The consequences of introducing an autophosphorylation site into the type I regulatory subunit of cAMP-dependent protein kinase

The type I and type II regulatory subunits of cAMP-dependent protein kinase can be distinguished by autophosphorylation. The type II regulatory subunits have an autophosphorylation site at a proteolytically sensitive hinge region, while the type I regulatory subunits have a pseudophosphorylation site. Only holoenzyme formed with type I regulatory subunits has a high affinity binding site for MgATP. In order to determine the functional consequences of regulatory subunit phosphorylation on interaction with the catalytic subunit, an autophosphorylation site was introduced into the type I regulatory subunit using recombinant DNA techniques. When Ala97 at the hinge region of the type I regulatory subunit was replaced with Ser, the regulatory subunit became a good substrate for the catalytic subunit. Stoichiometric phosphorylation occurred exclusively at Ser97. Radioactivity was incorporated primarily into the recombinant regulatory subunit when catalytic subunit and [gamma-32P]ATP were added to the total bacterial extract. Phosphorylation of the mutant regulatory subunit also occurred readily following polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose. Phosphorylation occurred as an intramolecular event in the absence of cAMP indicating that the hinge region of the regulatory subunit occupies the substrate recognition site of the catalytic subunit in the holoenzyme complex. Holoenzyme formed with both the wild type and mutant regulatory subunits was susceptible to dissociation in the presence of high salt; however, only the native holoenzyme was stabilized by MgATP. In contrast to the wild type holoenzyme, the affinity of the mutant holoenzyme for cAMP was not reduced in the presence of MgATP. Holoenzyme formation also was not facilitated by MgATP.     

Phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase in intact smooth muscle

A monoclonal antibody was used to quantitate changes in the extent of phosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase in intact bovine tracheal smooth muscle. The autophosphorylated and nonphosphorylated forms of the regulatory subunit (RII) were separated in sodium dodecyl sulfate-polyacrylamide gels and identified by immunoblot analysis. Addition of cAMP to tissue extracts resulted in rapid dephosphorylation of RII (t 1/2 = 20s at 4 degrees C) while addition of MgATP caused complete conversion to the phosphorylated form. Under basal conditions, 56% of RII in intact muscle was phosphorylated when the tissue was homogenized under conditions which fully inhibit protein kinase and phosphatase activities. Incubation with isoproterenol caused a dose-dependent decrease in the phosphorylation state of RII (EC50 = 5 X 10(-8) M). Incubation with high concentrations of isoproterenol, 1-methyl-3-isobutylxanthine, or forskolin caused maximal decreases in the phosphorylated form to 12-18% of the total RII. The effect of isoproterenol was rapid (t 1/2 = 15 s at 37 degrees C), reversible, and could be blocked with the antagonist propranolol. Contraction of the smooth muscle with K+ or low (less than 1 microM) concentrations of carbachol had no effect on the phosphorylation level. A decrease in the basal phosphorylation level to 41% was observed with 10 microM carbachol which was additive with the dephosphorylation produced by isoproterenol. The time course of isoproterenol-induced dephosphorylation of RII paralleled that of muscle relaxation, consistent with a role of cAMP-dependent protein kinase activation in relaxation of smooth muscle.     

Quantitative labeling of the regulatory subunit of type II cAMP-dependent protein kinase from bovine heart by a photoaffinity analog.

Several methods were compared for estimating the amount of regulatory subunit of an 800-fold purified Type II cAMP-dependent protein kinase from bovine heart. These methods included a reversable binding assay using either cAMP, or 8-N3-[32P]cAMP, photoaffinity labeling with 8-N3-[32P]cAMP, and autophosphorylation of the regulatory subunit of the enzyme. Although the regulatory subunit had a slightly lower affinity for 8-N3-cAMP than for cAMP, the total amount of regulatory subunit could be determined by each of the procedures examined. The results indicate that the photoaffinity analog 8-N3-[32P]cAMP is able to label quantitatively all cAMP-binding sites of the regulatory subunit of this cAMP-dependent protein kinase.     

A-kinase anchoring proteins: protein kinase A and beyond

Compartmentalization of kinases and phosphatases is a key determinant in the specificity of second messenger mediated signaling events. Localization of the cAMP-dependent protein kinase (PKA) and other signaling enzymes is mediated by interaction with A-kinase anchoring proteins (AKAPs). In the past year there have been many advances in our understanding of AKAPs, particularly in the field of the functional consequences of PKA anchoring.     

The role of autophosphorylation of cAMP-dependent protein kinase II in the inhibition of protein phosphatase-1

1. The inhibition of the catalytic subunit of protein phosphatase-1 (PP-1c) by the regulatory subunit of cAMP-dependent protein kinase II (RII) was studied. 2. Phosphorylation or thiophosphorylation of RII increased its inhibitory potency up to 4- and 6-fold and rendered it competitive with respect to the substrate of PP-1c, phosphorylase a. The Ki values for thiophospho-RII and phospho-RII were 200 and 500 nM, respectively. 3. Though PP-1c was able to release phosphate from phospho-RII, its activity once incubated with phospho-RII, remained inhibited even 80% of the phosphate was released from phospho-RII. 4. The catalytic subunit of cAMP-dependent protein kinase was effective in suspending the inhibition employed either before or after the addition of phospho-RII to PP-1c. 5. No exclusive bindings of thiophospho-RII and heat-stable protein inhibitors to the PP-1c could be proved by double inhibition studies, however some synergism was observed in their effect.     

The regulatory subunit of the type II cAMP-dependent protein kinase in rabbit ovaries is the RII beta isoform.

Based on RII autophosphorylation, photoaffinity labeling with 8-N3[32P]cAMP, and Western blot analysis we have identified the RII isoform found in rabbit corpora lutea as RII beta. The RII beta subunit found in rabbit corpora lutea differs from the RII beta found in rat follicles and corpora lutea in that it migrates at Mr 52,500 on SDS-PAGE and shifts to Mr 53,000 when phosphorylated.     

Identification of sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar to the type II regulatory subunit of PKA

The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A-kinase anchoring proteins (AKAPs). AKAPs contain an amphipathic helix domain that binds to the type II regulatory subunit of PKA (RII). Synthetic peptides containing this amphipathic helix domain bind to RII with high affinity and competitively inhibit the binding of PKA with AKAPs. Addition of these anchoring inhibitor peptides to spermatozoa inhibits motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P., and Carr, D. W. (1997) J. Biol. Chem. 272, 4747-4752). However, inhibition of the PKA catalytic activity does not mimic these peptides, suggesting that the peptides are disrupting the interaction of AKAP(s) with proteins other than PKA. Using the yeast two-hybrid system, we have now identified two sperm-specific human proteins that interact with the amphipathic helix region of AKAP110. These proteins, ropporin (a protein previously shown to interact with the Rho signaling pathway) and AKAP-associated sperm protein, are 39% identical to each other and share a strong sequence similarity with the conserved domain on the N terminus of RII that is involved in dimerization and AKAP binding. Mutation of conserved residues in ropporin or RII prevents binding to AKAP110. These data suggest that sperm contains several proteins that bind to AKAPs in a manner similar to RII and imply that AKAPs may have additional and perhaps unique functions in spermatozoa.     

Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II beta regulatory subunit

BACKGROUND: Cyclic AMP binding domains possess common structural features yet are diversely coupled to different signaling modules. Each cAMP binding domain receives and transmits a cAMP signal; however, the signaling networks differ even within the same family of regulatory proteins as evidenced by the long-standing biochemical and physiological differences between type I and type II regulatory subunits of cAMP-dependent protein kinase. RESULTS: We report the first type II regulatory subunit crystal structure, which we determined to 2.45 A resolution and refined to an R factor of 0.176 with a free R factor of 0.198. This new structure of the type II beta regulatory subunit of cAMP-dependent protein kinase demonstrates that the relative orientations of the two tandem cAMP binding domains are very different in the type II beta as compared to the type I alpha regulatory subunit. Each structural unit for binding cAMP contains the highly conserved phosphate binding cassette that can be considered the "signature" motif of cAMP binding domains. This motif is coupled to nonconserved regions that link the cAMP signal to diverse structural and functional modules. CONCLUSIONS: Both the diversity and similarity of cAMP binding sites are demonstrated by this new type II regulatory subunit structure. The structure represents an intramolecular paradigm for the cooperative triad that links two cAMP binding sites through a domain interface to the catalytic subunit of cAMP-dependent protein kinase. The domain interface surface is created by the binding of only one cAMP molecule and is enabled by amino acid sequence variability within the peptide chain that tethers the two domains together.     

Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79.

Postsynaptic densities (PSD) are a network of proteins located on the internal surface of excitatory synapses just inside the postsynaptic membrane. Enzymes associated with the PSD are optimally positioned to respond to signals transduced across the postsynaptic membrane resulting from excitatory synaptic transmission or neurotransmitter release. We present evidence suggesting that type II cAMP-dependent protein kinase (PKA) is anchored to the PSD through interaction of its regulatory subunit (RII) with an A-Kinase Anchor Protein (AKAPs). A cDNA for the human RII-anchoring protein, AKAP 79, was isolated by screening an expression library with radiolabeled RII. This cDNA (2621 base pairs) encodes a protein of 427 amino acids with 76% identity to bovine brain AKAP 75 and 93% identity to a carboxyl-terminal RII-binding fragment of murine brain AKAP 150. A bacterially expressed 92-amino acid fragment, AKAP 79 (335-427) was able to bind RII alpha. Disruption of secondary structure by site-directed mutagenesis at selected residues within a putative acidic amphipathic helix located between residues 392 and 408 prevented RII binding. Immunological studies demonstrate that AKAP 79 is predominantly expressed in the cerebral cortex and is a component of fractions enriched for postsynaptic densities. AKAP antisera strongly cross-react with a 150-kDa protein in murine PSD believed to be AKAP 150. Co-localization of the type II PKA in purified PSD fractions was confirmed immunologically by detection of RII and enzymologically by measuring cAMP-stimulated phosphorylation of the heptapeptide substrate Kemptide. Approximately 30% of the PSD kinase activity was specifically inhibited by PKI 5-24 peptide, a highly specific inhibitor of PKA. We propose that AKAP 79 and AKAP 150 function to anchor the type II PKA to the PSD, presumably for a role in the regulation of postsynaptic events.     

Conformational changes of type II regulatory subunit of cAMP-dependent protein kinase on cAMP binding

The effect of cAMP on the conformation of the regulatory subunit of type II cAMP-dependent protein kinase (RII) from bovine heart was investigated by UV-difference and circular dichroism (CD) spectroscopy. The UV-difference spectrum of RII with and without cAMP showed a positive band around 278 nm and a negative band at 256 nm. Similarly, cAMP enhanced the ellipticity of RII in the region between 280 and 300 nm and decreased that between 250 and 280 nm. In addition, cAMP transformed the far-UV CD spectrum of RII from that of a negative band minimally at 209 nm with a shoulder at 223 nm to one with two minima at 222 and 211 nm. These data show that cAMP induces conformational changes of RII upon binding. Such structural changes may be the basis of activation of cAMP-dependent protein kinases by cAMP.     

Expression cloning of a cDNA encoding the type II regulatory subunit of the cAMP-dependent protein kinase

We report here the isolation and sequence of a cDNA for the type II regulatory subunit of the cAMP-dependent protein kinase (cAMP-PK) from a lambda gt-11 cDNA library derived from a porcine epithelial cell line (LLC-PK1). The cDNA was detected by immunological screening using an affinity purified polyclonal antibody for bovine RII. DNA sequence analysis of the 467 bp EcoRI insert confirmed the identity of the clone, because the deduced amino acid sequence corresponded to the published sequence for the bovine RII protein. Northern analysis of total RNA from the LLC-PK1 cells indicated a single mRNA species of about 6.0 kb, probably derived from a single copy gene.     

Immunochemical properties of the regulatory subunit of cAMP-dependent protein kinase II]

Immunochemical analysis of the cAMP-dependent protein kinase regulatory subunit type II was performed with the use of two rabbit antisera elicited to a free R-subunit from pig brain and to a RcAMP complex. Quantitative precipitation of the homogeneous antigen revealed six determinants on the R-molecule. Of these at least one is localized in the R-fragment (37 kD), the others--in the N-terminal part of the R-molecule. The antigenic determinants seem to be remoted from the cAMP-binding centers, since the attachment of the affinity purified antibody Fab-fragments to the R-subunit did not influence the cAMP-binding activity of the latter. The antibodies to RcAMP caused dissociation of the holoenzyme. The antibody Fab-fragment binding to the R-subunit prevented its association with the catalytic subunit. The results of immunochemical analysis suggest that the R-subunit adopts different conformations when bound to cAMP or to the catalytic subunit.     

Interaction of the regulatory subunit of a type II cAMP-dependent protein kinase with mammalian sperm flagellum

We have reported previously (Horowitz, J. A., Toeg, H., and Orr, G. A. (1984) J. Biol. Chem. 259, 832-838) that most of the type II cAMP-dependent protein kinases in rat sperm are associated with the flagellum. We have now identified flagellar polypeptides which are capable of forming tight complexes with the regulatory subunit of type II cAMP-dependent protein kinase (RII). Flagellar RII-binding polypeptides were identified using an RII overlay/immunoblot procedure and had apparent subunit Mr of 120,000, 80,000, and 57,000 in rat and 120,000 and 57,000 in bovine flagella. RII is released from the flagellum by disulfide reducing agents, e.g. 1 mM dithiothreitol (DTT). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie Blue staining of the DTT-released material shows that a limited subpopulation of flagellar polypeptides are solubilized by disulfide-reducing agents. Neither tubulin, the dynein ATPase, or any of the RII-binding proteins are released by 1 mM DTT, and thin section electron microscopy revealed that the morphology of the flagellum is unaltered by reducing conditions. Our data established that RII is not linked to the flagellum via a direct disulfide bridge. We propose that RII is released from the flagellum, a highly disulfide cross-linked structure, due to structural changes in the flagellum which disrupts the interaction between RII and its binding proteins.     

A-kinase anchoring proteins: scaffolding proteins in the heart

The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network. Scaffolds that bind protein kinase A are called "A-kinase anchoring proteins" (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease.     

Unfolding of the regulatory subunit of cAMP-dependent protein kinase I.

The unfolding of the recombinant regulatory subunit of cAMP-dependent protein kinase I was followed by monitoring the intrinsic protein fluorescence. Unfolding proceeds in at least two stages. First, the quenching of fluorescence due to cAMP binding is abolished at relatively low levels of urea (less than 2 M) and is observed as an increase in intensity at 340 nm. The high-affinity binding of cAMP is retained in 3 M urea even though the quenching is lost. The second stage of unfolding, presumably representing unfolding of the polypeptide chain, is seen as a shift in lambda max from 340 to 353 nm. The midpoint concentration, Cm, for this process is 5.0 M. Cyclic AMP binding activity is lost at a half-maximal urea concentration of 3.5 M and precedes the shift in lambda max. Unfolding of the protein in the presence of urea was fully reversible; furthermore, the presence of excess levels of cAMP stabilized the regulatory subunit. A free energy value (delta GDH2O) of 7.1 +/- 0.2 kcal/mol was calculated for the native form of the protein when denaturation was induced with either urea or guanidine hydrochloride. Iodide quenching of tryptophan fluorescence was used to elucidate the number of tryptophan residues accessible during various stages of the unfolding process. In the native cAMP-bound form of the regulatory subunit, only one of the three tryptophans in the regulatory subunit is quenched by iodide while more than two tryptophans can be quenched with iodide in the presence of 3 M urea.     

Different phosphorylation behaviour of regulatory subunit isoforms of type 11 cAMP-dependent protein kinase from bovine heart

Two forms of the regulatory subunit of the type II cAMP-dependent protein kinase (RII₅₅ and RII₅₂) were identified from bovine heart by gel electrophoretic behaviour. After autophosphorylation the RII₅₅ isoform migrated more slowly (RII_55/57) while the migration of RII₅₂ isoform did not shift. Both isoforms showed different affinity for cAMP. The RII_55/57 isoform was eluted from a cAMP-agarose column at 10 mM cAMP at low ionic strenght whereas the RII₅₂ isoform required cAMP, plus 2 M NaCl. Partial proteolysis, using trypsin or formic acid, of autophosphorylated regulatory subunit isoforms resulted in different cleavage pattern as determined by peptide mapping. However, the V8¹²⁵I-peptides patterns of both isoforms are quite similar.Incubation of partially purified holoenzyme with 10 nM [-³²P]ATP (low ATP concentration) yielded a single band of Mr = 57,000 which corresponds to the RII_55/57 isoform. The incubation, however, at 20 µM [-³²P]ATP yielded two phosphobands corresponding to both RII_55/57 and RII₅₂ isoforms. The phosphorylation of RII₅₂ took place with a lower efficiency and was more sensitive to the cAMP than the corresponding phosphorylation of the RII_55/57.     

Overexpression of the type II regulatory subunit of the cAMP-dependent protein kinase eliminates the type I holoenzyme in mouse cells

Mammalian tissues and cell lines express two major types of cAMP-dependent protein kinase, PKA-I and PKA-II, which can be distinguished at the molecular level by the presence of either type I or type II regulatory subunits in the holoenzyme. An expression vector for the mouse type II regulatory subunit (RII alpha) was transfected into ras-transformed NIH3T3 (R3T3) cells, which contain approximately equal amounts of both holoenzymes, PKA-I and PKA-II. In RII alpha-overexpressing R3T3 cells, PKA-II levels were increased, and the level of PKA-I declined. The decrease in PKA-I was dependent on the amount of RII alpha expressed, and at high levels of RII alpha expression, PKA-I was completely eliminated. In contrast, overexpression of the type I regulatory subunit (RI alpha) did not alter PKA isozyme levels. We propose that competition between RII alpha and RI alpha for a limited pool of catalytic subunit results in preferential assembly of PKA-II and that significant amounts of PKA-I are formed only if catalytic subunit is present in excess of the RII alpha subunit. The PKA-I isozyme, which is absent in untransformed 3T3 cells, is not essential for the transformed phenotype of R3T3 cells. RII alpha-overexpressing R3T3 cells that are devoid of PKA-I continued to exhibit a transformed phenotype including anchorage-independent growth. Overexpression of RII alpha provides a genetic approach that may prove useful in demonstrating specific functions for the two PKA isozymes in cAMP-dependent signal transduction pathways.