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.     

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.     

Diastereomers of adenosine 3',5'-monothionophosphate (cAMP[S]) antagonize the activation of cGMP-dependent protein kinase

cGMP-dependent protein kinase contains four cGMP-binding sites which are homologous to the four cAMP-binding sites of cAMP-dependent protein kinase. The interaction of the diastereomers of adenosine 3',5'-thionophosphate, (PS)-cAMP[S] and (PR)-cAMP[S], with cGMP-dependent protein kinase has been studied. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 microM and 94 microM, respectively. cAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 microM. The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. KA 1 microM) and cAMP (approx. KA 98 microM) but neither by the (PR) nor (PS) stereoisomer of cAMP[S]. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively cAMP-or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 microM and 73 microM, respectively. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S] with IC50 values of 200 microM and 15 microM, respectively. These results show that both diastereomers of cAMP[S] bind to cGMP-dependent protein kinase. (PR)-cAMP[S] has properties of a pure antagonist whereas (PS)-cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP-binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.     

Inhibitory effect of the regulatory subunit of type I cAMP-dependent protein kinase on phosphoprotein phosphatase

The regulatory subunit of type I cAMP-dependent protein kinase (RI) from rabbit skeletal muscle inhibited the activity of a low molecular weight phosphoprotein phosphatase. The inhibition was concentration and time dependent. A maximum inhibition, about 70%, was observed at 2 microM of RI with an apparent Ki of 0.8 microM. Inhibition was associated with a decrease in Vmax with no change in Km for substrate, phosphorylase a. On the other hand, cAMP-dependent protein kinase holoenzyme or its catalytic subunit was without any effect. The inhibition of phosphoprotein phosphatase by RI may be of physiological significance since the dissociation of cAMP-dependent protein kinase by cAMP would result in a simultaneous increase in the phosphorylation and decrease in the dephosphorylation rates of target proteins.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

Purified cyclic GMP-dependent protein kinase catalyzes the phosphorylation of cardiac troponin inhibitory subunit (TN-1).

Cyclic AMP- and cGMP-dependent protein kinases catalyze the phosphorylation of cardiac troponin inhibitory subunit (TN-I). Unlike many substrates utilized by both kinases, TN-I is rapidly phosphorylated using relatively low concentrations of the cGMP-dependent protein kinase (0.01 to 0.1 micrometer). At low concentrations of cAMP- and cGMP-dependent protein kinases, approximately twice as much total phosphate is incorporated into TN-I using the cAMP-dependent enzyme. At higher enzyme concentrations, 1 mol of phosphate/mol of TN-I is found using either enzyme. Maximal levels of cAMP- and CGMP-dependent protein kinases do not catalyze additive phosphorylation, suggesting that the two enzymes catalyze the phosphorylation of the same site on TN-I. The results support the concept of overlapping substrate specificity for cAMP- and cGMP-dependent protein kinases, but suggest that cardiac troponin contains additional specificity determinants for the cGMP-dependent protein kinase not found in several other protein substrates.     

A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. II. Kinetic studies on its phosphorylation by guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases

Kinetic studies on the activity of purified cGMP-dependent protein kinase and catalytic subunit of cAMP-dependent protein kinase have been carried out using a protein termed G-substrate (see preceding paper) as the phosphate acceptor. Each enzyme catalyzed the phosphorylation of 2.0-2.1 mol of 32P/mol of G-substrate, with phosphorylation occurring primarily at threonine residues. When phosphorylation was carried out in the simultaneous presence of the two enzymes, the stoichiometry increased only slightly, to a value of 2.4, suggesting that both enzymes phosphorylated the same two sites. Initial rate studies on the phosphorylation of G-substrate by cGMP-dependent protein kinase yielded a Km of 0.21 microM and a Vmax of 2.2 mumol/min/mg. Similar studies with the cAMP-dependent protein kinase yielded a Km of 5.8 microM and a Vmax of 2.3 mumol/min/mg. cGMP-dependent protein kinase thus exhibited a high degree of specificity towards this substrate which was apparently based on selective substrate binding rather than catalytic efficacy. The activity of cGMP-dependent protein kinase towards G-substrate was maximal at pH 7.5-8.0 and a Mg2+ concentration of 1-3 mM. Activity declined sharply at high ionic strength (greater than 20 mM KCl).     

Phosphorylation of cardiac troponin by guanosine 3':5'-monophosphate-dependent protein kinase.

Homogeneous cGMP-dependent protein kinase catalyzes the rapid incorporation of phosphate, specifically into the inhibitory subunit of purified cardiac troponin with a maximal incorporation of 1 mol of phosphate/mol of troponin. When troponin was incubated in the presence of both cGMP- and cAMP-dependent protein kinases, a maximal incorporation of 1 mol of phosphate/mol of troponin was observed which suggested phosphorylation of the same site by the two kinases. Both cyclic nucleotide-dependent kinases had similar Km values for troponin, but the Vmax value for the phosphorylation reaction catalyzed by cAMP-dependent protein kinase was 12-fold greater than the value obtained for cGMP-dependent protein kinase.     

Inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3',5'-monophosphorothioates

The activation of the cGMP-dependent protein kinase and cAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monophosphorothioate, (Sp)-cGMPS and (Rp)-cGMPS, and 8-chloroguanosine 3',5'-monophosphorothioate, (Sp)-8-Cl-cGMPS and (Rp)-8-Cl-cGMPS, was investigated using the peptide Kemptide as substrate. The (Sp)-diastereomers, which have an axial exocyclic sulfur atom, bound to the cGMP-dependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers, which have an equatorial exocyclic sulfur atom, bound to the enzyme without stimulation of its activity. (Rp)-cGMPS and (Rp)-8-Cl-cGMPS antagonized the activation of the cGMP-dependent protein kinase with a Ki of 20 microM and 1.5 microM, respectively. (Rp)-cGMPS also antagonized the activation of cAMP-dependent protein kinase with a Ki of 20 microM. In contrast, (Rp)-8-cGMPS ws a weak inhibitor of the cAMP-dependent protein kinase with a Ki of 100 microM. (Rp)-8-Cl-cGMPS appears to be a rather selective inhibitor of the cGMP-dependent protein kinase and may be a useful tool for studying the role of cGMP in broken and intact cell systems.     

Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2-terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase.     

Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.     

PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 micromol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RIalpha) of cAMP-dependent protein kinase (KD = 10 nM) and the heat-stable protein kinase inhibitor (KD = 15 nM), but not with the type II regulatory subunit (RIIalpha, KD = 2.3 microM) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro, but only weakly by the RIIalpha subunit. The inhibition by the RIalpha subunit is reversed by addition of nanomolar concentrations of cAMP (Ka = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the Calpha subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RIalpha subunit and by phosphorylating the RIIalpha subunit in the absence of cAMP.     

Mechanism of autophosphorylation of the multifunctional Ca2+/calmodulin-dependent protein kinase

The multifunctional Ca2+/calmodulin-dependent protein kinase purified from rat brain cytosol undergoes a self-phosphorylation or autophosphorylation reaction. Our conclusion that this reaction is autocatalytic is based on the following lines of evidence: The autophosphorylation reaction and the protein kinase activity toward other substrates are absolutely dependent on the presence of both Ca2+ and calmodulin; autophosphorylation and phosvitin kinase activity show a similar time course and indistinguishable heat lability; the reaction is a consistent property of every preparation of rat brain kinase; the reaction is present in both crude and highly purified preparations of similar kinases or isozymes from rat lung, spleen, heart, bovine brain, and a neuronal tissue from Aplysia californica, a marine mollusk; phosphorylation of the kinase subunits is not mimicked by addition of cAMP, cGMP, Ca2+ plus diglyceride, or addition of the cAMP-dependent protein kinase, and is not blocked by the heat-stable inhibitor protein of the cAMP-dependent protein kinase; and the reaction is intramolecular. Autophosphorylation results in the stoichiometric incorporation of phosphate into both the 51,000- and 60,000-dalton subunits.     

Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites

Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.     

Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides.

Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C alpha subunit. Ki values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher Km for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (1991) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.     

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.     

Mechanism of self-phosphorylation of adenosine 3':5'-monophosphate-dependent protein kinase from bovine cardiac muscle.

The adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase purified from bovine cardiac muscle catalyzes the transfer of up to 2 mol of 32P from [lambda-32P]ATP to seryl residues in its cyclic nucleotide-binding protein component (Erlichman, J., Rosenfeld, R., and Rosen, O. M. (1974) J. Biol. Chem. 249, 5000-5003). We now present three lines of evidence to support our conclusions that the undissociated holoenzyme does not catalyze the phosphorylation of exogenous substrates but can undergo self-phosphorylation by an intramolecular reaction: (a) addition of either cAMP-binding protein or the protein kinase inhibitor (Walsh, D. A., Ashby C. D., Gonzales, C., Calkins, D., Fischer, E. H., and Krebs, D. G. (1971) J. Biol. Chem. 241, 1977-1985) does not inhibit self-phosphorylation as it does phosphorylation of exogenous substrates in the presence or absence of cAMP; (b) addition of catalytic subunit to an excess of cyclic nucleotide-binding protein results in phosphorylation equivalent to the amount of holoenzyme so generated; (c) the rate of self-phosphorylation is not affected by dilution of the holoenzyme.     

Phosphorylation of RII subunit and attenuation of cAMP-dependent protein kinase activity by proline-directed protein kinase.

Previous studies identified proline-directed protein kinase (PDPK) as a growth factor-sensitive serine/threonine protein kinase that is active in the cytosol of proliferative cells and tissues during interphase. In this communication, we report that the regulatory subunit (RII) of bovine cardiac muscle cAMP-dependent protein kinase (PKA) is a putative substrate for the multifunctional PDPK. Purified RII is readily phosphorylated by PDPK in vitro in a time-dependent, enzyme-dependent manner to a stoichiometry approaching 0.7 mol phosphate/mol RII subunit protein. The major RII phosphorylation site is identified as a threonine residue located within a large hydrophobic tryptic peptide that is predicted to contain the cAMP binding domains. In contrast to the reported effects of RII autophosphorylation, kinetic analysis of RII function following phosphorylation by PDPK indicates that the inhibitory potency of RII toward the catalytic subunit of PKA in a reassociation assay is increased in proportion to the degree of phosphorylation. Further studies indicate that the cAMP-dependent activation of the RII2C2 holoenzyme is inhibited by PDPK phosphorylation. Taken together, the results of these studies indicate that phosphorylation of RII by PDPK attenuates the activity of PKA. This antagonistic interaction suggests a biochemical mechanism by which a growth factor-activated signaling system may function to modulate cAMP-dependent cellular responses.     

Phospholipids and regulation of protein kinase reaction

Naturally occurring phospholipids such as phosphatidylinositol and phosphatidylserine inhibited cAMP-dependent protein kinase by interacting with the substrate protein (phosphate acceptor). This inhibition was observed both in the presence and absence of cAMP when histone H2B, protamine, and myelin basic protein were used, but was not detected when casein was used as the substrate. Other phospholipids such as phosphatidylethanolamine and phosphatidylcholine did not inhibit the kinase but did stimulate the kinase when protamine served as the substrate. Both cAMP-dependent and cGMP-dependent protein kinases were inhibited by phosphatidylserine when histone H2B was used as substrate. The substrate protein binding to phosphatidylinositol and phosphatidylserine was observed when these phospholipids were added to the incubation mixture, suggesting that direct interaction between the substrate protein and the phospholipids resulted in inhibition of cAMP and cGMP-dependent protein kinase. Thus the substrate protein for protein kinase probably plays an important role in regulating the kinase activity related to various phospholipids.     

Regulation of glycogen synthetase. Specificity and stoichiometry of phosphorylation of the skeletal muscle enzyme by cyclic 3':5'-AMP-dependent protein kinase.

Complete conversion of skeletal muscle glycogen synthetase from the I form to the D form requires incorporation of 2 mol of phosphate per enzyme subunit (90,000 g). Incubation of sythetase I with low concentrations of adenosine 3':5'-monophosphate(cAMP)-dependent protein kinase (10 units/ml) and ATP (0.1 to 0.3 mM) plus magnesium acetate (10 mM) results in incorporation within 1/2 hour of 1 mol of phosphate persubunit concomitant with a decrease in the synthetase activity ratio (minus glucose-6-P/plus glucose-6-P) from 0.85 to 0.25. Further incubation for 6 hours does not greatly increase the phosphate content of the synthetase or promote conversion to the D form. This level of phosphorylation is not increased by raising the concentration of protein kinase to 150 units/ml and is not influenced by the presence of glucose-6-P, UDP-glucose, or glycogen. However, at protein kinase concentrations of 10,000 to 30,000 units/ml a second mol of phosphate is incorporated per subunit, and the sythetase activity ratio decreases to 0.05 or less. In addition to the 2 mol of phosphate persubunit which are required for formation of sythetase D, further phosphorylation can be observed which is not associated with changes in synthetase activity. This phosphorylation occurs at a slow rate, is increased by raising the ATP concentration to 2 to 4mM, and is not blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. These data indicate that skeletal muscle glycogen synthetase contains multiple phosphorylation sites only two of which are involved in the synthetase I to D conversion.