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A Direct Interaction between the N Terminus of Adenylyl Cyclase AC8 and the Catalytic Subunit of Protein Phosphatase 2A

Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom

Received August 18, 2005; accepted October 28, 2005

	Abstract

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Although protein scaffolding complexes compartmentalize protein kinase A (PKA) and phosphodiesterases to optimize cAMP signaling, adenylyl cyclases, the sources of cAMP, have been implicated in very few direct protein interactions. The N termini of adenylyl cyclases are highly divergent, which hints at isoform-specific interactions. Indeed, the Ca²⁺-sensitive adenylyl cyclase 8 (AC8) contains a Ca²⁺/calmodulin binding site on the N terminus that is essential for stimulation of activity by the capacitative entry of Ca²⁺ in the intact cell. Here, we have used the N terminus of AC8 as a bait in a yeast two-hybrid screen of a human embryonic kidney (HEK) 293 cell cDNA library and identified the catalytic subunit of the serine/threonine protein phosphatase 2A (PP2A_C) as a binding partner. Confirming the highly specific nature of this novel interaction, glutathione-S-transferase fusion proteins containing the full-length N terminus of AC8 affinity precipitated catalytically active PP2A_C from both HEK293 and mouse forebrain membranes—the latter a normal source of AC8. The scaffolding subunit of PP2A (PP2A_A; 65 kDa) was also precipitated by the N terminus of AC8, indicating that AC8 may occur in a complex with the PP2A core dimer. The interaction between the N terminus of AC8 and PP2A_C was antagonized by Ca²⁺/calmodulin. However, PP2A_C and Ca²⁺/calmodulin did not share identical binding specificities in the N terminus of AC8. PKA-mediated phosphorylation did not influence either calmodulin or PP2A_C association with AC8. In addition, both PP2A_C and AC8 occurred in lipid rafts. These findings are the first demonstration of an association between adenylyl cyclase and any downstream element of cAMP signaling.

AC8 is predominantly expressed in brain areas associated with learning and memory (Mons et al., 1998). Indeed, AC8 knockout mice display deficits in hippocampal long-term potentiation, a cellular correlate of memory formation, which involves dynamic alterations in [Ca²⁺]_i and cAMP (Frey et al., 1993; Poser and Storm, 2001; Wang et al., 2003). The molecular basis whereby an increase in [Ca²⁺]_i regulates AC8 activity is becoming clearer (Gu and Cooper, 1999). The N terminus of AC8 contains an amphipathic -helical calmodulin-binding domain, which is absolutely essential for stimulation by Ca²⁺ in the intact cell (Gu and Cooper, 1999; Smith et al., 2002). Deletion of the N terminus results in an activity that, although still able to be stimulated by Ca²⁺/calmodulin in vitro, is no longer able to be stimulated by CCE in the intact cell (Smith et al., 2002). The N termini of all adenylyl cyclase isoforms are highly divergent and might be expected to function in isozyme-specific interactions. Indeed, recently, using a yeast two-hybrid approach, the N terminus of AC6 was found to interact with snapin, a component of the soluble N-ethylmaleimide-sensitive factor attached protein receptor complex (Chou et al., 2004). Because we were convinced that the N terminus of AC8 played a critical role in regulation in the intact cell, we adopted a yeast two-hybrid strategy to search for regulatory partners of AC8. We found that, in addition to associating with calmodulin, the N terminus of AC8 interacts with the catalytic subunit of protein phosphatase 2A (PP2A_C). The N terminus of AC8 pulled down PP2A_C not only from HEK293 cell lysates, but also from brain homogenates. In addition, AC8 and PP2A_C were both detected in lipid rafts, which suggests that PP2A may be situated to efficiently control downstream kinase activity, initiated by the Ca²⁺-dependent activation of AC8. These findings identify a potentially important direct interaction between adenylyl cyclase and a downstream component of the cAMP signaling cascade.

	Materials and Methods

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Materials. Monoclonal flotillin and PP2A catalytic subunit antibodies were obtained from BD Biosciences Transduction Laboratories (Erembodegem, Belgium). Monoclonal calmodulin antibody was from Upstate Biotechnology (Lake Placid, NY). Polyclonal PP2A structural subunit A and the polyclonal pan-PP2A_B antibody were a gift from Dr. Brian Wadzinski (Department of Pharmacology, Vanderbilt University, Nashville, TN). Polyclonal G_s-olf and PKA catalytic subunit (C-20) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from GE Healthcare (Little Chalfont, Buckinghamshire, UK), and horseradish peroxidase-conjugated goat anti-mouse IgG conjugated was from Promega (Madison, WI). Paraformaldehyde was obtained from TAAB Laboratories (Aldermaston, UK). Tissue culture media and mammalian protease inhibitor cocktail were purchased from Sigma Chemical (Poole, Dorset, UK). His-Select cobalt affinity gel was from Sigma, and glutathione-Sepharose was from GE Healthcare.

Yeast 2-Hybrid System. The N terminus of rat AC8 (residues 1–179) was PCR-amplified from a plasmid containing full-length AC8 (construct 8Nt). The following oligonucleotide primers were used: 5'-ccg aat tca tgg aac tct cgg atg tgc act gcc tta g-3' (primer r_AC8-Nt-F) and 5'-atg gat ccc tcc gat ttg cgc ctc tgg ccc agg aa-3' (primer r_AC8-Nt-R). The resulting PCR product was digested with EcoRI and BamHI restriction enzymes and subcloned between the EcoRI and BamHI sites of plasmid pGBK-T7 (BD Biosciences). The PCR-amplified insert (pGBK-8Nt) was sequenced to check for errors, and then the vectors were tested for self-activation in the yeast two-hybrid system alongside the empty transcription-activation domain vector pGAD-T7 (BD Biosciences). Plasmids pGBK-8Nt failed to self-activate. The AC8 N terminus encoding the DNA fragment was also subcloned between the EcoR1 and XhoI sites of plasmid pGAD-T7 (generating pGAD-8Nt1) to allow interactions detected in the screen to be confirmed in both directions. Yeast strain AH109 (genotype, MATa trp1-901 leu2–3,112 ura3-52 his3-200 gal4gal80 LYS2::GAL1_UAS-GAL1_TATA-HIS3 MEL1 GAL2_UAS-GAL2_TATA-ADE2, URA3::MEL1_UAS-MEL1_TATA-lacZ), a human kidney cDNA library (with 2.5 x 10⁶ independent clones and average cDNA size of 1.5 kilobases), as the source of cDNA and plasmids used in this study were obtained from BD Biosciences as part of their Matchmaker 3 system. For the stringent selection of interacting clones, media lacking histidine, leucine, tryptophan, and adenine were used.

Production and Expression of Recombinant Proteins. The N terminus of rat AC8 was PCR-amplified from a plasmid containing full-length AC8 from residues 1 to 179 (construct 8Nt). The following oligonucleotide primers were used: 5'-ccg aat tca tgg aac tct cgg atg tgc act gcc tta g-3' and 5'-cgc gtc gac tta ctc cga ttt gcg cct ctg g-3'. The resulting PCR product was digested with EcoRI and SalI restriction enzymes and subcloned between the EcoRI and SalI sites of plasmid pGEX4T (GE Healthcare) to produce a fusion protein between glutathione-S-transferase (GST) and NtAC8. The C terminus of rat AC8 was PCR-amplified from a plasmid containing full-length AC8, from residues 1106 to 1248 (construct C2bAC8). The following oligonucleotide primers were used: 5'-gcg agc tcg aca ttt ggg gta aaa ctg-3' and 5'-acg cgt cga ctt atg gca aat cgg att tg-3'. The resulting PCR product was digested with SacI and SalI restriction enzymes and subcloned between the SacI and SalI sites of plasmid pQE30 (QIAGEN, Valencia, CA) to produce a fusion protein containing 6x histidines at the Nt of C2bAC8. Both fusion proteins were expressed in Escherichia coli XL10 Gold and purified either on glutathione-Sepharose (GST-NtAC8) or cobalt affinity gel (His-C2bAC8).

Preparation of HEK293 Crude Membranes. HEK293 cells were detached with phosphate-buffered saline containing 0.03% EDTA and centrifuged at 195g for 5 min. The supernatant was removed and the pellet resuspended in hypotonic lysis buffer (10 mM Tris, 1 mM EDTA, 1 mM EGTA, and protease inhibitors, pH 8.0). After 10 min, cells were homogenized at 4°C by 50 strokes in a tight-fitting Dounce homogenizer followed by centrifugation (195g at 4°C for 5 min). The supernatant was centrifuged at 17,257g (15 min, 4°C). The supernatant was removed, and the pellet, representing crude membranes, was collected.

Preparation of Mouse Forebrain Crude Membranes. Adult mouse forebrains were dissected into ice-cold buffer consisting of 50 mM Tris, 1 mM MgCl₂, 1 mM EDTA, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 1 mM benzamidine, and 1 µg of DNAase, pH 7.4, and lysed by passing through a 0.22-gauge needle 20 times. After further centrifugation (195g at 4°C for 5 min) and dissociation, the lysate was centrifuged at 17,257g (15 min, 4°C). The supernatant was removed, and the pellet, representing crude membranes, was collected.

AC8 N- and C-Terminal Affinity Precipitation. Isolated crude membranes were solubilized in 2% SDS solubilization buffer (50 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, and protease inhibitors, pH 7.4) (Leonard et al., 1998) followed by centrifugation (17,257g at 4°C for 15 min). The supernatant was diluted (1:20) in binding buffer (50 mM phosphate buffer, pH 7.4, 150 mM NaCl, 0.2% Triton X-100, 1 mM EDTA, and 1 mM EGTA) and precleared for 30 min or more with 5 µl of a 50% suspension of GST glutathione-Sepharose. GST-NtAC8 glutathione-Sepharose that had been washed three times in dilution buffer was added to the precleared sample and rotated for 3 h at 4°C. Otherwise, for in vitro pull-downs, GST or cobalt affinity gels were used as controls, as noted in the figure legends. The Sepharose/cobalt beads were collected and washed four times in ice-cold binding buffer, resuspended in 2x boiling buffer (final concentration, 125 mM Tris, 300 mM dithiothreitol, 20% glycerol, and 0.004% bromphenol blue, pH 6.8) and heated at 100°C for 5 min. Supernatant was collected, and samples were stored at –80°C. The results shown are representative of at least five experiments with similar results.

Phosphorylation Assay. GST-NtAC8, His-C2bAC8, or myelin basic protein, as positive control), were incubated with either a cell-free extract of forskolin (20 µM) and prostaglandin E₁ (100 nM)-induced HEK293 cells or the catalytic subunit of PKA (Sigma) in phosphorylation buffer incubated with [-³²P]ATP (10 µCi; GE Healthcare) (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM EDTA, 10 µM ATP, 2 mM dithiothreitol, 0.01% Triton X-100, 20 µg/ml aprotonin, 20 µg/ml leupeptin, and 10 µg/ml pepstatin A), with or without six units of PKA (Sigma) at 30°C for 20 min. After separation by SDS-PAGE, phosphorylated proteins were detected by autoradiography.

Dephosphorylation Assay. myelin basic protein (250 µg) was phosphorylated as described above with 24 units of PKA in phosphorylation buffer incubated with [-³²P]ATP. The reaction was terminated by filtration in a Microcon YM-3 filter (Millipore Corporation, Billerica, MA) and washed with 500 µl of 20 mM Tris-HCl and 140 mM NaCl, pH 7.6. The phosphorylated myelin basic protein was resuspended in 100 µl of 12.5 mM Tris-HCl and 25 µM CaCl₂, pH 7.6, and 25-µl fractions were mixed with the resultant affinity precipitates (GST or GST-NtAC8) from Triton X-100-solubilized HEK293 cells. After incubation (30 min at 30°C), the reaction was terminated by the addition of 6 µl of 5x boiling buffer and heated at 100°C for 5 min. Samples were run on a 13% SDS-PAGE gel and exposed to Hyperfilm film for 17 h (GE Healthcare).

Immunofluorescence. Rat hippocampal neurons were cultured as described previously (Chawla et al., 2003) and transfected with N-terminally tagged GFP-AC8 (0.4 µg of cDNA) after 8 DIV using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Forty-eight hours after transfection, neurons were washed with phosphate-buffered saline (12.1 mM Na₂HPO₄, 4 mM KH₂PO₄, and 130 mM NaCl, pH 7.4) and fixed using 4% paraformaldehyde (1 h, 20°C). Otherwise, before fixing, neurons were prepermeabilized in 0.5% Triton X-100 at 4°C for 30 min (Hering et al., 2003). Coverslips were mounted in Antifade (Invitrogen) according to the manufacturer's procedures and the cells were visualized on a Zeiss Axiovert LSM510 confocal microscope (Carl Zeiss GmbH, Jena, Germany), using 40x and 63x oil immersion objectives. The scale bar in all images represents 10 µM.

Detergent-Resistant Membrane Preparation. Mouse forebrain crude membranes were incubated with ice-cold Triton X-100 in TNE buffer (150 mM NaCl, 5 mM EDTA, and 25 mM Tris) containing 0.1 M sodium carbonate, pH 11 (Ostermeyer et al., 1999). The suspension was transferred to a Dounce homogenizer and homogenized with 20 strokes and left on ice for 30 min. The homogenate was adjusted to 40% sucrose by the addition of 60% sucrose in TNE buffer, pH 7.4. The extract was placed below a 5 and 30% discontinuous sucrose gradient prepared in cold TNE buffer and centrifuged in a Beckman SW55 rotor at 24,000 rpm for 16 h at 4°C (Beckman Coulter, Fullerton, CA). Fractions (10 x 0.5 ml) were collected from the top of the gradient. The isolated fractions were diluted in TNE buffer and centrifuged in a Beckman SW55 rotor at 50,000 rpm for 1 h at 4°C. The pelleted membranes were resuspended in 1% SDS. Finally, samples were suspended in boiling buffer heated to 100°C for 5 min and stored at –80°C.

Immunoblotting. Proteins were resolved using SDS-PAGE 7.5% and 12% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. The polyvinylidene difluoride membrane was incubated in TBS blocking buffer (20 mM Tris, pH 7.5, 150 mM NaCl) containing 5% nonfat dry milk for 30 min, followed by two 10-min washes in TBS supplemented with 0.05% (v/v) Tween 20 (TTBS). Membranes were incubated overnight at room temperature with anti-PP2A_C mAb (1:5000), anti-PKAcat pAb (1:1000), anti-flotillin mAb (1:5000), anti-calmodulin mAb (1:5000), mAb anti-Gs-olf (1:1000), in TTBS containing 1% nonfat dry milk (antibody buffer). The membranes were washed (2 x 10 min) in TTBS and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5000 dilution of stock) or goat anti-mouse IgG conjugated to horseradish peroxidase (1:3000) in antibody buffer for 1 h. Finally, the membranes were washed in TTBS (2 x 10 min), rinsed in TBS, and treated with enhanced chemiluminescence plus reagent and exposed to Hyperfilm (GE Healthcare).

	Results

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The N Terminus of AC8 Interacts Specifically with PP2A Catalytic Subunit. AC8 lacking the N terminus is insensitive to regulation by Ca²⁺ in the intact cell, despite being targeted to lipid rafts, which is a property that is essential for regulation by CCE (Smith et al., 2002). However, AC8 is fully stimulated by Ca²⁺/calmodulin in vitro (Smith et al., 2002). This observation suggests that the N terminus of AC8 might engage with elements of the CCE regulatory apparatus. To determine whether such proteins would interact with the N terminus of AC8, amino acids 1 to 179 (i.e., the full-length N terminus) was cloned into a bait vector to screen an HEK293 cDNA library using the yeast two-hybrid system. The first screen identified 77 colonies that survived on synthetic media lacking histidine. The plasmids were recovered into E. coli and sequenced. Duplicate clones and common false-positives (transcription factors and "housekeeping" genes, such as those involved in metabolism) were discarded, leaving a pool of 22 genes of potential interest. The bait and prey vectors were reversed and tested positive in every case. Of these positive interacting plasmids, the one with most potential interest was a peptide sequence (Ser¹²⁰ to Leu³⁰⁹) corresponding to the C terminus of the -catalytic subunit of the serine/threonine PP2A_C. Other proteins that may yet be of some interest included a G protein, G₂, a laminin receptor, and a WD repeat domain protein; the others were rather obscure.

The finding that PP2Ac interacted with the N terminus of the Ca²⁺/calmodulin-stimulatable AC8 was deemed extremely interesting because PP2Ac participates in a number of signaling complexes (Lebrin et al., 1999; Boudreau et al., 2002). To be specific, PP2Ac interacts with the C-terminal region of the _1C L-type VGCC, which is known to be in a functional signaling complex in the rat forebrain with the 2-adrenergic receptor and an unidentified adenylyl cyclase isoform (Davare et al., 2000, 2001). No obvious candidates that might have been involved in regulating CCE were identified; therefore, further investigations centered on the association of the N terminus with PP2A_C.

PP2Ac is a member of the PPP family of serine/threonine phosphatases, which have conserved catalytic regions. Evidence from the crystal structure of the PPP family member PP1c suggests that regulation, either through post-translational modification or binding proteins, occurs at the C terminus (Egloff et al., 1997; Barford et al., 1998). In an effort to narrow down more precisely the domain of PP2Ac that interacted with the N terminus of AC8, two halves of the positive interacting PP2Ac fragment (Ser¹²⁰ to Arg²¹⁵) and (Arg²¹⁵ to Leu³⁰⁹) were expressed as histidine fusion proteins in E. coli. However, the peptide fragments were only present as an aggregated form within inclusion bodies, preventing further binding studies.

AC8 is predominantly neuronal in expression, with the highest levels of mRNA detected in the cortex, hippocampus, and cerebellum (Cali et al., 1994). Therefore, solubilized mouse cortical membranes were used to confirm the selectivity of the interaction between the N terminus of AC8 and PP2A_C, using a GST recombinant fusion protein containing the full-length N terminus of AC8 (GST-NtAC8). Initial GST pull-downs were carried out on cortical membranes solubilized with 1% Nonidet P-40 (NP40). Incubating GST or GST-NtAC8 with solubilized mouse forebrain membranes demonstrated that the full-length, native PP2A_C (36 kDa) was affinity-precipitated by GST-NtAC8, compared with a far less extent by a gross excess of GST (Fig. 1A).

View larger version (31K): [in this window] [in a new window]   Fig. 1. The N terminus of AC8 interacts with the catalytic subunit of protein phosphatase 2A. Mouse cortical membranes were solubilized by either 1% NP40 (A) or 2% SDS (B), precleared with an excess of GST, and incubated with a GST fusion protein containing the full-length N terminus of AC8 immobilized on glutathione-Sepharose beads. The affinity precipitates were resolved by SDS-PAGE and were immunoblotted with an antibody raised against the catalytic subunit of protein phosphatase 2A (anti-PP2AC). Aliquots from the same experiments were examined for protein loading by Coomassie staining of membranes or parallel in-gel silver nitrite staining. C, HEK293 membranes were solubilized by 2% SDS, precleared with an excess of GST, and incubated with a GST fusion protein containing the full-length N terminus of AC8 immobilized on glutathione-Sepharose beads. The affinity precipitates were resolved by SDS-PAGE and were immunoblotted with an antibody raised against the catalytic subunit of protein phosphatase 2A (anti-PP2AC). Aliquots from the same experiments were examined for protein loading by parallel in-gel silver nitrite staining.

GST-NtAC8 also affinity-precipitated PP2A_C from solubilized HEK293 membranes (Fig. 1C). We believe in vitro GST pull-downs to be a more accurate method than coimmunoprecipitation experiments to ascertain genuine interactions involving AC8 and native PP2A_C. This is because it is extremely difficult to solubilize adenylyl cyclases to any degree of relative purity. Just how difficult this can be is clearly demonstrated with Western blots of adenylyl cyclases, which typically run at a much higher than expected molecular mass. Indeed genuine interactions between adenylyl cyclase and interacting proteins are believed to be lost during solubilization procedures (Chou et al., 2004). Taken together, the results obtained with the yeast two-hybrid analysis, which depend on correctly folded protein fragments for the detection of positive interactions and results from the GST-NtAC8 pull-down experiments, which examine interactions with full-length native PP2A_C, clearly demonstrate that the N terminus of AC8 can efficiently interact with native PP2A catalytic subunit, even in the presence of a highly diverse range of membrane proteins.

The N Terminus of AC8 Interacts with the PP2A Core Enzyme. The core enzyme of PP2A is a dimer consisting of the catalytic subunit and a scaffolding subunit of 65 kDa (PR2A_A), referred to as PP2A_D (Janssens and Goris, 2001). A third regulatory B subunit, of which there are four separate families, can associate with the core enzyme to target the trimeric holoenzyme to specific subcellular locations. However, the core enzyme is functional without the B subunit (Kremmer et al., 1997). To determine whether the N terminus of AC8 associated with the PP2A core enzyme complex, GST-NtAC8 affinity precipitates were probed with antibodies to both PP2A_C and PP2A_A. GST-NtAC8 pulled down both PP2A_A (Fig. 2A) and PP2A_C (Fig. 2B) from the same solubilized brain membrane preparation, which demonstrated that the N terminus of AC8 interacts with the PP2A core enzyme complex. However, no immunoreactivity against the B subunit was detected from aliquots that were positive for the core enzyme (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 2. The N terminus of AC8 interact with the PP2A core enzyme. A GST fusion protein containing the full-length N terminus of AC8 immobilized on glutathione-Sepharose beads was incubated with GST precleared brain membranes. The affinity precipitates were resolved by SDS-PAGE and immunoblotted with an antibody raised against the scaffolding subunit of protein phosphatase 2A (anti-PP2AA) (A) or an antibody raised against the catalytic subunit of protein phosphatase 2A (anti-PP2AC) (B). The Coomassie-stained membrane is shown at the bottom.

View larger version (19K): [in this window] [in a new window]   Fig. 3. The N terminus of AC8 interacts with catalytically active PP2AC. GST or a GST fusion protein containing the full-length N terminus of AC8 immobilized on glutathione-Sepharose beads was incubated with Triton X-100-solubilized HEK293 cell lysates. The resulting washed affinity precipitates were incubated with myelin basic protein that had been previously phosphorylated with the catalytic subunit of PKA in buffer containing [-32P]ATP. A, proteins were resolved by SDS-PAGE and the level of myelin basic protein dephosphorylation determined by autoradiography. B, the Coomassie-stained membrane is shown at the bottom.

View larger version (34K): [in this window] [in a new window]   Fig. 4. A competitive interaction between Ca2+/calmodulin and PP2AC for association with the N terminus of AC8. A, GST or GST fusion proteins containing the full-length N terminus of AC8 immobilized on glutathione-Sepharose were incubated in the absence (–) or presence (+) of 0.5 µM calmodulin and 20 µMCa2+. The pull-downs were resolved by SDS-PAGE and immunoblotted with an antibody raised against calmodulin (anti-calmodulin). The Coomassie-stained membrane is shown at the bottom. B, a GST fusion protein containing the full-length N terminus of AC8 immobilized on glutathione-Sepharose beads was incubated with GST precleared brain membranes containing either 1 mM EDTA/EGTA, no exogenous additions, 20 µMCa2+, 0.5 µM calmodulin, or 20 µM Ca2+ and 0.5 µM calmodulin. The affinity precipitates were resolved by SDS-PAGE and immunoblotted with an antibody raised against the catalytic subunit of protein phosphatase 2A (anti-PP2AC). The Coomassie-stained membrane is shown at the bottom. C, a GST fusion protein containing the full-length N terminus of AC8 immobilized on glutathione-Sepharose beads was incubated with GST-precleared brain membranes containing either 1 mM EDTA/EGTA or 20 µM Ca2+ and 0.5 µM calmodulin. The affinity precipitates were resolved by SDS-PAGE and immunoblotted with an antibody raised against calmodulin (anti-calmodulin). The Coomassie-stained membrane is shown at the bottom.

The Amino Acids in the N Terminus of AC8 that Are Critical for Calmodulin Binding Are Not Critical for PP2A_C Binding. Point mutations in the helical calmodulin binding domain between amino acids 34 and 51 in the N terminus of AC8 prevent Ca²⁺ stimulation of AC8 in the whole cell (Smith et al., 2002). We generated a GST recombinant fusion protein containing the full-length N terminus of AC8 with the same six critical amino acids mutated to alanine (GST-Nt8M34). To verify that this prevented calmodulin binding, GST-Nt8M34, GST, and GST-NtAC8 were incubated in the presence of exogenous Ca²⁺ and calmodulin in vitro. GST-Nt8M34 clearly showed no interaction with calmodulin compared with GST-NtAC8 (Fig. 5A). To determine the relative amounts of fusion proteins present in the individual incubations, the blot was stripped and reprobed for GST immunoreactivity (Fig. 5A, bottom blot). Densitometric analysis of this immunoblot demonstrated that for equal levels of GST-fusion protein, GST-Nt8M34 is unable to interact with calmodulin compared with GST-NtAC8 (Fig. 5A). Therefore, GST-Nt8M34, which is unable to bind calmodulin in vitro, was examined for its ability to interact with endogenous PP2A_C. Solubilized mouse forebrain membranes were precleared with an excess of GST and divided into two aliquots. GST-NtAC8 was added to one and GST-Nt8M34 to the other. Equal volumes from the resulting pull-down were compared for PP2A_C immunoreactivity. Both GST-Nt8M34 and GST-NtAC8 could clearly affinity-precipitate PP2A_C (Fig. 5B). This demonstrates that the amino acids critical for binding calmodulin are not critical for binding PP2A_C. Therefore, if GST-Nt8M34 does not bind calmodulin, then the addition of exogenous calmodulin to solubilized mouse forebrain membranes would not be expected to prevent interaction of GST-Nt8M34 with PP2A_C. Indeed, including 0.5 µM calmodulin and 20 µM Ca²⁺ in the pull-down assay did not prevent the affinity precipitation of PP2A_C by GST-Nt8M34 (Fig. 5C). Taken together, these results demonstrate that calmodulin and PP2A_C have overlapping binding domains on the N terminus of AC8, but the precise amino acids that are essential for binding calmodulin are not essential for binding PP2A_C.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Amino acids critical for Ca2+/calmodulin association are not essential for binding PP2AC. A, GST, a GST fusion protein containing the full-length N terminus of AC8, or a GST fusion protein containing the full-length N terminus of AC8 with point mutations in the calmodulin binding region (8M34) that were immobilized on glutathione-Sepharose beads were incubated in the presence or absence of 20 µM Ca2+ and 0.5 µM calmodulin. After washing with 1% Triton X-100, the pull-downs were resolved by SDS-PAGE and immunoblotted with an antibody raised against calmodulin (anti-calmodulin). The same blot stripped and reprobed with an antibody raised against GST (anti-GST) is shown at the bottom. The relative amount of the three fusion proteins was quantified by densitometric analysis of the GST immunoblot. B, GST fusion proteins containing the full-length N terminus of AC8 or the full-length N terminus of AC8 with point mutations in the calmodulin binding region (8M34) immobilized on glutathione-Sepharose beads were incubated with equivalent volumes of GST precleared brain membranes. The affinity precipitates were resolved by SDS-PAGE and immunoblotted with an antibody raised against the catalytic subunit of protein phosphatase 2A (anti-PP2AC). The Coomassie-stained membrane is shown at the bottom. C, a GST fusion protein containing the full-length N terminus of AC8 with point mutations in the calmodulin binding region (8M34) immobilized on glutathione-Sepharose beads was incubated with GST precleared brain membranes containing either 1 mM EDTA/EGTA or 20 µM Ca2+ and 0.5 µM calmodulin. The affinity precipitates were resolved by SDS-PAGE and were immunoblotted with an antibody raised against the catalytic subunit of protein phosphatase 2A (anti-PP2AC). The Coomassie-stained membrane is shown at the bottom.

Calmodulin Binding to AC8 Is Not Regulated by Phosphorylation. The ability of calmodulin to bind to its target sequence is regulated by phosphorylation in a wide range of proteins (Hofmann et al., 1994; Williams and Coluccio, 1995; Enyedi et al., 1997; Turner et al., 2004). For instance, endothelial nitric-oxide synthase, which contains an -helical calmodulin binding domain similar to that present on the N terminus of AC8, contains a threonine residue, which, upon phosphorylation, prevents calmodulin binding (Fleming et al., 2001). In addition, this site is dephosphorylated by PP2A, and this reversible phosphorylation regulates Ca²⁺/calmodulin-stimulated endothelial nitric-oxide synthase activity (Fleming et al., 2001; Greif et al., 2002). AC8 contains two calmodulin binding domains, both of which are essential for in vivo stimulation (Gu and Cooper, 1999); therefore the possibility can be considered that reversible phosphorylation of either site could regulate calmodulin binding and, hence, activity. There are putative PKA-phosphorylation sites at positions 46 and 66 in the N terminus and positions 1156 and 1164 in the C terminus. The second calmodulin-binding domain of AC8 is an IQ motif that is situated in the C2b domain. We generated recombinant C2b domain protein containing a hexa-His tag at the N terminus (His-C2bAC8) to determine whether this would bind calmodulin in an in vitro assay as a prelude to conducting phosphorylation experiments. Incubating His-C2bAc8 with exogenous Ca²⁺ and calmodulin in vitro showed that the C2b domain specifically bound calmodulin (Fig. 6).

View larger version (43K): [in this window] [in a new window]   Fig. 6. The calmodulin binding domains of AC8 are not regulated by phosphorylation. Cobalt affinity gel (control) or a histine-fusion protein containing the C terminus of AC8 immobilized on cobalt affinity gel (C2bAC8) was incubated in the presence or absence of 20 µM Ca2+ and 0.5 µM calmodulin. The affinity precipitates were resolved by SDS-PAGE and immunoblotted with an antibody raised against calmodulin (anti-calmodulin). The Coomassie-stained membrane is shown at the bottom.

Lipid Raft Localization of AC8 and PP2A_C. If reversible phosphorylation of AC8 is not a major regulatory influence on calmodulin binding, the association of AC8 with PP2A might represent a novel mechanism for regulation and/or localization of the phosphatase. When heterologously expressed in nonexcitable cells, AC8 occurs in lipid rafts. This specific membrane compartmentalization is essential for regulating AC8 activity in vivo (Smith et al., 2002). In the whole animal, AC8 is largely confined to the brain, and we therefore examined whether in primary hippocampal neurons a membrane compartmentalization occurs similar to that observed in non-neuronal cells. Hippocampal neurons that were transfected with AC8 tagged with GFP at the N terminus (GFP-AC8) displayed predominant plasma membrane labeling at the soma, with fluorescence extending into the dendritic network (Fig. 7B). Higher magnification of axons demonstrated a clear plasma membrane localization of GFP-AC8 (Fig. 7B, inset). This was in obvious contrast to neurons transfected with GFP alone, in which fluorescence was clearly intracellular in the soma and confined to intracellular puncta throughout the processes (Fig. 7, A and inset).

View larger version (46K): [in this window] [in a new window]   Fig. 7. AC8 and PP2AC are present in lipid rafts. Hippocampal neurons at 10 DIV 48 h after transfection with GFP (A) or GFP-AC8 (B). C, hippocampal neurons at 10 DIV 48 h after transfection with GFP-AC8 were extracted with 0.5% Triton X-100 at 4°C. The scale bar in all images represents 10 µM. D, crude mouse forebrain membranes were incubated in cold Triton X-100 at a 10:1 detergent-to-protein ratio and prepared for sucrose equilibrium density gradient centrifugation as described under Materials and Methods. Fractions (3–10) were resolved by SDS-PAGE and immunoblotted with antibodies raised against flotillin (anti-flotillin), PKA catalytic subunit (anti-PKA), Gs-olf (anti-Gs), or the catalytic subunit of protein phosphatase 2A (anti-PP2AC).

This result clearly suggests that, as with nonexcitable cells, AC8 resides in lipid rafts in primary neurons. A component of cellular PP2A might be expected to occur in lipid rafts to permit interaction with the N terminus of AC8. PP2A is a highly abundant protein (Goldberg, 1999), and incubating hippocampal neurons with an antibody raised against PP2A_C demonstrated strong labeling throughout the entire neuronal structure, with no clear specific compartmentalization (data not shown). To identify whether PP2A_C was found in lipid rafts, a clearer method for the separation of cellular compartments was required. Lipid rafts resist extraction with cold Triton X-100 and have a higher lipid-to-protein ratio, which permits their isolation because of their increased buoyancy in sucrose density gradients (Pike et al., 2002).

Mouse brain membranes were fractionated at an optimized detergent-to-protein ratio as described in Materials and Methods. Sodium carbonate was included in the extraction procedure to reduce the binding of raft-associated proteins to high-density Triton-insoluble material that pellets during sucrose centrifugation, thus retaining a greater fraction of genuine raft proteins in the buoyant fraction (Arni et al., 1998). Flotillin immunoreactivity was used to identify the lipid raft fraction (Lang et al., 1998). At low detergent-to-protein ratios, flotillin was distributed in both the raft and nonraft fractions, whereas at ratios of 20:1 and greater, flotillin immunoreactivity was only observed in the pellet, demonstrating complete membrane solubilization (data not shown) (Lang et al., 1998). At a 10:1 detergent-to-protein ratio, flotillin was clearly enriched in fraction 4, which corresponded to 20% sucrose (Fig. 7D). G_s-olf was also enriched in the light membrane fraction (Fig. 7D) (Rybin et al., 2000). Because both of these lipid raft markers were clearly enriched in the buoyant membrane fraction, this indicated a high degree of separation between lipid rafts and bulk membranes. Lipid rafts constitute only a small amount of total protein, with the bulk of the protein content residing in fractions 6 to 10 (approximately 80% of total protein; data not shown). The catalytic subunit of PKA was present in lipid rafts (fraction 4) and in the bulk membrane, corresponding to fraction 7 ( 35% sucrose) (Fig. 7D) (Razani et al., 1999). This clear separation of lipid rafts allowed us to search for PP2A. Immunoblotting with an antibody raised against PP2A_C demonstrated PP2A_C in lipid rafts (Fig. 7D). However, as might be expected for a highly expressed protein with diverse regulatory subunits, PP2A_C immunoreactivity was also present in the bulk membrane and particulate fractions. It is noteworthy, however, that a fraction of PP2A_C occurs in an environment that would permit interaction with AC8.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
When heterologously expressed in nonexcitable cells, AC8 is exclusively regulated by CCE (Smith et al., 2002). This observation, along with other data, has led to the suggestion that AC8 and CCE channels reside in close proximity at the plasma membrane (Cooper et al., 1994; Chiono et al., 1995; Fagan et al., 1996, 1998, 2000a; Murthy and Makhlouf, 1998; Watson et al., 2000; Smith et al., 2002). The targeting of AC8 to specialized domains of the plasma membrane, enriched in cholesterol and sphingolipids, termed lipid rafts, is essential but not sufficient for enabling regulation by CCE (Smith et al., 2002). The molecular identity of CCE channels remains uncertain, although mammalian homologues of the Drosophila melanogaster transient receptor potential (TRP) proteins are putative candidates (Putney and McKay, 1999). Of the TRP proteins, TRP1 at least is enriched in lipid rafts (Lockwich et al., 2000). An N-terminally truncated form of AC8 is unresponsive to CCE regulation, although the enzyme remains localized in lipid rafts and is fully stimulated by Ca²⁺/calmodulin in vitro (Smith et al., 2002). This led us to consider that the N terminus of AC8 might associate with elements of the cellular CCE apparatus.

Therefore, in this study, we used the N terminus of AC8 in a yeast two-hybrid screen of an HEK293 cDNA library to try to identify interacting proteins that might contribute to CCE regulation of AC8. It turned out that none of the positive candidates identified were proteins that might obviously be involved in regulating CCE. However, the catalytic subunit of PP2A emerged as one compelling interacting protein. The failure to find more obvious candidates in this screen does not exclude the possibility that the N terminus targets AC8 directly to elements of the CCE apparatus, although it suggests that more indirect interactions should be countenanced.

Extending and confirming the interaction identified by the yeast two-hybrid screen, an N-terminal AC8 GST-fusion protein affinity precipitated the full-length PP2A_C from both brain—the natural source of AC8 —and HEK293 membranes. This underlined the highly specific nature of the interaction between PP2A_C and the N terminus of AC8 against a large background of other proteins.

PP2A is one of the four major types of serine/threonine protein phosphatase and associates in vivo with a scaffolding subunit of 65 kDa (PP2A_A). This core dimer can further associate with one of a class of regulatory B subunits, ranging in molecular mass from 55 to 130 kDa. The B subunits apparently play a role in specifying substrate selection and subcellular location (Goldberg, 1999; Janssens and Goris, 2001). We identified PP2A_A in pull-downs that were positive for PP2A_C, which suggested that the N terminus of AC8 interacted with the PP2A core dimer. However the possibility cannot be ruled out that PP2A_A and PP2A_C interact at individual sites on the N terminus of AC8. We did not detect any B subunits when immunoblots were probed with a pan-specific PP2A_B antibody. This may indicate that the core dimer preferentially associates with the N terminus of AC8 or that PP2A_B was not present in sufficient quantity to allow signal detection by enhanced chemiluminescence on immunoblotting with a pan-PP2A_B antibody.

Although the regulation of PP2A is believed to occur through the A and B subunits, additional proteins that bind specifically to the PP2A catalytic subunit have been identified. Axin, a component of the Wnt signal transduction system, PKC-, the 1_c subunit of the L-type VGCC, and the NR3A subunit of the N-methyl-D-aspartate receptor all display a specific interaction with PP2A_C (Hsu et al., 1999; Davare et al., 2000; Chan and Sucher, 2001; Boudreau et al., 2002). Such observations suggest that the catalytic subunit may itself confer a degree of regulation to PP2A activity. Among proteins shown to bind to PP2A_C, there seems to be no immediately obvious motif responsible for PP2A_C association. Indeed, it is possible that PP2A_C associates with low stringency to its respective binding sites (Ma and Sucher, 2004).

To begin exploring the putative physiological meaning of the interaction between PP2A_C and the N terminus of AC8, we investigated whether PP2A_C was catalytically active. Indeed, the fraction of PP2A_C that associated with the N terminus of AC8 dephosphorylated myelin basic protein that had been phosphorylated by PKA. This finding of an interaction between AC8 and catalytically active PP2A_C opens a very interesting possibility and a means of drawing together previous speculations that AC, VGCCs, PKA, 2-adrenergic receptors, and PP2A might form a regulatory complex (Davare et al., 2001).

Ca²⁺/calmodulin binding to the single helical domain in the N terminus of AC8 is an essential step in the activation of AC8 by increases in [Ca²⁺]_i and is considered to occur on a 1:1 ratio (Gu and Cooper, 1999; Smith et al., 2002). We found that the interaction of the AC8 N terminus with PP2A_C was prevented by Ca²⁺/calmodulin, which suggests that PP2A also interacted with the N terminus of AC8 on a 1:1 ratio. This competition between Ca²⁺/calmodulin and PP2A_C for the N terminus of AC8 is comparable with the binding of PP2A to the autoregulatory domain of CaMKIV, which also occurs in a mutually exclusive manner with respect to Ca²⁺/calmodulin (Anderson et al., 2004). However, there is no sequence homology between the autoregulatory domains of CaMKIV and the N terminus of AC8. Although binding of PP2A_C to the N terminus of AC8 was prevented by Ca²⁺/calmodulin, PP2A_C was not binding to the identical amino acid sequence that bound Ca²⁺/calmodulin, because mutations of amino acids within the helical domain that were essential for Ca²⁺/calmodulin association were not required for binding of PP2A_C. It is therefore likely that the respective domains overlap.

The association of calmodulin with its target sequence in proteins can often be regulated by dynamic phosphorylation of residues within or adjacent to the calmodulin binding domain (Black et al., 2004). When we explored the functional consequence of catalytically active PP2A association with AC8 in the context of regulating phosphorylation of the N-terminal or C2b calmodulin binding domains, we found that neither domain was phosphorylated by PKA or a forskolin- and prostaglandin E₁-induced HEK293 cell lysate. This suggests that PKA-mediated phosphorylation is not involved in regulating calmodulin binding to AC8. However, this does not rule out the possibility that other sites on AC8 are regulated by phosphorylation, either by PKA or additional kinases. Therefore, PP2A, in association with AC8, if not involved in the regulation of calmodulin binding to AC8, may be involved in regulating the phosphorylation status of proteins in the vicinity of AC8. For example, the association of PP2A with NR3A directly regulates the phosphorylation state, not of NR3A, but of the adjacent NR1 subunit (Chan and Sucher, 2001). In this context, both AC8 and PP2A_C are present in lipid raft microdomains in which PP2A_C is positioned in an environment with other signaling molecules, including putative CCE channels and voltage-gated calcium channels (Fagan et al., 2000a,b).

The association of PP2A_C with the NR3A subunit of the N-methyl-D-aspartate receptor is disrupted by Ca²⁺ entry through the receptor ion channel; however, the NR3A subunit has not been shown to bind calmodulin (Chan and Sucher, 2001). It is conceivable that a similar scenario occurs in relation to the association of the N terminus of AC8 with PP2A. AC8 selectively responds to increases in [Ca²⁺]_i through either CCE channels or L-type VGCC. These modes of Ca²⁺ entry are believed to give rise to microdomains of elevated [Ca²⁺]_i in the vicinity of the Ca²⁺-sensitive AC, an essential element for regulation within intact cells. Thus the high level of [Ca²⁺]_i at these sites would be expected to recruit calmodulin, which in turn may displace catalytically active PP2A_C from the N terminus of AC8, facilitating activation of the cyclase and the dephosphorylation of target proteins (Persechini and Cronk, 1999).

This study failed to detect any interaction between the N terminus of AC8 and putative CCE channel proteins, despite the fact that the N terminus is an essential component in enabling AC8 to respond to increases in [Ca²⁺]_i in vivo. Thus, it would seem that the N terminus of AC8 is not responsible for a direct association with CCE channels. However, the coimmunoprecipitation of an unidentified adenylyl cyclase isoform with an L-type VGCC has been described in rat forebrain membranes, which suggests a close association between endogenous Ca²⁺-sensitive adenylyl cyclases and L-type VGCCs. This latter interaction formed part of a larger signaling complex, in which PP2A_C, the 2-adrenergic receptor, PKA, and G_s were all identified (Davare et al., 2000, 2001). Thus, it is conceivable that the interaction of catalytically active PP2A_C with AC8 may function as part of a larger signaling complex coordinating increases in [Ca²⁺]_i with the generation of cAMP, with concomitant alterations in the phosphorylation levels of key signaling intermediates by either Ca²⁺- or cAMP-activated protein kinases. Despite the number of potential PP2A substrates, the in vivo regulation of PP2A by extracellular signals is not well understood; indeed, its regulation may rely on protein-protein interactions that position PP2A at sites of required activity (Hsu et al., 1999; Sim and Scott, 1999).

AC8 links [Ca²⁺]_i increases to elevations in cAMP and, as such, is intimately involved in hippocampal long-term potentiation (Wang et al., 2003). This first demonstration of the association of an adenylyl cyclase with an active protein phosphatase provides a key intermediate in the organization of a dynamic signaling network. This may be particularly important in neuronal contexts involving Ca²⁺/calmodulin, L-type VGCC, PKA, AC8, and PP2A, in which such signaling complexes may play an important role in regulating synaptic plasticity (Frey et al., 1993; Wang et al., 2003). Future experimental exploration of the physiological significance of the AC8/PP2A interaction described may be profitably considered in such neuronal contexts, in which AC8 naturally occurs. fluorescence resonance energy transfer studies involving cyan fluorescent protein- and yellow fluorescent protein-labeled components could provide a dynamic means for studying the interactions of the full-length proteins in living cells.

	Acknowledgements

 
We thank Dr. Brian Wadzinski for the PP2A_A and pan-PP2A_B polyclonal antibodies, Dr. Sangeeta Chawla and Johanna Belfield for culture and transfection of hippocampal neurons, and Dr. Debbie Willoughby for assistance with confocal imaging and useful comments on the manuscript.

	Footnotes

 
This work was funded by the Wellcome Trust and by National Institutes of Health grant GM32483 (to D.M.F.C.).

A.J.C. and A.C. contributed equally to this work.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.018275.

ABBREVIATIONS: PKA, protein kinase A; AC, adenylyl cyclase; CCE, capacitative Ca²⁺ entry; GFP, green fluorescent protein; GST, glutathione S-transferase; mAb, monoclonal antibody; NR, N-methyl-D-aspartate receptor subunit; PSD, postsynaptic density; PP2A, protein phosphatase 2A; PP2A_A, protein phosphatase 2A scaffolding subunit A; PP2A_B, protein phosphatase 2A regulatory subunit B; PP2A_C, protein phosphatase 2A catalytic subunit; TRP, transient receptor potential; VGCC, voltage-gated calcium channel; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; DIV, days in vitro; TNE buffer, NaCl/EDTA/Tris; TBS, Tris-buffered saline; TTBS, Tris-buffered saline/Tween 20; NP40, Nonidet P-40; TRP, transient receptor potential.

Address correspondence to: Dr. Dermot M. F. Cooper, Department of Pharmacology, University of Cambridge, Tennis Court Rd, Cambridge, CB2 1PD, United Kingdom. E-mail: dmfc2{at}cam.ac.uk

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