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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

The role of autophosphorylation in activation of the type II calmodulin-dependent protein kinase

Autophosphorylation of the type II calmodulin-dependent protein kinase is known to remove the dependence of this enzyme on Ca2+ and calmodulin. The enzymatic activity in the presence of Ca2+, on the other hand, was reported to be unaffected or decreased by this interconversion. The role of autophosphorylation in the kinase reaction was reinvestigated using short assay times and low ATP concentrations to decrease the extent and rate of this process. Under these conditions, the ATP dependence of the kinase reaction with syntide-2 as the substrate (but not the autophosphorylation reaction) exhibited kinetic cooperativity due to a lag in the progress curve of syntide-2 conversion. Partial autophosphorylation of the protein kinase prior to phosphorylation of the peptide substrate completely abolished this hysteretic response without affecting the final rate of substrate conversion. These observations suggest that autophosphorylation is an obligatory step in the response of this kinase to activation by calmodulin.     

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.     

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.     

Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

An expression vector has been constructed for the type I regulatory subunit of cAMP-dependent protein kinase. A cDNA clone for the bovine RI-subunit has been inserted into pUC7. When Escherichia coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-azidoadenosine 3':5'-mono[32P]phosphate following transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of isopropyl-beta-D-thiogalactopyranoside. R-subunit accumulated in large amounts only in the stationary phase of growth, and the addition of isopropyl-beta-D-thiogalactopyranoside during the log phase of growth actually blocked the accumulation of R-subunit. Maximum expression (20 mg/liter) was achieved when E. coli 222 was transformed with the RI-containing plasmid. E. coli 222 is a strain that contains two mutations; it is cya- and also has a mutation in the catabolite gene activator protein (crp) that enables the protein to bind to DNA in the absence of cAMP. The expressed RI-subunit was a soluble, dimeric protein, and no significant proteolysis was apparent in the cell extract. The purified RI-subunit bound 2 mol of cAMP/mol of R monomer, reassociated with C-subunit to form holoenzyme, and migrated as a dimer on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis, yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties, the expressed protein was indistinguishable from RI purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z' gene of the vector. This NH2-terminal sequence was confirmed by amino acid sequencing.     

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.     

The neural type II regulatory subunit of cAMP-dependent protein kinase is present and regulated by hormones in the rat ovary

In this study purified isoforms of rat ovarian regulatory subunit of type II cAMP-dependent protein kinase (R-II) were compared with R-II purified from rat brain. A special neural form of R-II has been previously described in bovine brain. Analysis by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved three isoforms of rat ovarian R-II (R-II54, Mr = 54,000; R-II52, Mr = 52,000; and R-II51, Mr = 51,000) compared to two R-II isoforms in rat brain (R-II54 and R-II52). Polychromatic silver-stained peptide maps of purified R-II subunits indicated that peptides generated from both rat ovarian R-II52 and R-II51 were similar (if not identical) to the peptides of the neural form, R-II52, purified from rat brain. These peptides differed markedly from those generated from R-II54 of either rat ovary, brain, or heart. Ovarian R-II52/51 photoaffinity labeled with 8-N3-[32P]cAMP and analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was shown to consist of three (rather than two) isoelectric variants, which were similar to three variants resolved from rat brain R-II and clearly distinct from that of rat heart R-II54. An antibody which recognized both the R-II54 and R-II52/51 isoforms of rat ovarian extracts also recognized both forms of rat brain R-II (R-II54 and R-II52) and similar forms in extracts of rat adrenal and parotid glands. These results strongly suggest that the R-II52 isoform previously designated as a neural specific form of R-II is present in high concentrations in a nonneural tissue, the rat ovary.     

Interactions between regulatory and catalytic subunits of the Candida albicans cAMP-dependent protein kinase are modulated by autophosphorylation of the regulatory subunit

The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser(180)) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser(180) is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [(32)P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.