Essential Role of A-kinase Anchor Protein 121 for cAMP Signaling to Mitochondria*

Adele AffaitatiDagger, Luca CardoneDagger, Tiziana de Cristofaro, Annalisa Carlucci, Michael D. Ginsberg§, Stelio Varrone, Max E. Gottesman§, Enrico V. Avvedimento, and Antonio Feliciello

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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).

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 beta -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).

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 RIIpartial (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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

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).


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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.

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).


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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.

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 - 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.

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- 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).

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 - 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.

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 (- 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).

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.


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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.

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).


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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.

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.


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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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.

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).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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