©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Adenylyl Cyclase by Protein Kinase A (*)

Gensho Iwami (2)(§), Jun-ichi Kawabe (1)(¶), Toshiaki Ebina (1)(¶), Paul J. Cannon (2), Charles J. Homcy (2) (3), Yoshihiro Ishikawa (2) (1)(**)

From the (1) Departments of Pharmacology and (2) Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032 and the (3) Medical Research Division of American Cyanamid-Lederle Laboratories, Pearl River, New York 10965

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The changing relationship between stimuli and responses after prolonged receptor stimulation is a general feature of hormonal signaling systems, termed desensitization. This phenomenon has been best exemplified in the covalent modification of the G protein-linked catecholamine receptors. However, other components within this signaling pathway can be involved in desensitization. Here we present evidence that desensitization occurs at the level of the effector enzyme itself through phosphorylation. Type V adenylyl cyclase (AC) is the major isoform expressed in the heart. Using purified enzymes, we demonstrate that protein kinase A (PKA) directly phosphorylates and thereby inhibits type V AC catalytic activity. This inhibition was negated in the presence of PKA inhibitor. Analysis of enzyme kinetics revealed that this inhibition was due to a decrease in the catalytic rate, not to a decrease in the affinity for the substrate ATP. Our results indicate that AC catalytic activity can be regulated through PKA-mediated phosphorylation, suggesting another mechanism of desensitization for receptor pathways which signal via increases in intracellular cAMP.


INTRODUCTION

Occupation of cell surface catecholamine receptors by norepinephrine released from the synaptic terminal evokes signaling via the stimulatory GTP regulatory protein, G, which in turn activates adenylyl cyclase (AC).() AC is a membrane-bound enzyme that catalyzes the conversion of ATP to cyclic AMP. Cyclic AMP, an intracellular second messenger, activates protein kinase A (PKA), which initiates an enzymatic cascade of phosphorylation reactions within the cell. Examples are various enzymes involved in myocyte contraction in the heart and those involved in glycolysis in the liver.

Besides initiating this downstream signaling cascade, PKA also phosphorylates proteins located upstream of this signaling pathway, i.e. catecholamine receptors, thereby mediating their desensitization (1) . Phosphorylation of the cytoplasmic domain of the receptor makes the molecule less efficient in coupling to the G protein. The receptor is also phosphorylated and uncoupled by another kinase, termed adrenergic receptor kinase (BARK). A unique feature of BARK is that it only phosphorylates the agonist-bound form of the receptor. BARK presumably recognizes changes in the tertiary structure of the receptor caused by ligand binding, thus phosphorylating only the activated form of the receptor. Nevertheless, in both cases receptor phosphorylation occurs only when the signaling pathway is activated; it is triggered either by agonist occupation of the receptor or by cyclic AMP formation as a result of receptor activation, thus forming a closed loop of negative feedback.

More recently, we have demonstrated that AC itself can also be regulated by phosphorylation (2) . This occurs through protein kinase C (PKC) in an isoenzyme-specific manner, and actually leads to an enhancement of catalytic activity. In contrast, the exact role of AC modification in the process of desensitization remains unknown. Amino acid sequence analysis of AC reveals the presence of multiple putative PKA-sensitive motifs within this molecule (3) . However, PKA-mediated phosphorylation of the AC molecule itself and consequent changes in catalytic activity have not been clearly demonstrated; these were the goals of the present study.


MATERIALS AND METHODS

Overexpression and Purification of Type V AC

Plasmid construction and purification of the recombinant hexa-histidine tagged type V AC were performed as described previously (2) . Briefly, High Five insect cells (1 10 cells) were infected with the recombinant baculovirus and harvested 60-70 h after infection. Cells were lysed by nitrogen cavitation at 800 p.s.i. for 30 min at 4 °C. Nuclei were removed by centrifugation at 500 g for 10 min. Membranes were harvested by centrifugation at 150,000 g at 4 °C for 40 min, followed by suspension in buffer A (20 mM Hepes (pH 8.0), 20% glycerol, 400 mM NaCl, 2 mM MgCl, 1 mM EDTA, 2 mM -mercaptoethanol) with protease inhibitors, and were recentrifuged at 150,000 g at 4 °C for 30 min. The membranes were then resuspended in buffer A with 0.8% dodecyl maltoside. After incubation and centrifugation at 150,000 g for 30 min, the supernatant was further incubated with forskolin-CH Sepharose 4B (Pharmacia Biotech Inc.) for 16 h at 4 °C. After washing, AC was eluted with 200 µM forskolin and 0.2% dodecyl maltoside in buffer A. The eluate was further incubated with nickel-nitrilotriacetic acid resin (Qiagen, CA) at 4 °C for 30 min. After washing, the enzyme was eluted with buffer A containing 0.1% dodecyl maltoside and 100 mM imidazole. The eluate was buffer changed and concentrated using Centricon-100 (Amicon, MA) and stored at -80 °C until use.

Phosphorylation of Type V AC by PKA

Type V AC was incubated in the presence or absence of forskolin (100 µM) or GTPS-G in a buffer containing 50 mM Hepes (pH 8.0), 5 mM MgSO, 1 mM EDTA, and 1 mM dithiothreitol at 30 °C for 10 min. Various amounts of PKA catalytic subunit (PKA-CS) (Sigma) were then added in a buffer containing 20 mM Hepes (pH 8.0), 10 mM MgCl, 1 mM dithiothreitol, 0.2 mM ATP, 1 mM creatine phosphate, 8 units/ml phosphocreatine kinase in the absence or presence of PKA regulatory subunit (PKA-RS) (Sigma) at 25 °C for 10 min. Cyclic AMP production, as a measurement of AC catalytic activity, was measured at 30 °C for 10 min as described previously (4) . Briefly, the mixture was assayed in a solution containing 1 mM creatine phosphate, 8 units/ml creatine phosphokinase, 20 mM Hepes (pH 8.0), 5 mM MgCl, 0.1 mM cAMP, 0.2 mM ATP, and [-P]ATP (0.2-5 mCi/assay tube) followed by the addition of 100 µl of 2% sodium dodecyl sulfate. [H]cAMP was used as an internal standard to measure overall recovery. Protein concentration was determined by staining with Amido Black (5) .

PKA Assay

Phosphorylation was monitored using a PKA assay system (Life Technologies, Inc.) with Kemptide as substrate. PKA-CS was incubated in 20 mM Hepes (pH 8.0), 10 mM MgCl, 1 mM dithiothreitol, 0.2 mM ATP, 1 mM creatine phosphate, 8 units/ml phosphocreatine kinase, 0.25 mg/ml bovine serum albumin, [-P]ATP (12-24 mCi/assay tube) with 50 mM Kemptide as substrate at 30 °C for 10 min. The reaction was quenched by spotting the sample mixture onto a phosphocellulose disc and the incorporation of P into Kemptide from [-P]ATP was measured using scintillation counting.

Phosphopeptide Mapping of Type V AC

Purified type V AC was phosphorylated either with 6 units/ml PKC or with 200 units/ml PKA-CS in the presence of [-P]ATP as described above and previously (2) . After separation on 8% SDS-PAGE, the phosphorylated type V AC was excised and rehydrated as described (6) . The phosphorylated protein was further digested at 37 °C for 18 h in a buffer containing 50 mM (NH)CO and 0.3 mg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma), followed by lyophilization. The trypsin digests were resuspended in a loading buffer containing 1 M acetic acid, 0.05 M NHOH, 6 M urea, 5% glycerol, and applied to an acidic 45% polyacrylamide gel designed for separating small peptides. The limit of resolution of this acidic gel electrophoresis is approximately 100 daltons (7) . After electrophoresis, the gel was dried and autoradiography was carried out.


RESULTS

PKA Inactivates Type V AC Catalytic Activity

As shown in Fig. 1, incubation of purified type V AC with PKA resulted in a decrease in both basal (85 ± 3% of control, mean ± S.E., p < 0.05, n = 4) and stimulated (with GTPS-G, 72 ± 3%; with forskolin, 57 ± 3%, p < 0.01, n = 4) AC catalytic activity. The two different preparations of PKA-CS, one partially purified from cardiac tissue (Sigma) and the other purified from Escherichia coli overexpressing recombinant PKA-CS (Upstate Biotechnology Inc., Lake Placid, NY), both produced a similar degree of inhibition. When analyzed on SDS-PAGE, the partially purified PKA showed a major band at 45 K, while that from E. coli showed a single band at the same molecular weight (data not shown). We also examined whether PKA affects any of the components in the cyclase assay buffer, such as creatine phosphokinase. After phosphorylating AC with PKA in the absence of the regeneration system, we started the cAMP production assay by adding the hot mixtures and regeneration system in the presence of an excess amount of PKA inhibitor. However, this did not alter the results (115 ± 9 nmol/min/mg in the absence of PKA, 66 ± 9 nmol/min/mg in the presence of PKA, n = 3, p < 0.05). This indicates that the inhibition of AC by PKA is not through affecting the regeneration system within the reaction mixture.


Figure 1: Effect of protein kinase A on the adenylyl cyclase catalytic activity. Purified type V AC was incubated in the presence or absence of various stimulators at 30 °C for 10 min. Various amounts of PKA catalytic subunit (0-200 units) were then added, followed by incubation at 25 °C for 10 min. Cyclic AMP production, as a measurement of AC catalytic activity, was measured at 30 °C for 10 min. Open circles, without any stimulators; closed circles, with 100 µM forskolin; triangles, with GTPS-G. The data are means ± S.E. from four independent experiments. Specific activities in the absence of PKA-CS were approximately 50 nmol/minmg (basal), 200 nmol/minmg (GTPS-G), and 250 nmol/minmg (forskolin). *, p < 0.05;**, p < 0.01 from that in the absence of PKA-CS.



In order to examine the specificity of this inactivation, we examined the effect of PKA-RS, a specific inhibitor of PKA-CS, on PKA-mediated inhibition of type V AC catalytic activity (Fig. 2). Addition of PKA-RS negated the inhibition by PKA in a concentration dependent manner (0-300 units/ml). We also performed a time course study on this inhibition. Type V AC was incubated in the presence or absence of PKA. The degree of phosphorylation was time-dependent and paralleled that of inhibition as assessed by the incorporation of P into type V AC (Fig. 3).


Figure 2: Effect of the protein kinase A regulatory subunits (PKA-RS) on protein kinase A-mediated inhibition of type V adenylyl cyclase catalytic activity. Forskolin-stimulated (100 µM), purified type V AC was incubated with 100 units/ml PKA-CS (Sigma) in the presence of increasing concentrations of PKA-RS (0-300 units/ml) (Sigma). AC catalytic activity was measured as described under ``Materials and Methods.'' Each point was determined in duplicate. The data are means ± S.E. of four independent experiments.




Figure 3: Time course study on adenylyl cyclase inhibition by protein kinase A. AC catalytic activity was plotted as a function of time. Type V AC was incubated in a buffer containing 20 mM Hepes (pH 8.0), 10 mM MgCl, 1 mM dithiothreitol, 1 mM creatine phosphate, 8 units/ml phosphocreatine kinase, 0.1 mM cAMP, 0.2 mM ATP, and [-P]ATP and 100 µM forskolin in the presence or absence of PKA (200 units/ml) (Sigma) or PKI (10 units/ml) (New England Biolabs) for 30 min. Small aliquots of the reaction mixture were removed at 2, 5, 10, 20, and 30 min after the initiation of the reaction, and were added to 2% SDS to terminate the reaction. The P-phosphorylation of AC was similarly performed except that [-P]ATP was used instead of [-P]ATP, followed by SDS-PAGE and autoradiography. Circles, without PKA and PKI; squares, with PKA without PKI; triangles, with PKA and PKI.**, p < 0.01, n = 4.



Enzyme Kinetic Analysis

We then examined whether this PKA-mediated inactivation of type V AC results from either a decreased AC catalytic rate (V) or decreased affinity for the substrate ATP (K ). Enzyme kinetic analysis using a Lineweaver-Burk plot revealed that this inhibition was due to a decrease in the catalytic rate (V = 410 ± 32 nmol/minmg without PKA-mediated phosphorylation, 231 ± 30 nmol/minmg with PKA-mediated phosphorylation, p < 0.01, n = 3) (Fig. 4). The K for ATP was unaltered (38 ± 9 µM without PKA-mediated phosphorylation, 39 ± 3 µM with PKA-mediated phosphorylation, p = not significant, n = 3).


Figure 4: Kinetic analysis of type V adenylyl cyclase catalytic activity in the presence or absence of protein kinase A-mediated phosphorylation. The AC catalytic activities were measured with 100 µM forskolin in the presence or absence of 200 units/ml PKA-CS at 30 °C for 30 min in the cAMP assay buffer described under ``Materials and Methods'' containing different concentrations of ATP. V, 410 ± 32 nmol/minmg without PKA-mediated phosphorylation (open circles); 231 ± 30 nmol/minmg with PKA-mediated phosphorylation (closed circles) (p < 0.01). K, 38 ± 9 µM without PKA-mediated phosphorylation; 39 ± 3 µM with PKA-mediated phosphorylation (p = NS). Means ± S.E. from three independent experiments are shown.



Phosphopeptide Mapping

We also compared the pattern of phosphorylation catalyzed by PKA to that of PKC. We had previously shown that PKC-mediated phosphorylation of type V AC leads to an enhancement of its catalytic activity (2) . Type V AC was phosphorylated in the presence of [-P]ATP either with PKA-CS or with PKC, followed by trypsin digestion. The digests were analyzed on SDS-PAGE. The phosphopeptide mapping pattern was then determined by SDS-PAGE and autoradiography. As shown in Fig. 5 , the sites phosphorylated by the two kinases were different.


Figure 5: Phosphopeptide mapping of type V adenylyl cyclase. Purified type V AC was phosphorylated either with 200 units/ml PKA-CS, or with 6 units/ml PKC, followed by trypsin digestion and separation on acidic SDS-PAGE as described under ``Materials and Methods.'' Similar results were obtained in three independent experiments.




DISCUSSION

The present study demonstrates that PKA directly phosphorylates and inactivates type V AC. Inactivation of type V AC results from a decreased catalytic rate, not from a decreased affinity for the substrate ATP. The sites of phosphorylation by PKA, as assessed by phosphopeptide mapping, were different from those phosphorylated by PKC. The degree of inhibition was more prominent when AC was stimulated with forskolin or with G. However, the basal form of AC was more heat-unstable, which has made it difficult to interpret these data. We did not see, on the other hand, gross difference in the degree and sites of phosphorylation in the phosphopeptide mapping studies between basal and stimulated AC. PKA-mediated phosphorylation and inactivation of AC may represent an alternative mechanism for heterologous desensitization of the G protein-coupled receptor pathways that lead to cAMP production.

Catecholamine-mediated desensitization has been attributed to a variety of mechanisms, particularly, uncoupling of the -adrenergic receptor from G by phosphorylation (1) . Indeed, regulation of the efficiency of receptor coupling to G proteins by phosphorylation is a well accepted mechanism to explain both homologous (by BARK) and heterologous (by PKA) desensitization. Desensitization may also result from sequestration and down-regulation of receptors. Multiple approaches have been utilized to study the role of kinases involved in desensitization of the -adrenoreceptor/cAMP signaling pathway. It was first demonstrated that in vitro phosphorylation of the purified receptor by the two kinases, PKA and BARK, diminishes receptor function as measured by GTPase activity (8) . Thereafter, utilizing a series of receptor mutants in which the phosphorylation sites had been removed, the same group demonstrated that these mutant receptors bound ligand and activated AC normally, but underwent markedly less agonist promoted desensitization upon stimulation (9, 10) .

While the vast majority of prior studies of desensitization have focused at the level of the receptor (11, 12) , alteration of other components within the -adrenoreceptor/cAMP production pathway has been suggested to result in heterologous desensitization. Earlier studies had suggested PKA-dependent modulation of G activity as a mechanism for regulation (13, 14, 15) . More recent studies have suggested that modulation of the catalyst AC may play a role in heterologous desensitization (16) . Treatment of chick hepatocytes with glucagon or 8-bromo-cAMP results in desensitization of receptor-stimulated AC activity. The addition of excess purified G to desensitized hepatocyte membranes, however, did not fully restore G-stimulated AC activity, pointing to hormone-induced desensitization at the level of catalyst as the mechanism.

The recent cloning of multiple mammalian ACs has finally allowed structure-function studies of this relatively poorly characterized component of the cAMP signaling pathway to be carried out (17) . We had identified an AC isoform from a canine cardiac cDNA library, designated as type V (18) . The expression of this isoform is restricted to the heart and brain. Interestingly, type V AC is the major isoform in the adult heart where catecholamine-mediated desensitization has been extensively investigated over the past decade in various pathophysiological conditions (19, 20) . We have more recently reported that type V AC is potently regulated through PKC-mediated phosphorylation (2) . The two PKC isoenzymes, PKC and PKC, directly phosphorylate type V AC at unique residues, leading to a 10-20-fold increase in catalytic activity. The degree of this activation is greater than that achieved by forskolin, the most potent AC agonist. Furthermore, the two PKC isoenzymes are additive in their capacity to activate AC. These data indicate that phosphorylation is a potent mechanism to activate type V AC, which, unlike other AC isoforms, is insensitive to other modulators, such as G protein subunits or calcium/calmodulin. In contrast, the present study demonstrates that PKA-mediated phosphorylation inhibits type V AC. Thus, type V AC is subject to dual regulation by phosphorylation: activation by PKC and inhibition by PKA, mediated via phosphorylation at unique residues within the type V molecule as demonstrated by phosphopeptide mapping. A similar dual regulation by these two kinases has been shown in the case of potassium channels (21, 22) . Within type V AC, there are 14 serine/threonine residues encompassed by a consensus sequence for PKA-mediated phosphorylation and 11 residues for PKC-mediated phosphorylation; however, we do not know the exact residues responsible for mediating the functional alteration in catalytic activity.

PKA-mediated inactivation of AC creates a feedback system within the cAMP signaling pathway, which is analogous to PKC-mediated inhibition of the phospholipase C pathway (23, 24) . Catecholamine stimulation in the heart activates both the phospholipase C/PKC pathway via -adrenoreceptors and the AC/PKA pathway via -adrenergic receptors. Thus, dual regulation of AC by PKC and PKA may play a major role in integrating these two principal signal transduction pathways and thereby modulate neuronal and hormonal input to the heart. We do not know, however, whether other AC isoforms are similarly regulated through PKA- and PKC-mediated phosphorylation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL-21006, HL-38070 and a grant from Lederle Laboratories. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Medicine, Kurume Medical School, Fukuoka, Japan.

Present address: Dept. of Medicine, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115.

**
To whom correspondence should be addressed: Dept. of Medicine, Brigham and Women's Hospital, Harvard Medical School, Rm. 3-310, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-278-0407; Fax: 617-264-6845.

The abbreviations used are: AC, adenylyl cyclase; PKA, protein kinase A; BARK, adrenergic receptor kinase; PKC, protein kinase C; GPTS, guanosine 5`-O-(3-thiotriphosphate); PKA-RS, PKA-regulatory subunit; PKA-CS, PKA-catalytic subunit; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. R. Iyengar (Mount Sinai Medical School) for helpful discussion.

Note Added in Proof-A similar PKA-mediate inhibition of AC was found in type VI (R. Iyengar, personal communication).


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.