Binding of regulatory subunits of cyclic AMP-dependent protein kinase to cyclic CMP agarose

The bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis as well as soluble guanylyl cyclase α(1)β(1) synthesize the cyclic pyrimidine nucleotide cCMP. These data raise the question to which effector proteins cCMP binds. Recently, we reported that cCMP activates the regulatory subunits RIα and RIIα of cAMP-dependent protein kinase. In this study, we used two cCMP agarose matrices as novel tools in combination with immunoblotting and mass spectrometry to identify cCMP-binding proteins. In agreement with our functional data, RIα and RIIα were identified as cCMP-binding proteins. These data corroborate the notion that cAMP-dependent protein kinase may serve as a cCMP target.     

Concentrations of cyclic AMP-dependent protein kinase subunits in various tissues.

The concentrations of the regulatory (R) and catalytic (C) subunits of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase(s) were measured in extracts of skeletal muscle, heart, liver, kidney, and brain. These concentrations were also estimated for the particulate fraction from brain, the only tissue in which a major part of the total activity was not readily extracted in a soluble form. Values for R were determined by measuring the amount of cyclic [3H]amp bound to protein in these tissue fractions under specified conditions; it was assumed that 1 mol of cyclic AMP binds to 1 mol of R. Values for C were determined from measurements of the specific protein kinase activity of the fractions utilizing the turnover number of pure C in the calculations. Turnover numbers for C were found to be identical for this subunit obtained in the pure form from rabbit skeletal muscle, rabbit liver, and beef heart. The methods used for measuring C were evaluated by kinetic studies and through the use of the specific heatstable protein inhibitor of cyclic AMP-dependent protein kinase(s). R and C were found to exist in a 1:1 molar ratio in all of the tissue fractions that were studied. the absolute concentrations of R and C ranged from 0.23 mumol/kg wet weight for liver to 0.78 mumol/kg wet weight for brain. For brain this value was based on the amount of each subunit in the particulate as well as the soluble fraction. For other tissues the values were based solely on the subunit content of the latter fraction. It was noted that the molar concentrations of R are close to those of cyclic AMP under basal conditions in the various tissues.     

Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase.

Mutations in the SRA1 or SRA3 gene eliminate the requirement for either RAS gene (RAS1 or RAS2) in Saccharomyces cerevisiae. We cloned SRA1 and SRA3 and determined their DNA sequences. SRA1 encodes the regulatory subunit of the cyclic AMP (cAMP)-dependent protein kinase and therefore is identical to REG1 and BCY1. This gene is not essential, but its deletion confers many traits: reduction of glycogen accumulation, temperature sensitivity, reduced growth rate on maltose and sucrose, inability to grow on galactose and nonfermentable carbon sources, and nitrogen starvation intolerance. SRA3 is homologous to protein kinases that phosphorylate serine and threonine and likely encodes the catalytic subunit of the cAMP-dependent protein kinase. The wild-type SRA3 gene either triplicated in the chromosome or on episomal, low-copy plasmids behaves like spontaneous dominant SRA3 mutations by suppressing ras2-530 (RAS2::LEU2 disruption), cdc25, and cdc35 mutations. These findings indicate that the yeast RAS genes are dispensable if there is constitutive cAMP-dependent protein kinase activity.     

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.     

Identification of electrostatic interaction sites between the regulatory and catalytic subunits of cyclic AMP-dependent protein kinase

Two classes of molecules inhibit the catalytic subunit (C) of the cyclic AMP-dependent protein kinase (cAPK), the heat-stable protein kinase inhibitors (PKIs) and the regulatory (R) subunits. Basic sites on C, previously identified as important for R/C interaction in yeast TPKI and corresponding to Lys213, Lys217, and Lys189 in murine Cα, were replaced with either Ala or Thr and characterized for their kinetic properties and ability to interact with RI and PKI. rC(K213A) and rC(K217A) were both defective in forming holoenzyme with RI but were inhibited readily with PKI. This contrasts with rC(R133A), which is defective in binding PKI but not RI (Wen & Taylor, 1994). Thus, the C-subunit employs two distinct electrostatic surfaces to achieve high-affinity binding with these two types of inhibitory molecules even though all inhibitors share a common consensus site that occupies the active site cleft. Unlike TPK1, mutation of Lys189 had no effect. The mutant C subunits that were defective in binding RI, rC(K213A) and rC(K217A), were then paired with three RI mutants, rRI(D140A), rRI(E143A), and rRI(D258A), shown previously to be defective in recognition of C. Although the mutations at Asp140 and Asp258 in RI were additive with respect to the C mutations, rC(K213A) and rRI(E143A) were compensatory, thus identifying a specific electrostatic interaction site between RI and C. The results are discussed in terms of the RI and C crystal structures and the sequence homology between the yeast and mammalian enzymes.     

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.     

Localization of cyclic AMP-dependent protein kinase subunits in rat hepatocyte nuclei by immunogold electron microscopy

We have applied the indirect colloidal immunogold technique to examine the ultrastructural localization of the catalytic subunit C and the regulatory subunits RI and RII of cyclic AMP-dependent protein kinase in rat hepatocyte nuclei before and after glucagon or dibutyryl cyclic AMP administration. The technique allowed the identification and localization of all three subunits in hepatocyte nuclei. Morphometric quantitation of the relative staining density of nuclear subunits indicated an increase of immunogold staining of nuclear catalytic subunit but not of the regulatory subunits after glucagon or dibutyryl cyclic AMP stimulation. The increase of catalytic subunit occurred in a biphasic manner with peak levels 2-30 min and 90-150 min after stimulation. Our experiments represent the first reported use of the immunogold procedure to identify and localize protein kinase subunits in the nucleus.     

An unusual cyclic AMP-dependent protein kinase from nematodes

The search for an unusual cyclic nucleotide-dependent protein kinase in nematodes represented an attempt to gain some insight into the proposed homology of the cAMP and cGMP-dependent protein kinases. Two species of protein kinase were found in high speed supernatants of the mycophagous nematode $\text{Aphelenchus}$$\text{avenae}$. One of the two, bound to DEAE cellulose and was eluted from it in a manner characteristic of the type I cAMP kinase. The enzyme had high affinity for cAMP and dissociated upon binding to the cyclic nucleotide, as judged by the fact that catalytic activity did not bind to a cAMP affinity column. The second enzyme did not bind to DEAE. Unexpectedly, it too had high affinity for cAMP and much lower affinity for cGMP (unlike the $\text{cAMP}\text{cGMP}$ kinase from insects). The holoenzyme bound tightly to the cAMP affinity column and required a high concentration of the cyclic nucleotide for elution. This latter enzyme is the only example of a cAMP-dependent protein kinase that does not dissociate upon activation.     

Subcellular localization and biological actions of activated RSK1 are determined by its interactions with subunits of cyclic AMP-dependent protein kinase

Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.     

Kv4.2 phosphorylation by cyclic AMP-dependent protein kinase

Recent evidence suggests that K(+) channels composed of Kv4.2 alpha-subunits underlie a transient current in hippocampal CA1 neurons and ventricular myocytes, and activation of the cAMP second messenger cascade has been shown to modulate this transient current. We determined if Kv4.2 alpha-subunits were directly phosphorylated by cAMP-dependent protein kinase (PKA). The intracellular domains of the amino and carboxyl termini of Kv4.2 were expressed as glutathione S-transferase fusion protein constructs; we observed that both of these fusion proteins were substrates for PKA in vitro. By using phosphopeptide mapping and amino acid sequencing, we identified PKA phosphorylation sites on the amino- and carboxyl-terminal fusion proteins corresponding to Thr(38) and Ser(552), respectively, within the Kv4.2 sequence. Kinetic characterization of the PKA sites demonstrated phosphorylation kinetics comparable to Kemptide. To evaluate PKA site phosphorylation in situ, phospho-selective antisera for each of the sites were generated. By using COS-7 cells expressing an EGFP-Kv4.2 fusion protein, we observed that stimulation of the endogenous PKA cascade resulted in an increase in phosphorylation of Thr(38) and Ser(552) within Kv4.2 in the intact cell. We also observed modulation of PKA phosphorylation at these sites within Kv4.2 in hippocampal area CA1. These results provide insight into likely sites of regulation of Kv4.2 by PKA.     

Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase.

Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.     

Phosphorylation of casein by cyclic AMP-dependent protein kinase.

The catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein transferase) has been tested on a variety of caseins. The B variant of β-casein was phosphorylated at a much greater rate than other β-caseins, α_s1-caseins, and κ-caseins. Whole casein homozygous for β-casein B was phosphorylated at 2.5 times the rate of commercial whole casein. Gel electrophoresis experiments indicate that β-casein is the predominant component phosphorylated in commerical casein. It is therefore suggested that phosphorylation of whole casein depends on its content of the specific genetic variant, β-casein B.     

Ezrin is a cyclic AMP-dependent protein kinase anchoring protein.

cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.     

Autophosphorylation of cyclic AMP-dependent protein kinase from pig brain

Autophosphorylation of cyclic AMP-dependent pig brain protein kinase has been detected. Up to 1,5 moles of gamma-32P are transferred from [gamma-32P]ATP to the dimer of the regulatory subunit. The autophosphorylation reaction is Mg2+-dependent and occurs at a high rate: more than 50% of the radioactive label is incorporated during the first minute of incubation at 30 degrees. The pH dependence of this reaction differs from that of the phosphotransferase reaction. The phosphoholoenzyme is more sensitive to cyclic AMP than the dephosphoholoenzyme; however, both forms bind up to 2 moles of 3H-cyclic AMP per 1 mole of the holoenzyme. The activation and dissociation constants for both forms of the holoenzyme have been calculated. The autophosphorylation reaction has been shown to occur via an intramolecular mechanism; the phosphorylation of the regulatory subunit can occur only within the holoenzyme. The increase in the concentration of cyclic AMP causes the latter to produce an inhibitory effect on autophosphorylation. The regulatory action of autophosphorylation on cyclic AMP-dependent protein kinases is discussed.     

Reversible inhibition of cyclic AMP-dependent protein kinase by insulin

Summary Extracts of fasted rat diaphragms, previously treated with or without insulin were assayed for glycogen synthase, protein kinase and cyclic [³H]-AMP binding. Treatment with insulin produced an elevation in the % of glycogen synthase I and a concurrent decrease in cyclic AMP-dependent protein kinase activity and cyclic [³H]-AMP binding. Analysis of extracts by disc gel electrophoresis demonstrated the inhibition of cyclic [3H]-AMP binding to involve the Type I protein kinase holoenzyme. Inhibition of protein kinase activity was most apparent in the presence of 0.2 µM cyclic AMP, with enzymatic activity of the insulin-treated extracts typically 60–65% of control. Higher assay concentrations diminished the difference between control and insulin-treated extracts and concentrations greater than 20 µm abolished it.The inhibition of cyclic AMP-dependent protein kinase activity after insulin was a transient and labile phenomenon. The effect was independent of ATP concentration in the assay, but was sensitive to the pH of tissue extraction, requiring a pH of 7.0 to 8.4 to be observed.Insulin-mediated inhibition of protein kinase activity was reversed upon preincubation of extracts at 0–2°. Relatively concentrated homogenates (<4 µl buffer/mg tissue) yielded extracts which exhibited little or no inhibition of protein kinase activity compared to extracts prepared from more dilute (6–10 µl/mg) homogenates. A model for the inhibition of the cyclic-AMP dependent protein kinase by an insulin-generated inhibitor which becomes directly associated with the Type 1 holoenzyme is proposed.Abbreviations cyclic AMP (cAMP) Adenosine 3,5-monophosphate - Tricine N-Tris (Hydroxy-methyl) methyl glycine - G-6-P glucose-6-phosphate - MES 2-[N-morpholino]ethane sulfonic acid A preliminary report was communicated to the 61st meeting of the F.A.S.E.B., April, 1977.     

Cyclic AMP-dependent protein kinase does not phosphorylate cyclic GMP-dependent protein kinase in vitro

The autophosphorylation reaction of purified cGMP-dependent protein kinase has been studied. Apparent initial rates of autophosphorylation in the absence of cyclic nucleotides and in the presence of cGMP and cAMP are 0.006, 0.04, 0.4 mol Pi incorp./min-1. mol cGMP-kinase subunit-1. In the presence of cGMP and cAMP approximately 1 and 2 mol Pi are incorporated/mol enzyme subunit. These values are independent of the enzyme concentration. Stimulation of autophosphorylation by cAMP is not due to activation of a contaminating cAMP-dependent protein kinase since: (a) addition of the heatstable inhibitor protein of cAMP-kinase does not inhibit autophosphorylation; and (b) catalytic subunit of cAMP-kinase added at a 10-fold excess over cGMP-kinase does not phosphorylate cGMP-kinase.