
peptide, Upstate Biotechnology Inc.) and 100 µM of [
-32P]ATP (2 Ci/mmol). After
incubation for 15 min at 30 °C, the reaction was terminated by
adding 10 µl of 25% (w/v) trichloroacetic acid. The samples were
centrifuged at 7,500 × g for 10 min. The supernatants were then spotted on 2 × 2-cm phosphocellulose squares (Whatman P-81), washed three times using 75 mM phosphoric acid, and
once using 75 mM sodium phosphate, pH 7.5. Radioactivity
retained on the P-81 papers were measured by scintillation counting.
PKC
activity was assayed as described above except that a
PKC
-specific pseudosubstrate peptide (sequence 113-129;
SIYRRGARRWRK-LYRAN) was added during the assay to block the PKC
activity. PKC
activity in PC12 cells was determined as the
difference between the PKC activity assayed in the absence and in the
presence of 300 µM PKC
-specific pseudosubstrate
peptide (17). PKC activity increased linearly for up to 30 min using up
to 30 µg of protein.
-glycerophosphate, 10 mM NaF, 1%
Triton X-100, 1 mM PMSF, 1 mM
Na3VO4, 2 µM aprotinin, 100 µM leupeptin, 2 µM pepstatin, and 0.5 µM OKA). Cell debris was removed by centrifugation at
7,500 × g for 10 min. The supernatant was utilized for
the Western blot analysis. Protein concentrations were determined using
the Bio-Rad Protein Assay Dye Reagent. Equal amounts of sample were
separated by SDS-polyacrylamide gel electrophoresis using 10%
polyacrylamide gels. The resolved proteins were then electroblotted
onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford,
MA). Membranes were blocked with 1% bovine serum albumin and incubated
with the desired primary antibody for 1 h at room temperature,
followed by the corresponding secondary antibody for 1 h at room
temperature. Blots were then washed and immunoreactive bands were
detected by enhanced chemiluminescence (Pierce) and recorded using
Kodak XAR-5 film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The A2A-R-selective agonist, CGS,
inhibits serum-deprived apoptosis in PC12 cells. A, CGS
prevented serum deprivation (24 h)-induced apoptosis as determined by a
DNA fragmentation assay in PC12 cells. B, CGS prevented
JNK1/2 phosphorylation after serum deprivation for 3 h.
C, a protein synthesis inhibitor, cycloheximide, attenuated
the protective effect of CGS in serum-deprived apoptosis in a
dose-dependent manner. Cell viability is expressed as
percentage of MTT metabolism observed in serum-treated cultures. Data
points are mean ± S.E. values from three independent experiments.
*, p < 0.05 compared with control (serum-free)
cultures treated with the indicated concentration of
cycloheximide.
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Fig. 2.
The protective effect of A2A-R
stimulation against serum-deprived apoptosis is
PKA-dependent. Cells (A, PC12 cells;
B, A123 cells) were pretreated with the indicated reagent
(KT-5720, 1 µM; H-89, 5 µM; or CSC, 10 µM) for 30 min before the addition of CGS (0.1 µM) or FK (5 µM) during serum deprivation.
The indicated inhibitor remained present during serum deprivation. Data
points represent mean ± S.E. values from three independent
experiments. a, p < 0.05 compared with
serum-deprived cells treated with CGS but not with CSC, KT-5720, or
H-89. b, p < 0.05 compared with cells treated
with FK but not with CSC, KT-5720, or H-89.
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Fig. 3.
The protective effect of A2A-R
stimulation against serum-deprived apoptosis is
PPase-dependent. Cells were pretreated with the
indicated concentration of PPase inhibitor (Caly A or OKA) for 30 min
before the addition of CGS (0.1 µM) or FK (5 µM) during serum deprivation. The indicated inhibitor
remained present during serum deprivation. Data points represent
mean ± S.E. values from three independent experiments.
A, p < 0.05 compared with serum-deprived
cells treated with CGS and not with Caly A or OKA. B,
p < 0.05 compared with serum-deprived cells treated
with FK and not with Caly A or OKA.
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Fig. 4.
Activation of MAPK is not important for the
protective effect of A2A-R stimulation. A,
PC12 cells were pretreated with PD98059 (20 µM) for 30 min before the addition of CGS (0.1 µM; 1 h) or FK
(5 µM; 15 min). Phosphorylated MAPK and total MAPK were
quantified by Western blot analysis. B, PC12 cells were
pretreated with PD98059 (20 µM) for 30 min before serum
deprivation in the presence or absence of CGS (0.1 µM).
PD98059 of the indicated concentration remained present during serum
deprivation. Data points are mean ± S.E. values from three
independent experiments. *, p < 0.05 compared with
serum-deprived control cells treated with or without PD98059.
A PKC inhibitor attenuates A2A-R-mediated protection against
apoptosis in serum-deprived PC12 cells
and
) play significant roles in the
desensitization of A2A-R-induced cAMP formation in PC12
cells (14). Moreover, two atypical PKC isozymes (aPKCs;
/
and
) and two conventional PKC isozymes (
and
) were also observed
in our line of PC12 cells (Fig. 5, B and C). To identify the PKC isozymes involved
in the protective effect of A2A-R, we treated PC12 cells
with a PKC-stimulating phorbol ester, PDD (100 nM), for
20 h to induce proteolysis and down-regulation of conventional and
novel PKCs. This treatment caused down-regulation of the conventional
PKCs (
and
; Fig. 5C) and novel PKCs (
and
; see
Ref. 14). Because aPKCs (
and
and
) are insensitive to
diacylglycerols and phorbol esters, long term PDD treatment did not
decrease levels of aPKCs in PC12 cells (Fig. 5B). Most
interestingly, long term PDD treatment did not alter the response to
A2A-R stimulation (Fig. 5A). Moreover, as shown
in Table II, stimulation of
A2A-R using CGS exerted a similar protective effect in PC12
variants expressing dominant-negative fragments of PKC
or PKC
.
Taken together, these data strongly suggest that conventional and novel
PKCs are not involved in A2A-R-mediated protection against
apoptosis in PC12 cells.
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Fig. 5.
Down-regulation of conventional and novel
PKCs does not interfere with the protective effect of A2A-R
stimulation. A, PC12 cells were pretreated with or
without PDD (100 nM) for 20 h and then subjected to
serum deprivation for 24 h in the presence of the indicated
reagent. Data points are mean ± S.E. values from three
independent experiments. *, p < 0.05 compared with
serum-deprived cells cultured without CGS. Expression of atypical
(B) and conventional (C) PKCs in PC12 cells after
treatment with PDD for 20 h were analyzed using the corresponding
antibody.
Stimulation of A2A-Rs promotes survival in PC12 cell lines that
express dominant negative fragments of novel PKCs
and PKC
/
immunoreactivities were markedly increased in the nuclear fraction
following treatment with CGS (Fig. 6C). FK also enhanced
aPKC activity in the nuclear fraction with a time course comparable to
that observed with CGS (Fig. 7). These
results suggest that PKA mediates increases in nuclear aPKCs during
A2A-R stimulation.
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Fig. 6.
Stimulation of A2A-R increases
nuclear aPKC activity and protein. PC12 cells were treated with or
without CGS (0.1 µM) for the indicated time and then
separated into nuclear (A) and non-nuclear fractions
(B) for determination of PKC activities. Data points
represent mean ± S.E. values from three independent experiments.
*, p < 0.05 compared with untreated control samples at
the same time point. C, Western analysis of PKC and
PKC
/
in the nuclear fractions after treatment with CGS for the
indicated time. Data shown are representative of results from three
independent experiments.
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Fig. 7.
Forskolin increases nuclear aPKC activity in
PC12 cells. PC12 cells were treated with or without FK (5 µM) for the indicated time and then were collected to
harvest nuclear fractions for determination of PKC activities. Data
points represent mean ± S.E. values from three independent
experiments. *, p < 0.05 compared with control samples
incubated for the same time.
pseudosubstrate
inhibitor to assess the role of PKC in the A2A-mediated
protection. As shown in Fig.
8A, the PKC
-specific inhibitor blocked A2A-R-mediated protection against
apoptosis. In addition, transient overexpression of a
dominant-positive PKC
(PKC
+, see Ref. 23) enhanced
the survival of serum-deprived PC12 cells by 39 ± 7% (Fig.
8B). Comparable results were obtained when control cells
transfected with empty vector and the GFP-expressing construct were
treated with CGS. Stimulation of A2A-R using CGS enhanced
the number of GFP-expressing cells by 51 ± 20% (mean ± S.E.; p < 0.05, Student's t test, seven
independent experiments). This observation is consistent with the
protective effect of A2A-R assessed by the MTT assay (Fig.
1C). These findings indicate that PKC
specifically
regulates A2A-R-mediated survival in PC12 cells.
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Fig. 8.
PKC mediates the protective effect of
A2A-R stimulation. A, PC12 cells were
pretreated with a cell-permeable PKC -specific inhibitor (2.5 µM) for 30 min before the addition of CGS (0.1 µM) or NGF (100 ng/ml) as indicated. Data points
represent mean ± S.E. values from three independent experiments.
*, p < 0.05 compared with the corresponding
serum-free-treated sample. #, p < 0.05 compared with the corresponding non-PKC inhibitor-treated sample.
B, PC12 cells were transiently transfected with a control
vector or with vectors encoding JNK1 or PKC
+ along with
one-seventh of the molar amount of a GFP vector as indicated. Two days
post-transfection, cells were subjected to serum deprivation for
24 h. Transfected cells were identified by GFP expression, and the
number of surviving GFP-expressing cells after 24 h serum
deprivation was quantified directly by counting. The percentage of
GFP-expressing cells transfected with the control vector was designated
as a survival index of 100%. Data points are mean ± S.E. values
from three independent experiments. A dominant-negative JNK1 (JNK1; see
Ref. 48) was included here as a positive control since it was reported
to increase survival upon serum deprivation in other cell types (12).
*, p < 0.05 compared with cells transfected with the
control vector. C, A123 cells were transiently transfected
with a control vector or a vector encoding PKC
+ along
with one-seventh of the molar amount of a GFP vector as indicated. Two
days post-transfection, cells were subjected to serum deprivation for
24 h. GFP-expressing cells were quantified by flow cytometry. The
percentage of GFP-expressing cells transfected with the control vector
was designated as a survival index of 100%. Data points are mean ± S.E. values from three independent experiments. *, p < 0.05 compared with cells transfected with the control vector.
,
and whether this regulation is PKA-dependent, we next
determined the activity of PKC
under our experimental conditions. We
found that nuclear PKC
activity was increased by CGS (Fig. 6) and FK (Fig. 7). Moreover, transient overexpression of PKC
+
enhanced the survival of serum-deprived A123 cells (a PKA-deficient PC12 variant) (Fig. 8C). Collectively, these results suggest
that the protective effect of A2A-R stimulation in
serum-deprived PC12 cells requires the sequential activation of PKA and
PKC
.
mediates the A2A-protective effect in PC12
cells. Interestingly, the myristoylated PKC
pseudosubstrate
inhibitor also partially suppressed the protective effect of NGF in
serum-deprived PC12 cells (Fig. 8A). Thus, distinct anti-apoptotic signals may converge on PKC
in PC12 cells. Because phosphatidylinositol 3-kinase (PI3K) has been implicated in the NGF-induced translocation of PKC
to the nucleus (24) and in suppression of apoptosis (25), we next investigated if the PI3K pathway
was involved in the protective effect of A2A-R stimulation. Although a PI3K inhibitor (LY294002) abolished the protective effect of
NGF against apoptosis in a dose-dependent manner, it did
not reduce CGS-mediated survival (Fig.
9A).
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Fig. 9.
The PI3K/Akt pathway is not required for the
protective effect of A2A-R stimulation. A,
cells were pretreated with the indicated concentration of LY 294002 for
60 min before the addition of CGS (0.1 µM) or NGF (100 ng/ml). Viability of PC12 cells was determined by MTT assay after
24 h of serum deprivation. Data points are mean ± S.E.
values from three independent experiments. *p < 0.05 compared with cells not exposed to LY 294002. Akt phosphorylated on
Ser-473 (B) or Thr-308 (C) and total Akt were
detected by Western analysis in PC12 cells treated with CGS (0.1 µM) or NGF (100 ng/ml) for the indicated time.
D, cells were transiently transfected with a control vector
or dnAkt along with one-seventh of the molar amount of a GFP vector as
indicated. Two days post-transfection, cells were subjected to serum
deprivation in the presence of CGS (0.1 µM) or NGF (100 ng/ml) for 24 h. GFP-expressing cells were quantified by flow
cytometry. The percentage of GFP-expressing cells transfected with a
control vector in the presence of the indicated anti-apoptotic reagent
(CGS or NGF) was used to define a survival index of 100%. Data points
are mean ± S.E. values from three independent experiments. *,
p < 0.05 compared with cells transfected with the
control vector.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) of the anti-apoptotic processes (Fig. 10).
-specific inhibitor (Fig.
8A), but not by down-regulation of conventional and novel
PKCs (Fig. 5 and Table II). BiM is widely used as a selective inhibitor
of PKCs. Since BiM acts as a competitive inhibitor of ATP binding, it
may also inhibit PKA at high concentrations (31). Although the in
vitro Ki value of BiM for PKC is 10 nM, the effective concentration for inhibiting PKC in
cultured cells appears to be higher. For example, in 3T3 fibroblasts,
maximal inhibition of PKC-dependent phosphorylation by BiM
occurs at 5 µM, which is a concentration that does not
inhibit PKA-mediated phosphorylation in those cells (31). In addition,
in rat basophilic leukemia (RBL-2H3) cells, 10 µM BiM
blocks PKC- but not PKA-evoked phosphorylation of phospholipase C (32).
BiM has therefore been routinely used to block PKC-mediated responses
at concentrations ranging from 1 to 10 µM in studies
employing various types of cultured cells (33, 34). Moreover, in our
study, we demonstrated that in addition to BiM, a PKC
-specific
pseudosubstrate peptide inhibitor also blocked the protective effect of
A2A-R, thus confirming the involvement of PKC
in this
process (Fig. 8A).
and
/
immunoreactivity in PC12 cells
(Fig. 6). This process appears to be mediated by PKA, since FK also
increases nuclear aPKC activity (Fig. 7). Increased nuclear aPKC may be
important for antagonizing apoptosis since it has been observed
following treatment with other mitogenic and differentiating factors
that promote cell survival (24, 35). Finally, overexpression of a
dominant-positive PKC
enhanced the survival of both wild-type and
PKA-deficient PC12 cells (Fig. 8, B and C). These
results indicate that aPKCs lie in a signal transduction pathway
downstream of PKA that mediates that protective effect of
A2A-R stimulation and that at least one aPKC, PKC
, is
critical for the prevention of serum-deprived apoptosis in PC12 cells.
in
conjunction with NF-
B and promote survival of PC12 cells during
serum deprivation (22). NGF, which promotes survival of PC12 cells,
also increases the abundance of nuclear PKC
in PC12 cells (24).
Results in the present study demonstrate that the myristoylated
PKC
-selective inhibitor partially suppressed the protective effect
of NGF (Fig. 9A), further suggesting that PKC
is an
important anti-apoptotic factor in PC12 cells. Since PKC
has been
implicated in the regulation of NF-
B (37), NF-
B might act
downstream of PKC
to inhibit apoptosis due to serum deprivation.
PKC
has been shown to protect human leukemia cells against
drug-induced apoptosis (38). We found that nuclear PKC
is increased
following A2A-R stimulation, suggesting that PKC
may
also contribute to important nuclear events that protect against apoptosis in PC12 cells. Further studies are required to clarify the
role of PKC
/
in the protective effect of A2A-R stimulation.
can be activated in a
PI3K-dependent manner (42). However, we found that whereas
a PI3K inhibitor blocked the ability of NGF to promote survival upon
serum deprivation, the protective effect of A2A-R
stimulation was PI3K-independent (Fig. 9A and
Fig. 10). In addition, Akt,
a kinase important for NGF-mediated survival (43), was not
involved in the protective effect of A2A-R stimulation.
These findings indicate that A2A-R agonists and NGF utilize
different signaling pathways to prevent apoptosis. A similar situation
exists in sympathetic neurons, where the PI3K/Akt pathway plays a
critical role in depolarization-mediated survival, but not in survival
mediated by the cAMP/PKA pathway (44). Our results also indicate that
in PC12 cells these two pathways converge on at least one common
anti-apoptotic factor, PKC
. The mechanism by which PKA activates
PKC
independent of PI3K requires further study.
View larger version (12K):
[in a new window]
Fig. 10.
Schematic representation of signaling
pathways that mediate A2A-R-mediated survival.
Our findings also implicate serine/threonine protein phosphatases in
the protective effect of A2A-R stimulation. Activation of
A2A-R increases the activity of a serine/threonine PPase in neutrophils (19). In the present study, we found that two
serine/threonine PPase inhibitors (Caly A and OKA) blocked the
protective effect of CGS and FK at nM concentrations.
Maximal inhibition was achieved using 1 nM of Caly A or 10 nM of OKA (Fig. 3). Caly A is a selective inhibitor of
PP1 and PP2A (Ki = 0.5-2
nM). In contrast, OKA is a relatively specific inhibitor of
PP2A with Ki values of 0.2 nM for PP2A and 20 nM for
PP1. Because 10 nM of OKA completely blocked
A2A-R-mediated protection against apoptosis (Fig.
3B), the PPase most likely involved in this process is a member of the PP2A family. Several important proteins
involved in apoptosis, such as IB kinase, MAP kinases, and cell
cycle regulators are substrates of PP2A (45). In a
cell-free model of apoptosis, OKA suppresses caspase-3 activation and
Akt cleavage, two key events in cell death (46). It remains to be
determined whether stimulation of A2A-R activates a
PP2A-like activity downstream of PKA that protects PC12
cells from apoptosis or whether basal PP2A activity is
merely necessary for A2A-R-mediated survival.
A2A-Rs are expressed in many areas of the brain (6) and in
various peripheral tissues (47). Previous work has suggested a role for
A2A-Rs in protection against cell death. Kobayashi and
Millhorn (9) reported that expression of A2A-Rs is
increased by hypoxia. Stimulation of A2A-Rs in PC12 cells
partially protects against cell death induced by hypoxia (9). In human
neutrophils, stimulation of A2A-Rs delays apoptosis,
presumably via a PKA-dependent mechanism (10). Our present
study demonstrates that multiple mechanisms downstream of PKA underlie
the action of A2A-Rs in preventing apoptosis of
serum-deprived PC12 cells. Our findings provide the first clear
evidence that PKC is a key downstream component of a
PKA-dependent, anti-apoptotic signaling pathway activated
by a GPCR. Further knowledge about protective mechanisms evoked by
A2A-R stimulation may help to facilitate the clinical application of A2A-R agonists in the treatment of
neurodegeneration-associated nervous system trauma and neurological disease.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Peter Parker (Imperial Cancer
Research Fund, London, UK) for the activated PKC plasmid; Dr. Alex
Toker (Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston) for the dominant-negative K179M-Akt mutant plasmid; and
D. Platt for reading and editing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Council Grants NSC88-2316-B001-008-M46, NSC89-2316-B001-008-M46, and NSC90-2316-B001-005-M46) and by Academia Sinica, Taipei, Taiwan, Republic of China.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence and reprint requests should be addressed. Tel.: 886-2-26523913; Fax: 886-2-27829143; E-mail: bmychern@ibms.sinica.edu.tw.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M008589200
2 C. H. Chen and Y. Chern, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
A2A-R, A2A adenosine
receptor;
CGS, CGS 21680;
CSC, 8-(3-chlorostyryl)-caffeine;
PKA, cAMP-dependent protein kinase;
JNK, c-Jun N-terminal
kinase;
aPKCs, atypical PKCs;
PP2A, protein phosphates 2A,
PDD, phorbol-12,13-didecanoate;
PBS, phosphate-buffered saline;
MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;
FK, forskolin;
PPase, protein phosphatase;
OKA, okadaic acid;
Caly A, calyculin A;
BiM, Bisindolylmaleimide I-HCl;
NGF, nerve growth factor;
NF-B, nuclear factor-
B;
PI3K, phosphatidylinositol 3-kinase;
PKC+, dominant-positive PKC;
dnAkt, a dominant-negative
Akt;
GFP, green fluorescent protein;
PMSF, phenylmethylsulfonyl
fluoride;
MAPK, mitogen-activated protein kinase;
DMEM, Dulbecco's
modified Eagle's medium;
ERK, extracellular signal-regulated
kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Daval, J.-L., Nehlig, A., and Nicolas, F. (1991) Life Sci. 49, 1435-1453[CrossRef][Medline] [Order article via Infotrieve] |
2. | Olah, M. E., and Stiles, G. L. (1996) Annu. Rev. Pharmacol. Toxicol. 35, 581-606[CrossRef][Medline] [Order article via Infotrieve] |
3. | Chern, Y. J., King, K., Lai, H.-L., and Lai, H. T. (1992) Biochem. Biophys. Res. Commun. 185, 304-309[Medline] [Order article via Infotrieve] |
4. | Chu, Y.-Y., Tu, K.-H., Lee, Y.-C., Kuo, Z.-J., Lai, H.-L., and Chern, Y. (1996) DNA Cell Biol. 15, 329-337[Medline] [Order article via Infotrieve] |
5. | Ferre, S., O'Connor, W. T., Fuxe, K., and Ungerstedt, U. (1993) J. Neurosci. 13, 5402-5406[Abstract] |
6. | Rosin, D. L., Robeva, A., Woodard, R. L., Guyenet, P. G., and Linden, J. (1998) J. Comp. Neurol. 401, 163-186[CrossRef][Medline] [Order article via Infotrieve] |
7. | Richardson, P. J., Kase, H., and Jenner, P. G. (1997) Trends Pharmacol. Sci. 18, 338-344[CrossRef][Medline] [Order article via Infotrieve] |
8. | Sebastiao, A. M., and Ribeiro, J. A. (1996) Prog. Neurobiol. 48, 167-189[CrossRef][Medline] [Order article via Infotrieve] |
9. | Kobayashi, S., and Millhorn, D. E. (1999) J. Biol. Chem. 294, 20358-20365[CrossRef] |
10. | Walker, B. A. M., Rocchini, C., Boone, R. H., Ip, S., and Jacobson, M. A. (1997) J. Immunol. 158, 2926-2931[Abstract] |
11. | Jones, P., A., Smith, R. A., and Stone, T. W. (1998) Neuroscience 85, 229-237[CrossRef][Medline] [Order article via Infotrieve] |
12. | Xia, Z., Dickens, M., Raingeaud, J., and Greenberg, M. E. (1995) Science 270, 1326-1331[Abstract] |
13. | Chern, Y., Chiou, J.-Y., Lai, H.-L., and Tsai, M.-H. (1995) Mol. Pharmacol. 48, 1-8[Abstract] |
14. |
Lai, H. L.,
Yang, T. H.,
Messing, R. O.,
Chin, Y. H.,
Lin, S. C.,
and Chern, Y.
(1997)
J. Biol. Chem.
272,
4970-4977 |
15. |
Ginty, D. D.,
Glowacka, D.,
DeFranco, C.,
and Wagner, J. A.
(1991)
J. Biol. Chem.
266,
15325-15333 |
16. |
Gerstin, E., Jr.,
McMahon, T.,
Dadgar, J.,
and Messing, R. O.
(1998)
J. Biol. Chem.
273,
16409-16414 |
17. | Wooten, M. W., Seibenhener, M. L., Boone, R. H., Matthews, L. H., Zhou, G., and Coleman, E. S. (1996) J. Neurochem. 67, 1023-1031[Medline] [Order article via Infotrieve] |
18. | Zhou, G., Seibenhener, M. L., and Wooten, M. W. (1997) J. Biol. Chem. 272, 3110-31137 |
19. |
Revan, S.,
Montesinos, M. C.,
Naime, D.,
Landau, S.,
and Cronstein, B. N.
(1996)
J. Biol. Chem.
271,
17114-17118 |
20. |
Seidel, M. G.,
Klinger, M.,
Freissmuth, M.,
and Holler, C.
(1999)
J. Biol. Chem.
274,
25833-25841 |
21. |
Creedon, D. J.,
Johnson, E. M., Jr.,
and Lawrence, J. C., Jr.
(1996)
J. Biol. Chem.
271,
20713-20718 |
22. | Wang, Y. M., Seibenhener, M. L., Vandenplas, M. L., and Wooten, M. W. (1999) J. Neurosci. Res. 55, 293-302[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Schonwasser, D. C.,
Marais, R. M.,
Marshall, C. J.,
and Parker, P. J.
(1998)
Mol. Cell. Biol.
18,
790-798 |
24. |
Neri, L. M.,
Martelli, A. M.,
Borgatti, P.,
Colamussi, M. L.,
Marchisio, M.,
and Capitani, A. S.
(1999)
FASEB J.
13,
2299-2310 |
25. |
Datta, S. R.,
Brunet, A.,
and Greenberg, M. E.
(1999)
Genes Dev.
13,
2905-2927 |
26. | Downward, J. (1998) Cell Biol. 10, 262-267 |
27. |
Toker, A.,
and Newton, A. C.
(2000)
J. Biol. Chem.
275,
8271-8274 |
28. |
Cichy, S. B.,
Uddin, S.,
Danilkovich, A.,
Guo, S.,
Klippel, A.,
and Unterman, T. G.
(1998)
J. Biol. Chem.
273,
6482-6487 |
29. | Rukenstein, A., Rydel, R. E., and Greene, L. A. (1991) J. Neurosci. 11, 2552-2563[Abstract] |
30. | Lambeng, N., Michel, P. P., Brugg, B., Agid, Y., and Ruberg, M. (1999) Brain Res. 821, 60-68[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Toullec, D.,
Pianetti, P.,
Coste, H.,
Bellevergue, P.,
Grand-Perret, T.,
Ajakane, M.,
Baudet, V.,
Boissin, P.,
Boursier, E.,
Loriolle, F.,
Duhamel, L.,
Charon, D.,
and Kirlovsky, J.
(1991)
J. Biol. Chem.
266,
15771-15781 |
32. |
Ali, H.,
Fisher, I.,
Haribabu, B.,
Richardson, R. M.,
and Snyderman, R.
(1998)
J. Biol. Chem.
272,
11706-11709 |
33. |
Taylor, S. C.,
Green, K. N.,
Carpenter, E.,
and Peer, C.
(2000)
J. Biol. Chem.
275,
26786-26791 |
34. |
Liu, H.,
Force, T.,
and Bloch, K. D.
(1997)
J. Biol. Chem.
272,
6038-6043 |
35. | Wooten, M. W., Zhou, G., Wooten, M. C., and Seibenhener, M. L. (1997) J. Neurosci. Res. 49, 393-403[CrossRef][Medline] [Order article via Infotrieve] |
36. | Berra, E., Municio, M. M., Sanz, L., Frutos, S., Diaz-Meco, M. T., and Moscat, J. (1997) Mol. Cell. Biol. 17, 4346-4354[Abstract] |
37. |
Lallena, M. J.,
Diaz-Meco, M. T.,
Bern, G.,
Paya, C. V.,
and Moscat, J.
(1999)
Mol. Cell. Biol.
19,
2180-2188 |
38. |
Murray, N. R.,
and Fields, A. P.
(1997)
J. Biol. Chem.
272,
27521-27524 |
39. |
Moule, S. K.,
Welsh, G. I.,
Edgell, N. J.,
Foulstone, E. J.,
Proud, C. G.,
and Denton, R. M.
(1997)
J. Biol. Chem.
272,
7713-7719 |
40. |
Polakiewicz, R. D.,
Schieferl, S. M.,
Gingras, A. C.,
Sonenberg, N.,
and Comb, M. J.
(1998)
J. Biol. Chem.
273,
23534-2341 |
41. | Klesse, L. J., Meyers, K. A., Marshall, C. J., and Parada, L. F. (1999) Oncogene 18, 2055-2068[CrossRef][Medline] [Order article via Infotrieve] |
42. | Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. H., Chen, C. S., Newton, A. C., Schaffhausen, B. S., and Toker, A. (1998) Curr. Biol. 8, 1069-1077[Medline] [Order article via Infotrieve] |
43. |
Crowder, R. J.,
and Freeman, R. S.
(1998)
J. Neurosci.
18,
2933-2943 |
44. | Crowder, R. J., and Freeman, R. S. (1999) J. Neurochem. 73, 466-475[CrossRef][Medline] [Order article via Infotrieve] |
45. | Millward, T. A., Zolnierowicz, S., and Hemmings, B. A. (1999) Trends Biochem. Sci. 24, 186-191[CrossRef][Medline] [Order article via Infotrieve] |
46. | Francois, F., and Grimes, M. L. (1999) J. Neurochem. 73, 1773-1776[CrossRef][Medline] [Order article via Infotrieve] |
47. | Dixon, A. K., Gubitz, A. K., Sirinathsinghji, D. J. S., Richardson, P. J., and Freeman, T. C. (1996) Br. J. Pharmacol. 118, 1461-1468[Abstract] |
48. | Li, Y., S., Shyy, J. Y. J., Li, S., Lee, J., Su, B., Karin, M., and Chien, S. (1996) Mol. Cell. Biol. 16, 5947-5954[Abstract] |