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.     

Inhibition of thyroidal cyclic AMP-dependent protein kinase by thyroid hormone.

Bovine thyroid cyclic AMP-dependent protein kinase was purified by DEAE-Sephadex and Sephadex G-200 chromatography. This preparation showed a 240-fold increase in specific activity over the initial 20,000 x g supernatant with histone as substrate and 1 micronM cyclic AMP in the assay mixture. In the presence of 2.5 X 10(-5)M L-triiodothyronine (T3), protein kinase activity was significantly reduced; 50% inhibition was achieved at 1 X 10(-4) M. Tests of diverse thyroid hormone analogs showed that T3 and its derivatives were more potent inhibitors than T4 and its derivatives which, in turn, were more potent than thyronine or diiodothyronine. Mono- and diiodotyrosine, tyrosine, and iodide were without effect. Triiodothyronine did not inhibit kidney, spleen, or lung protein kinase activity. The magnitude of the inhibition was the same whether or not cyclic AMP (1 micronM) was present in the incubation mixture, suggesting an effect on the catalytic, rather than the regulatory subunit of the enzyme. The inhibition of protein kinase by thyroid hormone was not influenced by Mg++ concentration but was overcome in a competitive manner by increasing ATP concentration. Increasing the histone concentration did not modify the inhibition. Although these studies suggest a novel cellular control mechanism, the high thyroid hormone concentrations required and the lack of concordance between inhibitory effects and biologic activity of the analogs tested precludes assumption of physiologic relevance.     

Uptake of alpha-aminoisobutyric acid and phosphate by membrane vesicles derived from growing and quiescent fibroblasts.

Membrane vesicles derived principally from the plasma membrane and endoplasmic reticulum of mouse 3T3 cells transformed by Simian virus 40 take up alpha-aminoisobutyric acid (AIB) and phosphate (Pi). When NaCl is added simultaneously with AIB or Pi, uptake rises two- to three-times above the equilibrium to accumulate AIB or Pi over the control value, in the presence of a Na+ gradient, is almost lost in membrane vesicles derived from benzpyrene-transformed 3T3 cells (BP3T3) arrested in the G1 phase of the cell cycle by serum starvation. When added to the membranes with NaCl and the uptake substrate, a combination of fibroblast growth factor (FGF) and epidermal growth factor EGF restores the ability of the membranes to accumulate AIB and Pi over the control value.     

Inhibition of rat liver cyclic AMP-dependent protein kinase by flavonoids.

Rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK), assayed using the synthetic peptide substrate, LRRASLG, is inhibited by a range of plant-derived flavonoids. In general, maximal inhibitory effectiveness (IC50 values 1 to 2 microM) requires 2,3-unsaturation and polyhydroxylation involving at least two of the three flavonoid rings. 3-Hydroxyflavone (IC50 value 4 microM), 3,5,7,2',4'-pentahydroxyflavone (IC50 = 10 microM) and 5,7,4'-trihydroxyflavone (IC50 = 7 microM) represent somewhat less active variations from this pattern. Flavonoid O-methylation or O-glycosylation greatly decreases inhibitory effectiveness, as does 2,3-saturation. Various flavonoid-related compounds, notably gossypol (IC50 = 10 microM), also inhibit cAK. Flavonoids and related compounds are in general much better inhibitors of cAK than of avian Ca(2+)-calmodulin-dependent myosin light chain kinase or of plant Ca(2+)-dependent protein kinase. Tricetin (IC50 = 1 microM) inhibits cAK in a fashion that is non-competitive with respect to both peptide substrate and ATP (Ki value 0.7 microM). When histone III-S is used as a substrate, inhibition of cAK requires much higher flavonoid concentrations.     

Inhibition of cerebral protein kinase activity and cyclic AMP-dependent ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia

Studies were carried out to elucidate the mechanisms underlying the diminished phosphorylation of cerebral ribosomal protein in experimental hyperphenylalaninaemia [Roberts & Morelos (1980) Biochem. J.190, 405-419]. Administration of N(6),O(2)'-dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine, which increased phosphorylation of the S6 protein of cerebral 40S ribosomal subunits in control infant rats, did not counteract the decreased phosphorylation of this ribosomal protein resulting from intraperitoneal administration of a loading dose of l-phenylalanine. N(2),O(2)'-Dibutyryl cyclic GMP had no effect on cerebral ribosomal-protein phosphorylation in either control or hyperphenylalaninaemic animals. The phenylalanine-induced decrease in ribosomal-protein phosphorylation was associated with decreased protein kinase activity in cerebral cytosolic and microsomal preparations. However, the maximal protein kinase response to cyclic AMP added in vitro was unaltered by prior administration of phenylalanine in vivo. The heat-stable protein inhibitor of cyclic AMP-dependent protein kinases decreased the activity of these enzymes by about 90% and eliminated the phenylalanine-induced difference in protein kinase activity in the absence of added cyclic AMP. Intracisternal administration of doses of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine which increased the cyclic AMP-dependent protein kinase activity ratio in control infant rats was without effect on this index in phenylalanine-treated animals. Dibutyryl cyclic GMP had no effect on the protein kinase activity ratio in either group of animals. These results suggest that inhibition of cerebral cyclic AMP-dependent protein kinases by abnormally high concentrations of phenylalanine may contribute to the decrease in cerebral ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.     

Inhibition of mammary gland cyclic AMP-dependent protein kinase by arginine-rich histones.

The cyclic AMP-dependent protein kinase activity from lactating bovine mammary gland efficiently phosphorylates lysine-rich histones but not arginine-rich histones. It is shown that arginine-rich histones in fact inhibit phosphorylation of lysine-rich histones. Polyarginine and a range of low molecular weight cationic molecules are also inhibitors. Inhibition of histone H2b phosphorylation by histones H4 and H3 is competitive with respect to H2b. This inhibition behaviour may be tissue-specific since the protein kinase activity in crude extracts from lactating bovine mammary gland, although heterogeneous, may be completely inhibited (>95%) by arginine-rich histones and polyarginine.     

Phosphofructokinase from Dirofilaria immitis. Stimulation of activity by phosphorylation with cyclic AMP-dependent protein kinase

Phosphofructokinase has been partially purified from the filariid helminth, Dirofilaria immitis, using ion exchange and affinity chromatography. The D. immitis phosphofructokinase cross-reacted with antibodies prepared against the phosphofructokinase from Ascaris suum. These antibodies had been bound to agarose beads. The enzyme was eluted from the immobilized antigen-antibody complex by denaturing agents, and the subunit molecular weight determined by sodium dodecyl sulfate gel electrophoresis was identical to that of the ascarid enzyme, 90,000. At pH 6.8, substrate saturation curves of the filarial phosphofructokinase with ATP revealed that the enzyme was inhibited by ATP. The fructose-6-P saturation curve was sigmoid at all ATP levels tested. Phosphorylation of the D. immitis phosphofructokinase by the catalytic subunit of beef heart cyclic AMP-dependent protein kinase resulted in incorporation of 0.8 mol of phosphate/mol of subunit and in a 3-4-fold increase in catalytic activity when measured at pH 6.8 at inhibitory levels of ATP. Additional kinetic studies revealed that the phosphorylated enzyme was less susceptible to ATP inhibition than was the nonphosphorylated form. It is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of carbohydrate metabolism in the filarial as well as the intestinal-dwelling nematodes.     

Endogenous substrates for in vitro phosphorylation by cyclic AMP-dependent protein kinase from Dictyostelium discoideum

Endogenous proteins which could serve as substrates for cyclic AMP-dependent protein kinase in vitro were measured in cytosolic fractions at four stages of development. A peak of cyclic AMP-dependent phosphorylation occurred at the slug stage, coincident with the appearance of cyclic AMP-dependent protein kinase. After partial purification of the slug-stage extracts by DE-52 cellulose and Sephacryl S-300 chromatography, cyclic AMP dependency of six proteins was observed. The apparent subunit molecular weights of the proteins were greater than 200,000, 110,000, 107,000, 91,000, 75,000 and 69,000. Upon further purification of the cyclic AMP-dependent protein kinase by chromatofocusing, the endogenous substrates were separated from the enzyme. In addition, the enzyme separated into catalytic and regulatory subunits. If the purified catalytic subunit was added to heated S300 fractions, proteins with apparent molecular weights of 91,000 and 107,000 were specificity phosphorylated. The results show the stage-dependent appearance of a cyclic AMP-dependent protein kinase and point out several in vitro substrates for the enzyme.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts.

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743-745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L6 myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system. We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f2b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10(-5) M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase. Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems. The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane phosphorylation as a regulator of membrane functions including those that may control cellular differentiation.     

Activation and phosphorylation of the 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase.

Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense-vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.     

Evidence for phosphorylation of bovine adrenal tyrosine hydroxylase by cyclic AMP-dependent protein kinase.

Direct phosphorylation of bovine adrenal tyrosine hydroxylase with an associated increase in enzyme activity by cyclic AMP-dependent protein kinase was demonstrated by gel filtration on Sephadex G-200.     

Active phosphate ion transport in plasma membrane vesicles isolated from mouse fibroblasts.

Inorganic phosphate accumulated 8-fold in plasma membrane vesicles derived from simian virus 40-transformed 3T3 mouse fibroblasts when a NaCl gradient (external greater than internal) was artificially imposed across the membrane. Preincubation with Na+ or addition of monensin markedly reduced phosphate accumulation. Na+-stimulated phosphate transport was not affected by addition of either dicarboxylic acids, antimycin A, or ouabain and persisted after addition of proton ionophores. The coupling of phosphate transport to Na+ gradients was pH-dependent, with maximal stimulation by Na+ below pH 7. These findings suggest that monovalent phosphate anion moves across the plasma membrane in co-transport with sodium ion.     

In vitro phosphorylation of type I collagen by cyclic AMP-dependent protein kinase

Both the triple-helical and denatured forms of nonfibrillar bovine dermal type I collagen were tested as substrates for the catalytic subunit of cAMP-dependent protein kinase in an in vitro reaction. Native, triple-helical collagen was not phosphorylated, but collagen that had been thermally denatured into individual alpha chains was a substrate for the protein kinase. Catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured collagen to between 3 to 4 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Pepsin-solubilized and intact collagens were phosphorylated similarly, as long as each was in a nonhelical conformation. The first 2 mol of phosphate incorporated into type I collagen by the protein kinase were present in the alpha 2(I) chain. The alpha 1(I) chain was only phosphorylated during long incubations in which the stoichiometry exceeded 2 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Phosphoserine was the only phosphoamino acid identified in collagen that had been phosphorylated to any degree by the protein kinase. The 2 mol of phosphate incorporated into the alpha 2(I) chain were localized to the alpha 2(I)CB4 cyanogen bromide fragment. The catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured pepsin-solubilized collagen with a Km of 8 microM and a Vmax of approximately 0.1 mumol/min/mg of enzyme. Denatured, but not triple-helical, type I collagen was also phosphorylated by cGMP-dependent protein kinase, although it was a poorer substrate for this enzyme than for the cAMP-dependent protein kinase. Collagen was not a substrate for phospholipid-sensitive Ca2+-dependent protein kinase. These results suggest the potential for nascent alpha chains of type I collagen to be susceptible to phosphorylation by cAMP-dependent protein kinase in vivo prior to triple-helix formation. Such a phosphorylation of collagen could be relevant to the action of cAMP to increase the intracellular degradation of newly synthesized collagen.     

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.     

Inhibition of cyclic AMP-dependent protein kinase by analogues of a synthetic peptide substrate.

Analogues of the synthetic substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in which the serine is replaced by other amino acids inhibited the activity of the catalytic subunit of cyclic AMP-dependent protein kinase from beef skeletal muscle (Peak I). All of the analogues were competitive with respect to peptide substrate but apparent Ki values varied depending on the particular amino acid that was substituted for serine. Inhibition was also competitive with respect to mixed histone as determined in experiments utilizing one of the analogues. Acetylation of the terminal amino group of Leu-Arg-Arg-Ala-Ser-Leu-Gly lowered the Km for this substrate from 16 micrometer to 3 micrometer, but a similar modification of the inhibitory analogue Leu-Arg-Arg-Ala-Ala-Leu-Gly resulted in no major change in the Ki value. An amount of inhibitory peptide sufficient to inhibit the cyclic AMP-dependent protein kinase by 90% caused less than 10% inhibition of several cyclic AMP-independent protein kinases indicating a high degree of specificity of inhibition by the peptide analogues. The experiments show that synthetic peptide analogues could be useful in identifying phosphorylation reactions catalyzed by cyclic AMP-dependent protein kinase as distinguished from other protein kinase reactions.     

Evidence against phosphorylation of a cyclic AMP-dependent protein kinase from bovine tracheobronchial smooth muscle.

The data presented in this report are evidence against the autophosphorylation of the cyclic AMP-dependent protein kinase isolated from bovine tracheobronchial smooth muscle. This suggests that there may be a fundamental difference in the regulation in vivo of the protein kinases from bovine heart and tracheobronchial smooth muscle.     

Evidence for phosphorylation of rat brain guanylate cyclase by cyclic AMP-dependent protein kinase

Direct phosphorylation of purified rat brain guanylate cyclase by cyclic AMP-dependent protein kinase is demonstrated. In the presence of [γ-³²P]ATP, ³²P was incorporated into the protein to the extent of 0.8 to 0.9 mol/mol of guanylate cyclase. The presence of ³²P in the guanylate cyclase molecule was demonstrated by gel-filtration and by autoradiography after gel electrophoresis. The phosphorylation was accompanied by an increase in enzyme activity, characterized by an increase of V_M. These results suggest that the activity of guanylate cyclase may be regulated in vivo by phosphorylation.     

Liver isozyme of rabbit glycogen synthase. Amino acid sequences surrounding phosphorylation sites recognized by cyclic AMP-dependent protein kinase

Glycogen synthase, the rate-limiting enzyme in glycogen biosynthesis, has been postulated to exist as isozymes in rabbit liver and muscle (Camici, M., Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1984) J. Biol. Chem. 259, 2466-2473). Both isozymes share a number of properties including multiple phosphorylation of the enzyme subunit. In the present study, we determined the amino acid sequences surrounding phosphorylation sites in the rabbit liver isozyme recognized by cyclic AMP-dependent protein kinase. Two dominant phosphopeptides (P-1 and P-2) were generated from tryptic digestion. Amino acid sequences of the purified peptides were determined by automated Edman degradation using a gas-phase sequenator. The locations of phosphorylated residues were identified by measuring 32Pi release during Edman degradation cycles. The NH2-terminal sequence of peptide P-1 is S-L-S(P)-V-T-S-L-G-G-L-P-Q-W-E-V-E-E-L-P-V-D-D-L-L-L-P-E-V. This sequence exhibits a strong homology to the site 2 region in the NH2 terminus of the muscle isozyme. The NH2-terminal sequence of peptide P-2 is M-Y-P-R-P-S(P)-S(P)-V-P-P-S-P-L-G-S-Q-A. This sequence shows strong homology to the site 3 region in the COOH terminus of the muscle isozyme. However, some interesting sequence differences were revealed in this region. For example, substitution of serine for alanine at position 6 of peptide P-2 created a new phosphorylation site for cyclic AMP-dependent protein kinase. Phosphorylation of the proline/serine-rich site 3 region correlated with inactivation of the liver isozyme and suggests an important role for this segment of the molecule in the regulation of glycogen synthase. No phosphorylation sites corresponding to sites 1a and 1b of the muscle isozyme were detected. In addition, the results provide definitive chemical proof that glycogen synthase from rabbit liver and muscle are isozymes encoded by distinct messages.