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J. Biol. Chem., Vol. 277, Issue 18, 15721-15728, May 3, 2002

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Protein Kinase C Inhibits Type VI Adenylyl Cyclase by Phosphorylating the Regulatory N Domain and Two Catalytic C1 and C2 Domains^*

Ting-Hui Lin^§¶, Hsing-Lin Lai^¶, Yu-Ya Kao, Chung-Nan Sun, Ming-Jing Hwang, and Yijuang Chern^**

From the  Institute of Biomedical Sciences, Academia Sinica, Taipei 115, ^§ Department of Life Sciences, Chung Shan Medical University, Taichung 402, and the  Institute of Neuroscience, National Yang-Ming University, Taipei 112, Taiwan, Republic of China

Received for publication, December 4, 2001, and in revised form, January 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We previously showed that phosphorylation of Ser¹⁰ of the N terminus domain of the type VI adenylyl cyclase (ACVI) partly mediated protein kinase C (PKC)-induced inhibition of ACVI. We now report that phosphorylation of the other two cytosolic domains (C1 and C2), which form the catalytic core complex of ACVI, also contributes to PKC-mediated inhibition. In vitro phosphorylation by PKC of the recombinant C1a and C2 domains, and of the synthetic peptides representing potential PKC phosphorylation sites, suggests that Ser⁵⁶⁸ and Ser⁶⁷⁴ of the C1 domain and Thr⁹³¹ of the C2 domain might act as substrates for PKC. We next created several full-length ACVI mutants in which one or more of the four likely PKC phosphorylation sites (Ser¹⁰, Ser⁵⁶⁸, Ser⁶⁷⁴, and Thr⁹³¹) were mutated to alanine. Simultaneous mutation of at least two of the three likely residues located in the N and C1 domains (Ser¹⁰, Ser⁵⁶⁸, and Ser⁶⁷⁴) was required to render ACVI variants completely insensitive to PKC treatment. In contrast, a single mutation of Thr⁹³¹ was sufficient to create a functional ACVI mutant that exhibited no detectable PKC-mediated inhibition, demonstrating the essentiality of Thr⁹³¹ to PKC-mediated regulation. Based on these results, we propose that the three cytosolic domains of ACVI might form a regulatory complex. Phosphorylation of this regulatory complex at different sites might induce a fine-tuning of the catalytic core complex and subsequently lead to alternation in the catalytic activity of ACVI.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genes of nine mammalian membrane-bound adenylyl cyclases (ACs)¹ have been isolated and characterized (1-3). All of these ACs contain two hydrophobic spans, composed of six transmembrane helices, and three large cytoplasmic domains (N, C1a/b, and C2, see Fig. 1A). The crystal structure of the catalytic domains of AC has been resolved and analyzed in detail (4, 5). It was clearly demonstrated that two cytoplasmic domains (C1a and C2) of ACs form the catalytic core. In addition, G_s and/or forskolin increase the affinity between C1a and C2 and activate cyclase by changing the relative orientation of the C1 and C2 domains to an active conformation (4, 5). The functional roles of the C1b domain of ACs are relatively more variable. For ACII and ACVII, the C1b domain suppresses the catalytic activity by holding ACs in their basal non-stimulated state (6). Moreover, the C1b domain modulates calmodulin-elicited activation of ACI- and PKA-evoked inhibition of ACVI (7, 8). The N terminus domains of ACs are highly variable among ACs and have been demonstrated to play a regulatory role (9, 10). The twelve transmembrane segments are responsible for linking together the two catalytic domains (C1a and C2) to achieve a proper functional conformation and are important for membrane targeting of ACs (4, 11).

We have previously reported that prolonged activation of the A_2A adenosine receptor (A_2A-R) in PC12 cells significantly inhibits the activity of type VI adenylyl cyclase (ACVI), which in turn causes a lower response to subsequent stimulation by A_2A-R (12, 13). In addition, stimulation by A_2A-R activates the calcium-independent protein kinase C (PKC) that phosphorylates and inhibits ACVI. Suppression of ACVI by protein phosphorylation thus produces a lower response of ACVI to subsequent stimulation by A_2A-R in PC12 cells (14). Further biochemical analyses reveal that the apparent maximal forskolin- and G_s-stimulated activities of PKC-treated ACVI are significantly lower than those of the non-treated enzyme. However, treatment with PKC does not alter the K_m of the substrate, nor does it markedly affect the EC₅₀ values of forskolin or G_s (10). Interestingly, the glycosylation state of ACVI affects the ability of ACVI to be inhibited by PKC (15), further suggesting that post-translational modification of ACVI is important for its activity. Analysis of the amino acid sequence of rat ACVI (16) using the Genetics Computer Group program (Madison, WI) reveals that there are 11 potential PKC phosphorylation sites that lie within the three predicted large intracellular cytoplasmic domains (N, C1a/b, and C2; see Fig. 1A). We earlier reported that PKC phosphorylates the highly variable N terminus domain of ACVI at Ser¹⁰. The N terminus domain therefore appears to mediate, at least partly, the inhibition and phosphorylation of ACVI by PKC (10). Notably, the ACVI mutant lacking amino acids 1-86 or Ser¹⁰ can still be phosphorylated by PKC (10), suggesting that PKC also phosphorylates residues located in the C1 or C2 domain of ACVI.

In the present study, we have sought to further elucidate the molecular mechanism underlying the inhibitory effect of PKC on ACVI. Detailed mutational characterization suggests that PKC phosphorylates at least four residues (Ser¹⁰, Ser⁵⁶⁸, Ser⁶⁷⁴, and Thr⁹³¹) located in the three large cytosolic domains (N, C1, and C2) of ACVI and subsequently exerts an inhibitory effect on the catalytic activity of ACVI. Moreover, phosphorylation of ACVI at multiple sites located in different cytosolic domains is required to achieve the maximal inhibitory effect of PKC, indicating that the three large cytosolic domains might interact with each other to acquire an active conformation, which is subject to phosphorylation-driven regulation by PKC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Adenylyl Cyclases in Sf21 Cells-- The cDNA of rat ACVI was kindly provided by Dr. Iyengar (Department of Pharmacology, Mount Sinai School of Medicine, NY) (16). Expressions of wild-type and mutant ACVI were carried out in a recombinant baculovirus-driven Sf21 cell system following the manufacturer's protocol (BD PharMingen, San Diego, CA). Membrane fractions were collected from Sf21 cells infected with the indicated virus carrying the desired ACVI variant 68-72 h after infection.

Adenylyl Cyclase Assay-- AC activity was assayed as described previously (13). Briefly, cells were resuspended in lysis buffer (0.4 mM EDTA, 1 mM EGTA, 25 mM Tris-HCl, 250 mM sucrose, 0.1 mM leupeptin, and 40 µM PMSF, pH 7.4), and sonicated using a W-380 sonicator (Ultrasonics, Farmingdale, NY) at a setting of 20% output power for a total of 45 s. The homogenate was centrifuged at 50,000 × g for 30 min to collect the P1 membrane fractions. The AC activity assay was performed at 37 °C for 10 min in a 400-µl reaction mixture containing 1 mM ATP, 100 mM NaCl, 50 mM Hepes, 0.5 mM 3-isobutyl-1-methylxanthine, 6 mM MgCl₂, 1 µM GTP, and 20 µg of membrane protein. Reactions were stopped by addition of 0.6 ml of 10% trichloroacetic acid. The cAMP formed was isolated by Dowex chromatography (Sigma) and assayed as described previously (13). The ACVI activity was determined as the difference between cyclase activities measured in membrane fractions collected from Sf21 cells infected with the ACVI virus and with the control Autographa californica multicapsid nuclear polyhedrosis virus. Endogenous cyclase activities in Sf21 cells represented ~20% of the total activity. The enzyme activity was linear for up to 30 min with up to 40 µg of membrane proteins.

Site-directed Mutagenesis-- Potential PKC phosphorylation sites of rat ACVI were identified using the Genetics Computer Group program (Madison, WI). Site-directed mutagenesis of Ser¹⁰, Ser³²⁵, Ser⁵⁶⁸, Ser⁶⁷⁴, and Thr⁹³¹ of ACVI was carried out using the Altered Sites II in vitro Mutagenesis Systems (Promega) following the manufacturer's protocol. Mutations were confirmed by DNA sequencing. Mutant cDNAs were cloned into a baculovirus transfer vector (pVL1392) and expressed in Sf21 cells as described above.

Recombinant G_SProtein Expression-- The expression construct (pQE60/H6-G_s) of G_s protein was a generous gift from Dr. W.-J. Tang (University of Chicago, Chicago, IL). The hexahistidine-tagged G_s protein was expressed in Escherichia coli and purified using the His·Bind metal chelation resin (Novagen, Madison, WI) as described elsewhere (17). To stimulate AC activity, the indicated concentration of G_s protein was activated by GTPS (20 µM) in a 100-µl reaction mixture containing 10 mM MgCl₂, 20 mM Tris, 1 mM EDTA, and 1 mM dithiothreitol for 20 min at 20 °C.

Polymerase Chain Reaction-- DNA fragments encoding the C1a, C2S, and C2L domains of ACVI were produced using the PCR technique. DNA amplification was carried out in a solution containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 0.001% (w/v) gelatin, 0.2 mg of the desired primers, 0.2 mM of each deoxynucleoside triphosphate (dNTP), a DNA template, and 2 units of DynaZyme thermostable DNA polymerase (Finnzymes, Espoo, Finland) per 50 µl of reaction solution. Primers for ACVI (16) were as follows: for the C1a domain (amino acids 356-599), 5'-GCATATGTCGGTGTTGCCCCAGCAT-3' and 5'-GGATCCGTCAGGAACCCAGCGGGG-3'; and for the C2S domain (amino acids 946-1173), 5'-CATATGAAGGAGGAGATGGAGGAG-3' and 5'-GAGATGACCACCTACTTCTAGGGATCCTAT-3'. The reactions proceeded for 40 PCR cycles (95 °C, 1 min; 68 °C, 1 min; 72 °C, 4 min). Primers for the C2L domain (amino acids 925-1180) were 5'-CATATGCAACAGGTGGAATCT-3' and 5'-GGATCCCTAACTGCTGGGGCCCCCATT-3'. The reactions proceeded for 40 PCR cycles (95 °C, 1 min; 55 °C, 1 min; 72 °C, 4 min). The amplified DNA fragments were ligated to the C terminus of a hexahistidine tag, confirmed by DNA sequencing, and subcloned into pET11d (Novagen) for expression in E. coli. Because the expression levels of the recombinant C1a and C2S domains were very low in E. coli, the DNA fragment encoding C1a or C2S was subcloned into pVL1393 for expression in Sf21 cells. Expressions of these domain proteins were carried out in a recombinant baculovirus-driven Sf21 cell system following the manufacturer's protocol (BD PharMingen).

SDS-PAGE and Western Blotting-- We determined protein concentrations by a simple colorimetric assay based on the Bradford dye-binding procedure (18) using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). For SDS-PAGE, membrane fractions were combined with 2× sample buffer containing 125 mM Tris-HCl (pH 6.8), 20% glycerol, 1% SDS, 15% 2-mercaptoethanol, and 0.01% bromphenol blue, boiled for 5 min, centrifuged to remove the insoluble material, and then separated on 8% separating gels according to the method of Laemmli (19). Following electrophoresis, the gel was transferred to a polyvinylidene difluoride membrane, blocked with 5% skim milk in PBS, then incubated with an anti-ACVI antibody (AC6D, 1:5000 dilution (14)) at 4 °C overnight. After three washes of 5 min each in PBS, the membranes were incubated with peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences, Inc., Buckinghamshire, UK) at a 1:5000 dilution for 1 h at room temperature. The membranes were washed three times with PBS, and the immunoreactive bands were stained using a light-emitting non-radioactive method (ECL, Amersham Biosciences, Inc.). The polyclonal AC6D was raised against the recombinant C2 domain (amino acids 987~1180) of ACVI (14). Because the C1a and C2 domains of ACVI are highly homologous (44% similarity), AC6D recognized both the C1a and C2 domains of ACVI.

Immunoprecipitation and in Vitro Phosphorylation-- To carry out the protein phosphorylation study, ACVI variants expressed in Sf21 cells were purified by immunoprecipitation using AC6D. In brief, 0.25 mg of P1 membrane fractions collected from Sf21 cells infected with the indicated virus were solubilized in 0.25 ml of ice-cold radioimmune precipitation buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mM leupeptin, 40 µM PMSF, and 50 mM Tris, pH 8) at 4 °C for 30 min. Immunoprecipitation was initiated by addition of AC6D (1:50 dilution). The reaction mixture was incubated with gentle agitation at 4 °C for 90 min. Immunocomplexes were purified using Sephadex-conjugated protein A (Sigma) and then washed three times with ice-cold radioimmune precipitation buffer. Phosphorylation by PKC was carried out in a final volume of 0.1 ml of a reaction mixture containing 10 mM MgCl₂, 1 mM CaCl₂, 0.25% bovine serum albumin, 12 µM N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89), 12 µM ATP, 50 µCi of [-³²P]ATP (6000 Ci/mmol, 1 Ci = 37 GBq, Amersham Biosciences, Inc., Piscataway, NJ), 0.1 mM leupeptin, 40 µM phenylmethylsulfonyl fluoride (PMSF), 30 nM okadaic acid, 0.2 mM sodium vanadate, and 20 mM Tris (pH 7.5). The phosphorylation reaction was initiated by addition of PKC (0.1 milliunit (mU)) purified from rat brain (Roche Molecular Biochemicals) for 30 min at 4 °C and was terminated by the addition of 2× SDS sample treatment buffer. Samples were then boiled for 5 min then analyzed by SDS-PAGE (8%) and Western blotting using AC6D. To visualize the phosphorylation of ACVI by PKC, immunoblots were rinsed twice with PBS, air-dried, and autoradiographed.

Peptide Phosphorylation-- Peptides were synthesized and purchased from Genosys (Woodlands, TX). The composition and amount of each peptide were confirmed by amino acid analysis (Analytical Biotechnology Services, National Taiwan University, Taipei, Taiwan). Names and sequences of the peptides are shown in Table I. Phosphorylation of each peptide was carried in a final volume of 50 µl of a reaction mixture containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 0.5 mM CaCl₂, 0.1 mg/ml phosphatidylserine, 20 µg/ml diolein (Sigma), 0.25 mM [-³²P]ATP (500 cpm/pmol), 1 mM peptide (MBP4-14, Sigma), and 0.01 mU of PKC. After a 30-min incubation at 30 °C, the reaction was terminated by boiling for 5 min. The phosphorylated peptides were segregated from [-³²P]ATP by 20% acrylamide SDS-polyacrylamide gel electrophoresis as described elsewhere (20). Gels were fixed with two changes of 30% (v/v) methanol followed by two changes of 10% (v/v) acetic acid after which they were exposed to the film at 80 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In addition to phosphorylation of the highly variable N domain at Ser¹⁰, our previous study (10) suggests that PKC might phosphorylate other cytosolic domains (C1a/b and C2, Fig. 1A). Because the C1 and C2 domains form the catalytic core complex (5), we examined whether the activation state of the catalytic core complex affects the ability of ACVI to be regulated by PKC. Activation of ACVI was first achieved by pretreating ACVI with activated G_s proteins (240 nM), which enhanced the activity of ACVI by ~5 fold (Fig. 2). As shown in Fig. 2, ACVI in both the basal/non-stimulated and G_s-activated states could be inhibited by PKC treatment to a similar extent.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Schematic representation of rat ACVI (amino acid sequence derived from M96160.Gb_Ro). A, 11 potential PKC phosphorylation sites located on the three large intracellular domains are indicated as open circles. B, schematic representation of the C1a, C2S, and C2L domains. Amino acid residues that define these domains are shown.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Inhibition of ACVI by PKC is independent of the activation status of ACVI. Membrane fractions of Sf21 cells infected with ACVI baculoviruses were collected. Activation of ACVI by Gs was carried out by treating the desired membrane fractions with Gs (240 nM)/GTPS (5 µM) for 10 min at 20 °C as described under "Experimental Procedures." Activated Gs proteins were then removed by centrifugation, followed by PKC treatment of the membrane fractions as indicated. Activity of ACVI was then stimulated by forskolin (5 µM). Values represent the means ± S.E. of three determinations. The standard deviations are too small to be visible in the figure. The results are from one representative example of four independent experiments performed. Statistical significance was evaluated by one-way analysis of variance (ANOVA). Specific comparisons between the PKC-treated and the control group of each activation status were performed using the Dunnett method (33). *, p < 0.05.

Based on the available crystallographic structure of VC1a/IIC2 (5), we built a computer model of the catalytic core composed of the C1a and C2 domains of ACVI (Fig. 3). According to the model, two potential PKC phosphorylation sites, Ser⁴⁸⁹ and Ser¹¹²⁶, are located in the catalytic core of the C1a·C2 complex of ACVI. Ser⁴⁸⁹ and Ser¹¹²⁶ are very conserved among eight other AC isoforms, and lie close to the forskolin and ATP binding sites, respectively (Fig. 3). Because these two residues are most likely buried inside the core catalytic complex at all times (basal or activated), they are less likely to be accessible to PKC-mediated phosphorylation.

View larger version (73K): [in this window] [in a new window]   Fig. 3.   A computer model of the VIC1a·VIC2 complex. The model was predicted based on the x-ray complex structure of VC1a and IIC2 (5) and using the homology-modeling package of InsightII (MSI, San Diego, CA). Of the possible PKC phosphorylation sites identified in Fig. 1A, only Ser489 and Ser1126 are located within the catalytic core. The two residues are not accessible to solvent according to the model (calculation not shown). Note that Ser568 is three residues beyond the last C1a residue (Leu565) of the model, and Ser674 is many more residues further beyond. Because Ser568 and Ser674 may come close to the Gi binding site, their PKC phosphorylation may thus directly or indirectly interfere with Gi binding. This figure was prepared using MOLSCRIPT (35).

We next determined whether the other putative PKC phosphorylation residues located in the C1 (Ser³²⁵, Thr³⁷², Ser⁵⁶⁸, Ser⁶¹⁹, and Ser⁶⁷⁴) and C2 (Thr⁹³¹) domains of ACVI could be phosphorylated by PKC. Because synthetic peptides have been used to assess the most likely phosphorylation sites (21, 22), peptides (Table I) representing the potential PKC phosphorylation sites of the C1 domain were treated with PKC to screen for the most likely PKC phosphorylation sites in the C1 domain. As shown in Fig. 4A, peptides containing Ser³²⁵, Ser⁵⁶⁸, or Ser⁶⁷⁴, but not the others, were phosphorylated by PKC. In addition, the recombinant C1a domain (amino acids 356-599) could be effectively phosphorylated by PKC (Fig. 4B). PKC, therefore, might phosphorylate the C1 domain of ACVI, and the likely phosphorylated residues of the C1 domain by PKC are Ser³²⁵, Ser⁵⁶⁸, and Ser⁶⁷⁴.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Phosphorylation of at least two amino acid residues (Ser568 and Ser674) located in the C1 domain of ACVI by PKC. A, PKC phosphorylated synthetic peptides representing Ser325, Ser568, and Ser674. Peptides (1 mM) representing potential PKC phosphorylation sites in the C1 domain as listed in Table I were incubated with PKC (0.01 mU) for 30 min at 30 °C in the presence of [-32P]ATP. At the end of incubation, samples were analyzed by 20% acrylamide SDS-polyacrylamide gel electrophoresis followed by autoradiography. The results are from one representative example of four independent experiments performed. B, PKC phosphorylated the recombinant C1a domain (containing Thr372, Ser489, and Ser568). The recombinant C1a proteins were immunologically purified as described and were incubated with or without PKC (0.1 mU) as indicated for 30 min at 4 °C in the presence of [-32P]ATP. At the end of incubation, samples were analyzed by Western blotting (lower panel) using AC6D (1:5000 dilution) and by autoradiography (upper panel). C, site-directed mutation of Ser568 or Ser674 reduced the phosphorylation levels of ACVI by PKC. The indicated AC variant protein was expressed in Sf21 cells and purified using AC6D as described. Purified protein was then incubated with or without PKC (0.1 mU) as indicated for 30 min at 4 °C in the presence of [-32P]ATP. At the end of incubation, samples were analyzed by Western blotting (lower panel) using AC6D (1:5000 dilution) and by autoradiography (upper panel). D, values for phosphorylation extent are expressed as percentages of phosphorylation of the wild-type protein of type VI AC. i.e. (integrated absorbance units of phosphorylation signal  protein signal of the indicated protein) × 100. Data were generated by quantitative computing densitometry of autoradiograms from 4 to 12 independent experiments using the image analysis software package, ImageQuaNT version 3.15 (Molecular Dynamics). Phosphorylation levels and protein levels of PKC-treated ACVI variants were determined using relatively short-exposure films. Because no phosphorylation signal was detected in the absence of PKC, we did not determine the phosphorylation levels for the control (non-treated) ACVI proteins. Values represent the means ± S.E. Statistical significance was evaluated by one-way ANOVA. Specific comparisons between the indicated treatment and the control group were performed using the Dunnett method (33). *, p < 0.05.

We next proceeded to individually mutate the three most likely phosphorylation sites (Ser³²⁵, Ser⁵⁶⁸, or Ser⁶⁷⁴) of the C1 domain into alanine and then expressed the resultant ACVI mutants in insect cells using a baculovirus-driven system. As shown in Fig. 4C, each ACVI mutant carrying a single point mutation exhibited similar molecular weights as that of the wild-type ACVI. In addition, mutation of Ser⁵⁶⁸ or Ser⁶⁷⁴ led to a statistically significant reduction in the phosphorylation levels of ACVI by PKC (Fig. 4D). Because multiple residues of ACVI might be phosphorylated by PKC, it is not surprising to find that PKC was able to cause a substantial level of phosphorylation in both ACVI-S568A and ACVI-674. In contrast, mutation of Ser³²⁵ did not diminish the PKC-mediated phosphorylation levels. In some of the experiments performed, we even observed a slight increase in PKC-mediated phosphorylation of ACVI-S325A compared with that of the wild-type (WT, Fig. 4C). However, the difference in the PKC-mediated phosphorylation levels between the WT and the ACVI-S325A mutant was not statistically significant (Fig. 4D). Collectively, at least two residues (Ser⁵⁶⁸ and Ser⁶⁷⁴) located in the C1 domain of ACVI might be phosphorylated by PKC.

To assess the functional relevance of Ser⁵⁶⁸ and Ser⁶⁷⁴ in PKC-mediated inhibition of ACVI, we next examined the effect of PKC treatment on the activities of ACVI-S568A and ACVI-S674A. As shown in Table II, mutation of Ser⁵⁶⁸ or Ser⁶⁷⁴ diminished PKC-mediated inhibition when relative activities of the PKC-treated WT and PKC-treated ACVI-mutant groups were compared. Reduced PKC-evoked inhibition of both ACVI mutants correlated well with the decreased phosphorylation levels of ACVI-S568A and S674A. Ser⁵⁶⁸ and Ser⁶⁷⁴, therefore, might be critical for PKC-mediated inhibition of ACVI activity. It is interesting to note that individual mutations of these two residues only partially relieved PKC-mediated inhibition, further supporting our hypothesis that multiple residues contribute to the phosphorylation and suppression of ACVI by PKC.

We have previously reported that mutation of Ser¹⁰ located in the N terminus domain of ACVI into alanine partially relieved PKC-evoked inhibition of ACVI (10). We next simultaneously mutated two of the three residues (Ser¹⁰, Ser⁵⁶⁸, and Ser⁶⁷⁴) located in the N and C1 domains to analyze their contributions to PKC-mediated phosphorylation and inhibition. As shown in Fig. 5A, phosphorylation levels of the ACVI mutants carrying mutations of two residues were significantly reduced. Most importantly, activities of these ACVI mutants containing double mutations could not be inhibited by PKC treatment (Table II). Simultaneous phosphorylation of at least two of these three residues of the C1 and N domains therefore appears to be important for PKC-mediated inhibition of ACVI activity. Likewise, as expected, activity of the ACVI mutant (ACVI-S10A/S568A/S674A) that contained mutations of all three residues was not sensitive to PKC-mediated inhibition (Table II), although, at very low levels, ACVI-S10A/S568A/S674A could still be phosphorylated by PKC (Fig. 5B, Table II). Other residues, likely to be those located in the C2 domain, thus might also contribute to PKC-mediated phosphorylation and inhibition of ACVI. Note that each of these residues contributes to only a small portion of the overall PKC-induced phosphorylation level of ACVI, and inevitably, there are slight variations among independent experiments. Although phosphorylation levels of single ACVI mutants appear to be higher than those of ACVI variants with multiple mutations, the differences are not statistically significant (Table II). Quantitation of phosphorylation levels therefore only allows us to conclude that PKC-mediated phosphorylation levels of all mutants examined are lower than that of the wild-type.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Further reduction in PKC-mediated phosphorylation of ACVI by multiple mutations of Ser10, Ser568, or Ser674. Recombinant wild-type (WT) and the indicated mutants of ACVI were prepared in Sf21 cells and purified using AC6D as described in the text. Purified protein was then incubated with PKC (0.1 mU) as indicated for 30 min at 4 °C in the presence of [-32P]ATP. At the end of incubation, samples were analyzed by Western blotting (lower panel) using AC6D (1:5000 dilution) and by autoradiography (upper panel). Values for the phosphorylation extent of these ACVI variant proteins were determined as described in Fig. 4 and are listed in Table II.

The C2 domain of ACVI contains two potential PKC phosphorylation sites (Thr⁹³¹ and Ser¹¹²⁶; Fig. 1A). Ser¹¹²⁶ is located inside the catalytic core of ACVI (Fig. 3) and is therefore less likely to be accessible by PKC. To investigate this conjecture, two recombinant C2 domain proteins (C2L, amino acids 925-1180; C2S, amino acids 946-1173; Fig. 1B) of ACVI were prepared and used to determine whether PKC phosphorylates these two domains. As shown in Fig. 6, PKC effectively phosphorylated the recombinant C2L protein, which contains Thr⁹³¹ and Ser¹¹²⁶, but not the recombinant C2S domain containing only Ser¹¹²⁶ (Fig. 6, A and B). We then substituted Thr⁹³¹ with alanine to examine the involvement of Thr⁹³¹ in PKC-elicited inhibition of ACVI. PKC-evoked phosphorylation levels of the resultant ACVI mutant (ACVI-T931A) were reduced when compared with those of the wild-type ACVI (Fig. 6C). Moreover, mutation of Thr⁹³¹ resulted in a functional ACVI mutant that exhibited no PKC-mediated inhibition. Thr⁹³¹ thus appears to play a critical role in PKC-mediated inhibition of ACVI.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   PKC phosphorylation of the C2 domain of ACVI at Thr931. A and B, PKC phosphorylated the recombinant C2L domain (containing Thr931 and Ser1126) but not the C2S domain (containing Ser1126). The indicated recombinant proteins were immunologically purified as described and incubated with or without PKC (0.1 mU) as indicated for 30 min at 4 °C in the presence of [-32P]ATP. At the end of incubation, samples were analyzed by Western blotting (lower panel) using AC6D (1:5000 dilution) and by autoradiography (upper panel). C, site-directed mutation of Thr931 reduced the phosphorylation levels of ACVI by PKC. The indicated AC variant protein was expressed in Sf21 cells and purified using AC6D as described. Purified protein was then incubated with PKC (0.1 mU) as indicated for 30 min at 4 °C in the presence of [-32P]ATP. At the end of incubation, samples were analyzed by Western blotting (lower panel) using AC6D (1:5000 dilution) and by autoradiography (upper panel). Values for the phosphorylation extent of these ACVI variant proteins were determined as described in Fig. 4 and are listed in Table II.

Except for Ser⁴⁸⁹ and Ser¹¹²⁶, all other potential PKC phosphorylation sites are located outside of the catalytic core of ACVI as defined by the computer model (Fig. 3). As expected, mutation of the potential PKC phosphorylation sites examined in the present study did not markedly alter the general enzymatic properties or protein expression levels of ACVI (data not shown). The variation observed in the apparent maximal AC activity evoked by forskolin (V_m', Table II) was likely to have been caused by the passages and ages of Sf21 cells employed for expression as previously observed in Sf9 cells (23). To determine the effect of PKC, AC activities of the indicated ACVI variant were measured in the absence or presence of PKC in the same experiment using the same preparation of plasma membranes. The observed variation in the V_m' values resulting from different batches of plasma membranes therefore did not interfere with our analysis of the effect of PKC.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we demonstrate that PKC suppresses the activity of ACVI in both the basal/non-stimulated and G_s-stimulated states. Phosphorylation studies and mutational analyses reveal that at least four amino acids (Ser¹⁰, Ser⁵⁶⁸, Ser⁶⁷⁴, and Thr⁹³¹) located in the regulatory N domain and two catalytic domains (C1 and C2) of ACVI might contribute to PKC-evoked inhibition and phosphorylation. Such a conclusion is based on several lines of evidence (Ref. 10 and this report). First, synthetic peptides encoding the potential phosphorylation sites of Ser¹⁰, Ser⁵⁶⁸, and Ser⁶⁷⁴ are substrates of PKC. Second, the recombinant N domain (containing Ser¹⁰), C1a domain (containing Ser⁵⁶⁸), and C2L domain (containing Thr⁹³¹) were all effectively phosphorylated by PKC. Last and most importantly, both single and multiple mutations of these four residues reduced the ability of ACVI to be phosphorylated and regulated by PKC. The goal of the present study was neither to identify all phosphorylated residues of ACVI by PKC nor to exclude the involvement of residues other than the four identified above. Instead, results presented herein attribute the PKC-mediated inhibition of ACVI to phosphorylation of the three large cytosolic domains and further underscore the importance of domains outside the catalytic core in determining the activity of ACVI. Because phosphorylation of all cytosolic domains is required to achieve maximal inhibition of ACVI by PKC, a delicate fine-tuning by intramolecular interactions among the three large cytosolic domains must play a critical role in the regulation of ACVI.

Phosphorylation is one of the most important regulatory mechanisms of the superfamily of ACs. Except for ACVI, residues of ACs phosphorylated by various kinases reported so far are located in only one cytosolic domain (either C1b or C2 (8, 24-26)). The one or more mechanisms underlying the modulation of different ACs by various kinases are distinct. For example, phosphorylation of the C1b domain of ACI at Ser⁵⁴⁵ and Ser⁵⁵² by Ca²⁺/calmodulin-dependent kinase II affects calmodulin-induced activation of ACI without altering basal catalytic activity (24), whereas phosphorylation of multiple residues of ACVI by PKC reduces forskolin-induced catalytic activity (Ref. 10 and this report). Regulation of ACVI by phosphorylation is intriguing, because this AC isozyme can be regulated by at least three different kinases, including PKA, PKC, and a tyrosine kinase (8, 10, 27). We note with particular interest that PKC phosphorylates the C1b domain of ACVI at Ser⁶⁷⁴, which is the same residue phosphorylated by PKA (8). Phosphorylation of ACVI by PKA reduces the low affinity stimulation by G_s protein (8), while phosphorylation of ACVI by PKC causes a reduction in its catalytic activity without changing its affinities toward stimuli (i.e. forskolin and G_s (10)). This difference in the effects of PKC- and PKA-mediated phosphorylation on enzymatic properties of ACVI might be due to PKC phosphorylating at least three more residues in addition to Ser⁶⁷⁴, which is the only site phosphorylated by PKA. Because ACVI is mostly expressed in neurons (28), regulation of ACVI by different kinases might provide an important mechanism to render neurons capable of detecting simultaneous stimulation by multiple neurotransmitters.

The importance of phosphorylation of the highly variable N terminus domain of ACVI at Ser¹⁰ has been thoroughly discussed elsewhere (10). In the C1 domain, Ser⁵⁶⁸ and Ser⁶⁷⁴ are located in relatively conserved regions. Structural information on Ser⁵⁶⁸ in the C1a domain is currently unavailable, because it is located at the crystallographically disordered C terminus of the C1a domain (Fig. 3). Ser ⁶⁷⁴ is located in a short and conserved region of the C1b domain, which interacts with the C1a and C2 domains (6). We were surprised to find that, although single mutations of Ser¹⁰, Ser⁵⁶⁸, or Ser⁶⁷⁴ caused only partial relief of PKC-mediated inhibition of ACVI, simultaneous mutation of two or all three of these residues gave rise to ACVI mutants insensitive to PKC treatment (Table II). The present study therefore supports and extends our previous findings (10) and suggests that intramolecular interactions between the N domain and the catalytic core complex are important for the activity of ACVI. Regulation mediated by phosphorylation of multiple residues, each with a small but significant contribution, has other precedent examples (29).

Another residue contributing significantly to PKC-mediated inhibition of ACVI is Thr⁹³¹. Thr⁹³¹ is located in a relatively conserved region at the beginning of the C2 domain of ACVI (Fig. 1A), and, therefore, is in an ideal position to influence the dynamic conformation of the catalytic core complex. In contrast to what was observed for other single mutants of ACVI (ACVI-S10A, ACVI-S568A, and ACVI-S674A), substitution of Thr⁹³¹ with alanine resulted in a functional ACVI insensitive to PKC-mediated inhibition. Phosphorylation of the C2 domain at Thr⁹³¹ thus is essential for regulation of ACVI by PKC. This result is in agreement with the observation that the catalytic cleft of ACs is mainly constructed by the C2 moiety (30). In addition, given the positions of Ser⁶⁷⁴ and Thr⁹³¹ (Fig. 1A), our finding is consistent with the concept that regions linking transmembrane domains and cytosolic loops are important for the regulation of ACs (31). In the equivalent position of Thr⁹³¹, four other AC isozymes (ACV, ACVI, ACVIII, and ACIX) also possess a potential PKC phosphorylation site. Note that ACV is phosphorylated and stimulated by PKC (32). Whether the Thr⁹³¹-equivalent site of ACV is phosphorylated by PKC is currently unknown. It will be of interest to examine whether ACVIII and ACIX can be regulated by PKC.

Because the catalytic core complexes of ACV and ACVI are highly homologous, the fact that PKC has an opposite effect on ACV than it does on ACVI is intriguing. There are totally 12 potential PKC phosphorylation sites in the three intracellular cytosolic domains of ACV (Genetics Computer Group program, Madison, WI). At least two different PKC isozymes can phosphorylate ACV (probably at multiple sites) and markedly enhance its catalytic activity (32). Among these potential PKC phosphorylation sites, five are in the same equivalent positions as those of ACVI (Ser¹⁴⁵, Ser³²⁵, Ser⁴⁸⁹, Ser⁵⁶⁸, and Thr⁹³¹). At least two major PKC phosphorylation sites (Ser¹⁰ and Ser⁶⁷⁴) of ACVI are missing in ACV. The importance of Ser⁶⁷⁴ is obvious, because it can be phosphorylated by both PKC and PKA in ACVI (Ref. 8 and this report) and is conserved as the only potential PKA phosphorylation site in ACV which can be inhibited by PKA-mediated phosphorylation (34). Negative charges caused by phosphorylation at Ser⁶⁷⁴ of ACVI and its equivalent site of ACV therefore might suppress the catalytic core activity. The absence of PKC-mediated phosphorylation at the Ser⁶⁷⁴-equivalent site of ACV might partially contribute to the lack of a negative effect of PKC on ACV. Importantly, unlike the catalytic core, the N terminus domains of ACV and ACVI show little sequence homology. For ACVI, we have reported that PKC phosphorylates its N domain at Ser¹⁰, a PKC site missing in ACV, and that the N domain might interact with its catalytic core to confer an active conformation sensitive to PKC-mediated phosphorylation (Ref. 10 and this report). Given the regulatory roles of the N domain that have been elucidated for two AC isozymes (9, 10), it is tempting to speculate that the N domain of ACV, which contains six potential PKC phosphorylation sites, might be phosphorylated by PKC and that it plays a role in the PKC-evoked enhancing effect on the catalytic activity of ACV.

Together with our previous study on the N terminus (10), we have demonstrated that PKC phosphorylates at least four residues distributed in the three large cytosolic domains, and phosphorylation of these resides all contributes to PKC-mediated inhibition. The complexity of the regulation led us to hypothesize that the three cytosolic domains of ACVI might form a regulatory complex that can, through phosphorylation at multiple sites, fine-tune the relative orientations of the C1 and C2 domains to modulate the enzyme's catalytic activity. Based on the boundaries defined by the structural model of the catalytic core (Fig. 3) and the likely spatial location of Ser⁵⁶⁸ and Ser⁶⁷⁴, it also seems reasonable to hypothesize that PKC phosphorylates the C1 domain at a region opposite to the G_s binding site (Figs. 3 and 5), perhaps by interfering with the G_i binding site (23). In this context, it is of interest to note that deglycosylation of ACVI elicits a similar neutralizing effect, although to different extents, on the inhibition of the enzyme mediated by a G_i protein-coupled receptor or PKC (15).

	ACKNOWLEDGEMENTS

We thank Te-Hsun Yang for creating mutant ACVI-S674A and Ya-Wen Lin for her effort in maintaining the Sf21 cell culture. We are also grateful to Yi-Sheng Cheng and Yuh-Shin Jiang for generating the computer model and Fig. 3.

	FOOTNOTES

^* This work was supported by grants from the National Science Council (NSC89-2320-B001-011; NSC90-2320-B001-009) and from Academia Sinica, Taipei, Taiwan, Republic of China.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Both authors contributed equally to this work.

^** To whom correspondence should be addressed. Tel.: 886-2-2652-3913; Fax: 886-2-2782-9143; E-mail: bmychern@ibms.sinica.edu.tw.

Published, JBC Papers in Press, February 27, 2002, DOI 10.1074/jbc.M111537200

	ABBREVIATIONS

The abbreviations used are: AC(s), adenylyl cyclase(s); ACVI, type VI adenylyl cyclase; A_2A adenosine receptor, A_2A-R; PKA, protein kinase A; PKC, protein kinase C; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; GTPS, guanosine 5'-3-O-(thio)triphosphate; WT, wild-type.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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