From the Dipartimento di Biologia e Patologia Molecolare e Cellulare, BioGeM Consortium, Istituto di Endocrinologia ed Oncologia Sperimentale CNR, Facoltà di Medicina, Universitá "Federico II," via S. Pansini 5, 80131 Napoli, Italy and the § Institute of Cancer Research, Columbia University, New York, New York 10032
Received for publication, September 27, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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A-Kinase anchor proteins (AKAPs) immobilize and
concentrate protein kinase A (PKA) isoforms at specific subcellular
compartments. Intracellular targeting of PKA holoenzyme elicits rapid
and efficient phosphorylation of target proteins, thereby increasing
sensitivity of downstream effectors to cAMP action. AKAP121 targets PKA
to the cytoplasmic surface of mitochondria. Here we show that
conditional expression of AKAP121 in PC12 cells selectively enhances
cAMP·PKA signaling to mitochondria. AKAP121 induction stimulates
PKA-dependent phosphorylation of the proapoptotic
protein BAD at Ser155, inhibits release of
cytochrome c from mitochondria, and protects cells from
apoptosis. An AKAP121 derivative mutant that localizes on mitochondria
but does not bind PKA down-regulates PKA signaling to the mitochondria
and promotes apoptosis. These findings indicate that PKA anchored by
AKAP121 transduces cAMP signals to the mitochondria, and it may play an
important role in mitochondrial physiology.
Binding of extracellular ligand to G-protein-coupled receptors at
the cell membrane activates adenylate cyclase and increases cAMP levels
at discrete points along the membrane. cAMP binds the regulatory (R)
subunits of protein kinase A
(PKA),1 dissociating the
holoenzyme and releasing free catalytic subunit (C-PKA).
Phosphorylation of nuclear and cytoplasmic substrates by PKA controls
multiple cell functions (1-7).
PKA is concentrated in particulate membranes and cellular organelles
through interaction with a family of distinct but functionally homologous A-kinase anchor
proteins (AKAPs) (8-11). Although the preferred ligand is
RII/PKAII, several AKAPs also bind RI/PKAI (12-14). AKAPs enhance the
efficiency of cAMP signal-transducing pathways by localizing PKA near
sites of cAMP generation or at PKA targets (15-26).
S-AKAP84 and AKAP121 derive from alternatively spliced products of the
same gene. They are expressed in the male germ cell lineage as well as
in several other tissues (27-31). Hormones that activate the
cAMP·PKA pathway induce accumulation of S-AKAP84/AKAP121 transcripts
and protein (28). This suggests a positive feedback loop between
membrane-generated signals and downstream effector molecules of
cAMP·PKA signals. All splice variants share the same 525-amino acid
NH2-terminal core, which includes the anchoring domain and
the R-binding domain but diverge significantly at the COOH
terminus. The first 30 NH2-terminal residues mediate the targeting of S-AKAP84/AKAP121 to the outer membrane of mitochondria, both in male germ cells and in transfected heterologous cells (27, 30).
However, other localization sites have been observed. D-AKAP1, an alternative splice product of S-AKAP84 carrying
additional NH2-terminal residues, colocalizes with both
mitochondria and endoplasmic reticulum (32, 33). Furthermore,
S-AKAP84/AKAP121 also interacts with microtubules and associates with
mitochondria in interphase and mitotic spindles during metaphase
transition (34). This suggests that the same anchor protein might focus cAMP·PKA signaling to distinct subcellular compartments in a cell cycle-dependent manner.
Its location on the outer surface of mitochondria implies a role for
AKAP121 in cAMP-mediated reactions at or within mitochondria. Mitochondria are the seat of a number of major cellular functions, including essential pathways of intermediate metabolism, amino acid
biosynthesis, fatty acid oxidation, steroid metabolism, apoptosis, and
oxidative energy metabolism (35). Several of these functions are
constitutive, whereas others are finely regulated at the
post-translational level. BAD is a BH3-proapoptotic Bcl-2 family member
that acts at a key nodal point in the mitochondrial apoptotic pathway.
Unphosphorylated BAD binds and inactivates antiapoptotic Bcl-2
homologs. This allows release of cytochrome c from
mitochondria and consequent activation of the apoptotic pathway
(36-41). Phosphorylation by PKA blocks BAD association with BCL-2 and
inhibits apoptosis (42-45). Previous observations suggested a role of
mitochondria-anchored PKA in the inhibition of apoptosis (46).
Treatment with a synthetic peptide spanning the RII-binding domain of
thyroid AKAP (Ht-31) decreased BAD phosphorylation at
Ser112 and increased apoptosis of FL12.5 cells. However, it
was not clear whether BAD phosphorylation necessitates PKA anchored to mitochondria by AKAP121 or whether it can be carried out by PKA anchored in other membrane compartments (46). To further analyze this
mechanism and investigate the role of AKAP121 in the cAMP signaling to
the mitochondria, we have established a conditional PC12 cell line in
which the expression of AKAP121 is regulated. AKAP121 induction
stimulates BAD phosphorylation at Ser155 and increases cell
survival. Conversely, expression of an AKAP121 mutant that binds to
mitochondria but does not anchor PKA activates the mitochondrial
caspase pathway and provokes apoptosis.
Antibodies--
Anti-caspase-3 was purchased from Pharmingen;
anti-caspase-9, anti-BAD, and anti-phospho-specific BAD antibodies were
purchased from Cell Signaling; anti-phospho-ERK, anti-ERK,
anti-p21/Waf, anti-RII, and anti-cytochrome c
antibodies were purchased from Santa Cruz Biotechnology, Inc.; and
anti-manganese superoxide dismutase were purchased from Bender
MedSystems. Anti-RII and anti-AKAP121 polyclonal antibodies were
purchased from Santa Cruz Biotechnology, Inc.; CREB and
anti-phospho136CREB antibodies were purchased from Upstate
Biotechnology Inc.
Cell Culture--
PC12 wild-type or PC12-tet off
(Clontech) cells were maintained in DMEM
supplemented with 10% heat-inactivated horse serum, 5% fetal bovine
serum, 1 mM glutamine, and 1% penicillin-streptomycin at
37 °C and 5% CO2. PC12-tet off cells stably expressing
the doxycyclin-regulated transcription factor (tTA) were grown in the
presence of G-418 (100 µg/ml, Invitrogen). Cells were cultured on
poly-L-lysine-coated plates. SK-N-BE neuroblastoma cells
(American Type Culture Collection (ATTC)) were maintained in RPMI 1640 medium (Invitrogen) containing 16% fetal bovine serum, 100 µg/ml
glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin in 5%
CO2 at 37 °C.
Plasmid and Transfections--
AKAP121 cDNA was excised from
pCEP4-AKAP121 vector using SacII/BamHI
restriction enzymes. AKAP121 cDNA was subcloned into pTRE-vector
(Clontech) predigested with the same restriction
enzymes. Mutant AKAP121 (AKAP121m) was generated by using the
QuikChangeTM site-directed mutagenesis kit (Stratagene).
Oligonucleotide primers containing point mutations are as follows:
forward, 5'-GCT GCC TTC CAG CCC ATC TCC CAG GTG ATC CCG GAA GCA ACT-3';
reverse, 5'-AGT TGC TTC CGG GAT CAC CTG GGA GAT GGG CTG GAA GGC AGC-3'.
Amplification products were sequenced, and cDNA vectors encoding
wild-type or mutant AKAP121 were prepared and purified with Qiagen
Plasmid Maxi kit tips columns (Qiagen, Chatworth, CA) and stably
transfected in PC12-Tet-off cells (Clontech) using
the Lipofectin procedure (Lipofectin reagent, Invitrogen). Positive
clones were selected in complete medium containing G-418 (100 µg/ml),
hygromycin (200 µg/ml), and doxycyclin (10 ng/ml). Removal of
doxycyclin for 2 days induces accumulation of significant amounts of
AKAP121. Transient transfections using CRE-CAT or pBD-Elk/GAL4-CAT
(Stratagene) vectors were performed using the calcium phosphate
procedure (47). CRE-CAT expression is enhanced by phosphorylated CREB
(15, 18). pBD-Elk1 is a fusion protein between the activation domain of
Elk1, a substrate of MAPK, and the DNA-binding domain of the yeast GAL4
transcription factor. GAL-CAT is a reporter plasmid carrying CAT
downstream to a GAL-4 synthetic promoter. The promoter
contains five tandem repeats of GAL4-binding sites. Transfection
efficiency was analyzed by co-transfecting RSV- Apoptosis and Fluorescent-activated Cell Sorter Analysis
(FACS)--
Apoptosis was analyzed by double staining with propidium
iodide and annexin (Apoptosis detection kit, Medical and
Biological Laboratories). Briefly, cells were harvested at indicated
times after treatment, washed twice with 1× PBS, and incubated for 10 min with propidium iodide (50 ng/ml in 1× PBS, Sigma) and annexin. After three washes with PBS, the cells were analyzed by fluorescence microscopy using an Axiovert microscope IX70 (Olympus) or by FACS analysis. For microscopy, apoptosis was quantified by scoring the
percentage of cells stained with propidium iodide (red) and annexin
(green) in the adherent cell population. To avoid unbiased counting,
plates were coded, and the cells scored blind without knowledge of the
treatment performed. Four to six independent experiments made in
triplicate were performed for each treatment. For FACS analysis, cells
were harvested in 1× PBS containing trypsin and 20 mM
EDTA. 3 × 106 cells were resuspended in PBS,
fixed with cold 100% ethanol, and treated with RNase-DNase-free enzyme
(50 µg/ml). Cells were stained with propidium iodide (50 µg/ml) and
annexin in a dark room for 20 min and analyzed by flow cytometry using
a BD Biosciences FACScan apparatus.
Immunoblot Analyses--
Cells were homogenized in lysis
buffer (20 mM Tris-HCl, pH 7.9, 150 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.05%
Tween 20, 0.02% sodium azide) containing protease inhibitor (5 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin, and 0.5 mM phenylmethylsulfonyl fluoride) and phosphatase inhibitor
(10 mM sodium fluoride, 20 mM
Assay for RII Binding Activity--
Protein samples were
size-fractionated by 8% SDS-PAGE gel as described above. Resolved
polypeptides were transferred to polyvinylidene difluoride membranes
(Immobilon P, Millipore). RII probes were labeled with
[32P]ATP and PKA catalytic subunit (1 unit) (Sigma), as
described previously (30).
Immunofluorescence--
PC12 cells were rinsed in PBS and fixed
in 3.7% formaldehyde for 20 min. After permeabilization with 0.5%
Triton X-100 in PBS/5 min, the cells were incubated with 1× PBS
containing 0.1 mg/ml bovine serum albumin for 1 h at room
temperature. Double immunofluorescence was carried out with the
following antibodies: anti-superoxide dismutase monoclonal antibody
(1/250) and anti-AKAP121 (dilution 1/250) or RII cAMP·PKA Signaling Suppresses Apoptosis Induced by Serum
Deprivation--
To analyze the role of PKA in cell survival, we
investigated the effects of a cAMP analogue in neuronal cells deprived
of growth factors. In human neuroblastoma (SK-N-BE) cells, prolonged serum starvation triggers the apoptotic program that includes stress-activated protein kinases and caspases (48, 49). Cells were
grown to subconfluence, starved in medium containing 0.1% serum, and
harvested at different times. The percentage of apoptotic cells was
determined by fluorescent microscopy (see "Materials and Methods").
As shown in Fig. 1A, serum
deprivation induced a time-dependent increase in cell
death. As a second monitor of apoptosis, we followed the accumulation
of p17, the cleaved active caspase-3 fragment. The kinetics of
caspase-3 activity roughly coincided with cellular apoptosis (Fig.
1B). Activation of the cAMP·PKA signal transduction
pathway can suppress or promote apoptosis, depending on cell type
(50-55). As shown in Fig. 1, C and D, treatment with CPT-cAMP, a potent cAMP analogue, increased survival of
serum-deprived SK-N-BE cells and inhibited caspase-3 activation. The
protective effects of PKA activation toward trophic factor withdrawal
were also demonstrated in the PC12 pheochromocytoma cell line (Fig. 1,
E and F).
Conditional Expression of AKAP121 Enhances PKA Targeting to
Mitochondria--
We wished to determine the role of AKAP in
transmitting cAMP signals to mitochondria. Accordingly, we used the
tet-off inducible system to regulate expression of AKAP121, an AKAP
that binds and targets PKA to the cytoplasmic surface of mitochondria
(Fig. 2A). Doxycyclin
down-regulates transcription of a target gene in this system by
inactivating the tetA transcription factor. We isolated several PC12
stable transfectants (PC-A121) that expressed AKAP121 when cultured in
the absence of doxycycline for 48 h, as shown by RII overlay (Fig.
2B, left panel). Doxycyclin down-regulated AKAP121 accumulation in PC-A121 clones number 3 and 11 to the levels of
PC12 controls, as demonstrated by immunoblot analysis with anti-AKAP121
antibody (Fig. 2B, right panel). Expression of
the transgene was reversible since readdition of doxycyclin to the
medium reduced AKAP121 concentrations (data not shown). AKAP121
expressed in PC-A121 localized principally on mitochondria, as shown by
double labeling with antibody to manganese superoxide dismutase, a
protein that selectively accumulates in mitochondria (Fig.
2C) (56). AKAP121 accumulation coincided with increased targeting of RII subunit to mitochondria, as demonstrated by immunoblot analysis of proteins extracted from partially purified mitochondria (Fig. 2D). The total cellular content of RII was unaffected
by AKAP121 expression (Fig. 2D).
Expression of AKAP121 Protects PC12 Cells Against
Apoptosis--
We then assessed the biological effects of AKAP121
accumulation and increased association of PKA with mitochondria. First, we determined the growth rates of PC12 and PC-A121 cells in the presence and absence of doxycylin. As shown in Fig.
3A, cells expressing AKAP121
increased more rapidly than controls over a 96-h period. AKAP121
expression did not accelerate the cell cycle since FACS analysis and
[3H]thymidine incorporation showed no significant
difference in the number of cells in G1, S, or
G2/M when compared with controls (data not shown). We thus
considered the possibility that AKAP121 might enhance cell viability.
We measured the effects of AKAP121 on sensitivity to trophic factor
deprivation. Cells were grown to semiconfluence ± doxycyclin and
then serum-starved and harvested at different times. Fig. 3B
shows that control cultures (PC12 ± dox and PC-A121 + dox) became
apoptotic more rapidly than the experimental culture (PC-A121
To demonstrate that AKAP121 acted through PKA, these experiments were
repeated in the presence of the PKA inhibitor, H89. As shown in Fig.
3C, H89 abrogated the enhancement of survival by AKAP121 in
serum-deprived cells. Parallel experiments were performed using
hydrogen peroxide (H2O2) as a proapoptotic
stimulus. About 30% of PC12 and PC-A121 + dox cells treated with
H2O2 (200 µM) became apoptotic 1 day after treatment. In contrast, 20% of cells expressing
AKAP121 (PC-A121 AKAP121 Selectively Increases PKA-dependent
Phosphorylation of Endogenous BAD at Ser155--
These
data indicate that the AKAP121·PKA pathway mediates, at least in
part, the protective effects of cAMP on cell survival. Based on
previous reports, we thought it likely that the downstream effector of
PKA was likely to be BAD. BAD, a proapoptotic protein, binds and
inactivates Bcl, an antiapoptotic protein located on the outer
mitochondrial membrane. Phosphorylation by PKA at Ser155
blocks association of BAD with Bcl (45). To determine the
phosphorylation pattern of BAD, total proteins isolated from control or
cAMP-treated cells were size-fractionated by denaturing gel
electrophoresis and immunoblotted using specific antibodies to BAD
phosphorylated at Ser112, Ser136, or
Ser155. As shown in Fig.
4A, growing PC-A121 cells
contain significant amounts of BAD phosphorylated at one or more of
these three sites. After 6 h of serum deprivation, little or no
phosphorylated BAD could be detected. Addition of CPT-cAMP 24 h
after serum deprivation increased phosphorylation of BAD at
Ser155. Phosphorylation could be detected at 15 min and was
more extensive at 30 min. Expression of AKAP121 (PC-AKAP121
We next asked whether expression of AKAP121 modulates BAD
phosphorylation induced by other signaling pathways. Using the
phospho-BAD specific antibodies described above, we performed
immunoblot analysis on total proteins from serum-deprived PC-A121 cells
stimulated with neurotrophin (NGF), phorbol ester (TPA), or CPT-cAMP
(Fig. 4, C-E). NGF stimulated Ser155 and
Ser112 phosphorylation 2-fold in both control cells (+ dox)
and in cells that expressed AKAP121 (
AKAP121 specifically stimulated phosphorylation of mitochondrial PKA
substrates. Fig. 5A shows that
the transient increase in phospho-CREB in cells treated with CTP-cAMP
was unaffected by AKAP121 induction. Similarly, CREB-directed
expression of a CRE-CAT fusion was not enhanced by AKAP121 induction
during CPT-cAMP exposure (Fig. 5B), nor did AKAP121 affect
NGF-dependent phosphorylation of ERK and activation of the
MAPK signaling pathway (Fig. 5, C and D) (55,
57). Furthermore, stimulation of the MAPK signaling pathway by cAMP, as
shown by ERK phosphorylation, was likewise independent of
AKAP121 (Fig. 5E). These data indicate that expression of
AKAP121 selectively up-regulates PKA signaling to the mitochondria without affecting the rate or magnitude of PKA-dependent or
MAPK-dependent signaling to the nucleus.
Anchoring of PKA on Mitochondria Is Critical for Survival--
The
experiments described above suggest that localization of PKA on
mitochondria mediated by AKAP121 plays a critical role for
PKA-dependent inhibition of apoptosis. To further support this notion, we generated a mutant carrying L313P and L319P
substitutions within the R-binding domain of AKAP121 (AKAP121m). These
mutations disrupt an amphipathic helix that is required for AKAP·PKA
interaction in vitro and in vivo (58). The mutant
transgene was subcloned into a eucaryotic expression vector under the
control of the TetR promoter and stably transfected in
PC12-tet-off cells as described above for wild-type AKAP121. Expression
of AKAP121m was induced by growing the cells for 48 h in the
absence of doxycyclin. Total cellular proteins were then extracted and
assayed for AKAP121m by immunoblot and RII overlay analyses. As shown
in Fig. 6A, wild-type and
mutant AKAP121 accumulate to comparable levels after doxycyclin removal. As predicted, the affinity of AKAP121m for RII was
significantly lower than wild-type AKAP121. The mutant protein remains
associated with mitochondria (Fig. 6B). Expression of the
mutant protein provokes the movement of PKA from mitochondria to the
cytosol without significantly altering the total concentration of the kinase (Fig. 6, C and D).
The phenotype of PC12 cells that express AKAP121m is shown in Fig.
7. Expression of AKAP121m correlates with
increased apoptosis, as shown by reduced cell viability and activation
of mitochondrial pro-caspase 9, and the extent of apoptosis is directly
related to the amount of AKAP121m expressed (Fig. 7A). The
proapoptotic effects of AKAP121m are evident both in growing cells
(Fig. 7B) and in cells deprived of serum (Fig. 7,
C-E). Furthermore, AKAP121m impedes
cAMP-dependent phosphorylation of endogenous BAD at
Ser155 (Fig. 7F). The data indicate that
AKAP121m protein acts in a dominant-negative fashion by displacing
AKAP121/PKA from mitochondria and down-regulating cAMP signaling to
these organelles.
PC12 cells deprived of serum and trophic factors undergo apoptosis
(48, 49). Activation of cAMP·PKA signaling prevents apoptosis and
induces their differentiation toward neuronal cells (50-52, 59-61).
We have sought to identify the PKA targets involved in this response.
Note that PKA phosphorylates and modulates a great variety of cellular
substrates localized in distinct cellular compartments. We report here
that reversible phosphorylation of PKA substrates on or within
mitochondria inhibits apoptosis. This has been achieved by establishing
a PC12 line (PC-A121) that conditionally expresses AKAP121, a scaffold
protein that anchors PKA to the outer membrane of mitochondria
(27-29). AKAP121 induction in PC-A121 cells stimulates translocation
of PKA to mitochondria. When the induced cells are treated with cAMP,
there is enhanced phosphorylation of BAD at Ser155.
Phosphorylation of BAD correlates with inhibition of
cytochrome c release from mitochondria and reduced
apoptosis. Our system is uniquely suited to analyze the effects of PKA
and cAMP on mitochondrial physiology and apoptosis: 1) expression of
AKAP121 is efficient, reversible, and temporally modulated; 2) AKAP121
increases PKA targeting to mitochondria without affecting the total
concentration of cellular PKA holoenzyme; 3) AKAP121 facilitates
PKA-cAMP signaling to mitochondria without affecting cAMP signaling to
the nucleus or activation of MAPK signaling by cAMP or NGF. Conversely,
a mutant of AKAP121 that does not bind PKA but localizes on
mitochondria (AKAP121m) acts as dominant-negative. Induction of
AKAP121m activates mitochondrial caspase-9 and promotes apoptosis, even
in the presence of trophic factors. The mutant protein displaces
endogenous AKAP121·PKA complexes from mitochondria sites, thus
impairing physiological flux of cAMP signals from cell membrane to
organelles. In particular, cAMP-dependent phosphorylation
of endogenous BAD at Ser155 is down-regulated. A similar
mechanism has been postulated for the Biochemical and genetic studies indicate that Ser155 of BAD
is the PKA high affinity site (43-45). However, most of these studies were performed supplying BAD as a substrate. BAD phosphorylation was
measured with recombinant protein in vitro or expressing
exogenous BAD in vivo. In this work, we have explored
site-specific phosphorylation of endogenous BAD following activation of
distinct signaling pathways. We found that PKA specifically
phosphorylates BAD at Ser155 in intact cells. This effect
is potentiated by AKAP121 and inhibited by AKAP121m.
Ser112, another potential phosphorylation site, was
efficiently phosphorylated after activation of the MAPK or protein
kinase C signaling pathways. No phosphorylation of Ser136
was observed under any of our experimental conditions (43). Different
signals converge to inactivate BAD through phosphorylation at various
serine resides (42-45). The specificity of the responding serines and
the extent to which they are modified may be a critical element to
discriminate the pathway activated and the intensity of the signal. In
this respect, BAD may be similar to other key signaling molecules where
many pathways converge, such as the cyclin-dependent kinase
(CDK) inhibitor p27 (62).
Our studies demonstrate that AKAP121·PKA complexes play a unique role
in mediating cAMP signaling to mitochondria. The cAMP pathway
influences mitochondrial physiology at multiple points, and AKAP121
appears to be an important multifaceted mediator of these effects. For
example, we recently found that AKAP121 binds the
3'-untranslated region of mRNA encoding mitochondrial
proteins and that this interaction is stimulated by PKA phosphorylation of AKAP121.2 Thus AKAP121
assembles protein kinases, mRNA, and possibly protein phosphatases
on the mitochondrial surface in proximity to heterogeneous PKA
substrates and other macromolecules critical for mitochondrial function(s).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase
vector. 24 h after transfection, the cells were washed twice with
phosphate-buffered saline and serum-starved overnight. Drug
concentrations were: CPT-cAMP (50 or 250 µM, Sigma),
nerve growth factor (NGF) (100 ng/ml, Roche Molecular Biochemicals) or
phorbol ester (TPA, 220 ng/ml, Sigma), H89 (5 µM,
Calbiochem), H2O2 (200 µM, CarloErba).
-glycerol-phosphate). Total lysate (0.1 mg) was cleared by centrifugation at 15,000 × g for 15 min, resolved by
SDS-PAGE gel electrophoresis, and immunoblotted as described previously (34). Chemiluminescent (ECL) signals were quantified by scanning densitometry (Amersham Biosciences). For caspase-9, total extracts were
prepared using lysis buffer (50 mM PIPES/KOH, pH 6.5, 2 mM EDTA, 0.1% Chaps, 5 mM dithiothreitol, and
protease inhibitors) provided by Cell Signaling. Cytosolic fractions
for anti-cytochrome c were prepared as described (48). Cell
fractionation for cytosolic and mitochondrial fractions were prepared
as described (27).
(dilution 1/250) polyclonal antibodies. Fluorescein- or
rhodamine-tagged anti-rabbit and anti-mouse IgG (Technogenetics)
secondary antibodies were used. Coverslips were analyzed as described
(34).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Inhibition of apoptosis by
cAMP·PKA signaling. A, apoptosis induced by serum
deprivation of neuroblastoma cells (SK-N-BE). B, immunoblot
analysis for caspase-3 on total proteins from growing (0) or
serum-deprived neuroblastoma cells. C, apoptosis induced by
serum deprivation of neuroblastoma cells (SK-N-BE) ± CPT-cAMP
(100 µM). As shown in D, total extracts from
control or serum-deprived neuroblastoma cells ± CPT-cAMP were
immunoblotted with anti-caspase-3 antibody. E, apoptosis
induced by serum deprivation of PC12 cells ± CPT-cAMP. As shown
in F, CPT-cAMP-treated PC12 cells were serum-deprived for
48 h in the presence or absence of the PKA inhibitor, H89 (5 µM), and monitored for apoptosis. Data are expressed as
the mean ± S.E. of 3-6 independent experiments made in
duplicate.
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Fig. 2.
Expression of AKAP121 in
rat pheochromocytoma cells (PC12) by a Tet-off vector.
A, schematic diagram of AKAP121 protein. KH,
RNA-binding motif; RBD, R-binding domain; MT,
mitochondrial targeting domain. B, RII overlay (left
panel) and immunoblot (IB) analysis of total proteins
extracted from control (C) or PC12 cells stably transfected
with AKAP121 vector (clones number 3 and 11). Doxycyclin was removed
from the medium for 48 h. A proteolytic RII-binding fragment of
AKAP121 of ~80 Kda was sometimes present in the extracts.
C, double immunofluorescence of PC-A121 dox cells
using specific antibody directed against AKAP121 (a) and
manganese superoxide dismutase (b). A field containing
several cells is shown. The punctate pattern of AKAP121 colocalizes
with mitochondria, as shown by the merge of both signals
(c). PC-A121 + dox cells were stained with anti-AKAP121 and
used as control (d). A higher magnification of a single
PC-A121
dox cell is also shown (inset).
Bars, 5 µm. As shown in D, protein extracts
from total lysate (clone number 11) or from purified mitochondria
(mito) (clones number 11 and 3) were immunoblotted with
anti-RII or anti-manganese superoxide dismutase antibodies. Lower
panels represent densitometric analyses. The data are relative to
values from control cells set as 2 for mitochondrial fractions (clone
number 3 + dox) and set as 50 for total lysate (clone number 11 + dox) and represent the mean ± S.E. of three independent
experiments that gave similar results. MnSOD, manganese
superoxide dismutase.
dox). We conclude that AKAP121 expression protects cells against
apoptosis induced by serum deprivation.
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Fig. 3.
Expression of AKAP121 increases survival of
PC12 cells. A, growth curve of control (PC12) or
PC-AKAP121 cells. The data are expressed as the mean ± S.E. of
five independent experiments made in triplicate. B,
apoptosis following serum deprivation of control (PC12) or PC-A121
cells ± dox. C, same as in panel B, except
that H89 (5 µM) was added to the medium of PC-A121
cells dox cells. As shown in D, control (PC12) or
PC-A121 cells ± dox were treated with 200 µM
H2O2, harvested 24 h later, and counted.
*, p < 0.001 when compared with control PC-A121 + dox
cells. As shown in E, cytosolic fractions from growing (0)
or serum-starved (6 and 24 h) PC-A121 ± dox cells were
subjected to immunoblot analysis with anti-cytochrome c
(upper panel) or anti-p21/waf (lower panel)
specific antibody. F, densitometric analysis of the
experiments presented in panel E. Data are expressed as the
mean ± S.E. of three independent experiments that gave similar
results.
dox) showed H2O2-induced
apoptosis (Fig. 3D). The release of cytochrome c
from mitochondria is a critical step in the activation of downstream
effectors of the apoptotic pathway. The binding of released cytochrome
c to Apaf-1 induces the formation of the "apoptosome"
complex and the sequential activation of the caspase cascade (38). As
shown in Fig. 3, E and F, deprivation of trophic
factors in PC-A121 + dox cells induced a time-dependent
translocation of cytochrome c from mitochondria to cytosol.
Expression of AKAP121 (PC-A121
dox) delayed cytochrome c release (Fig. 3, see 6 and 24 h). Under these
conditions, activation of pro-caspase-3 was partly inhibited by AKAP121
expression (data not shown).
dox) stimulated basal and cAMP-induced phosphorylation of BAD
Ser155 at both time points. Under these conditions,
phosphorylation of BAD at Ser136 and Ser112 was
undetectable, even after a 60-min exposure of cells to CPT-cAMP (Fig.
4, A and B, and data not shown).
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Fig. 4.
AKAP121 stimulates PKA-dependent
phosphorylation of BAD at Ser155. As shown in
A, serum-starved PC-A121 cells were treated with CPT-cAMP
(200 µM) for 15 or 30 min. Total proteins were extracted
and immunoblotted with anti-phospho-BAD or anti-BAD specific
antibodies. B, cumulative data relative to
phospho-Ser155. The data are expressed as relative
densitometric units ± S.E. of three independent experiments. As
shown in C, PC-A121 ± dox cells were serum-deprived
for 24 h and treated with NGF (100 ng/15 min), TPA (220 ng/30
min), or CPT-cAMP (50 µM or 250 µM/30 min).
Where indicated, cells were pretreated with H89 (5 µM).
Total proteins were extracted and immunoblotted with antibody to
phosphorylated BAD Ser155. Total BAD protein was detected
as above. D, densitometric analysis of the experiment shown
in panel C. The data represent the mean of three independent
experiments. E, densitometric analysis of BAD phosphorylated
at Ser112. The data represent a mean ± S.E. of four
independent experiments.
dox). TPA enhanced
Ser112 phosphorylation but had no effect on
Ser155. As shown above, CTP-cAMP induced phosphorylation of
Ser155 but not Ser112, and this was enhanced by
AKAP121 expression. AKAP121 enhancement of BAD Ser155
phosphorylation was detectable even at very low concentrations of
CPT-cAMP (50 µM). As expected, CPT-cAMP stimulation of
BAD phosphorylation was sensitive to the PKA inhibitor, H89.
Ser136 phosphorylation was unaffected by any of the stimuli
applied (data not shown).
View larger version (49K):
[in a new window]
Fig. 5.
AKAP121 does not influence CREB- and
MAPK-dependent signaling. As shown in A,
serum-deprived PC-A121 ± dox cells were treated with CPT-cAMP
(200 µM) for the indicated times, harvested, and lysed.
Total proteins were immunoblotted with either
anti-phospho-Ser133 of CREB or anti-CREB antibodies.
P-CREB, phospho-CREB. As shown in B, PC-A121 ± dox cells were transiently transfected with a CRE-CAT reporter
cDNA vector, serum-deprived for 24 h, and treated with
CPT-cAMP (200 µM) for the indicated times. CAT activity
is expressed as relative units and represents a mean ± S.E. of
three independent experiments. C, immunoblot analysis of
total proteins extracted from serum-starved or NGF-stimulated
PC-A121 ± dox cells by using anti-phospho-ERK or anti-ERK
antibody. P-ERK, phospho-ERK. As shown in D,
PC-A121 ± dox cells were transiently co-transfected with pBD-Elk1
and Gal-CAT cDNA vectors, serum-deprived overnight, and stimulated
with NGF (100 ng/ml). CAT activity is expressed as relative units and
represents a mean ± S.E. of three independent experiments.
E, immunoblot analysis of total proteins extracted from
serum-starved or CPT-cAMP-stimulated PC-A121 ± dox cells by using
anti-phospho-ERK or anti-ERK antibody.
View larger version (37K):
[in a new window]
Fig. 6.
AKAP121m, which does not anchor PKA,
displaces PKA from mitochondria. As shown in A, total
proteins from control (C) or PC12 cells expressing AKAP121m
(L313/319P) were immunoblotted (IB) with anti-AKAP121
antibody (left panel) or subjected to RII binding assay
(right panel). B, double immunofluorescence of
PC-A121m dox cells using anti-AKAP121 (AKAP121m,
green) and anti-superoxide dismutase antibodies
(MnSOD, red). A merge (yellow) of both
signals is also presented. Bar, 5 µm. As shown in
C, total lysates, purified mitochondria (mito),
or cytosolic fractions from PC-A121 ± dox cells were
immunoblotted with anti-RII antibody, anti-manganese superoxide
dismutase, or anti-ERK antibodies. D, densitometric analyses
of the experiments indicated in panel C.
View larger version (34K):
[in a new window]
Fig. 7.
AKAP121m down-regulates cAMP signaling to
mitochondria and promotes apoptosis. A, immunoblot
analysis on total extracts from PC-A121m cells grown for 48 h in
the absence ( dox) or presence of the indicated amount of doxycyclin
(0.5 ng/ml or 10 ng/ml). B, growth curve of PC12 cells
expressing wild-type (PC-A121) or mutant AKAP121 (PC-A121m). As shown
in C, viable cells following serum deprivation
(S.D.) were harvested and counted. Cumulative data are
expressed as the mean ± S.E. of 4-6 independent experiments made
in duplicate. D, immunoblot analysis for caspase-9 of total
extracts from serum-deprived PC-A121m ± dox cells.
Asterisks indicate processed, activated caspase-9.
E, immunoblot analysis for caspase-9 of total extract from
serum-deprived PC-A121m cells. F, BAD phosphorylation in
AKAP121m expressing cells treated with CPT-cAMP (200 µM/30 min). The densitometric analysis is expressed as
the mean of two independent experiments that gave similar results.
P-BAD, phospho-BAD.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-adrenergic receptor where
expression of an AKAP79 mutant, which does not bind PKA but still
associates with the receptor, down-regulates PKA-dependent
phosphorylation of the receptor (26). The use of such
proline-derivative AKAP mutants represents a novel and useful approach
to dissect and selectively manipulate signaling pathways traveling from
cell membrane to target organelles.
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ACKNOWLEDGEMENTS |
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Special thanks go to Drs. John Scott, Susan Taylor, and Charles Rubin for helpful suggestions and critical discussion. Mouse AKAP121 cDNA was kindly provided by Dr C. Rubin. We thank Rita Cerillo for technical support. We are in debt to Prof. Corrado Garbi for valuable help in fluorescence microscopy and to Franco D'Agnello for the artwork.
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FOOTNOTES |
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* This work was supported by grants from "Associazione Italiana per la Ricerca sul Cancro" (AIRC) and Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST) (Italian Department of University and Research).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.
These authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 39-081-7463614; Fax: 39-081-7463252; E-mail: feliciel@unina.it.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M209941200
2 M. D. Ginsberg, A. Feliciello, E. V. Avvedimento, and M. E. Gottesman, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: PKA, cAMP-dependent protein kinase; AKAP, A-kinase anchor protein; CAT, chloramphenicol acetyl transferase; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; CRE, cAMP-response element; CREB, CRE-binding protein; CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate; NGF, nerve growth factor; dox, doxycyclin; TPA, 12-O-tetradecanoylphorbol-13-acetate; PIPES, 1,4-piperazinediethanesulfonic acid; RSV, Rous sarcoma virus.
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