Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase.

The present study was undertaken in order to identify the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKI) and to synthesize a peptide that could serve as a useful inhibitor of the enzyme. Digestion of purified PKI by mast cell proteinase II yielded a peptide fragment that retained inhibitory activity. A sequence of 20 amino acids of the peptide, (sequence in text) revealed the presence of a "pseudosubstrate site" (Arg-Arg-Asn-Ala-Ile) for the cAMP-dependent protein kinase in which alanine replaces the seryl or threonyl residue that is normally phosphorylated. Digestion of PKI with various other proteinases implicated the involvement of arginyl and hydrophobic residues as determinants for the inhibitory activity. The assumption that this region is part of the inhibitory site was confirmed by the synthesis of a corresponding duodecapeptide that displayed strong inhibitory activity. Inhibition by the peptide was competitive with a Ki of 0.8 microM as measured against a number of protein substrates. The sequence of this fragment bears a strong resemblance to the autophosphorylation site in the type II regulatory subunit of cAMP-dependent protein kinase, a region also postulated to interact with the catalytic subunit, and the analogous region of type I regulatory subunit. Neither intact PKI nor the synthetic peptide inhibit the cGMP-dependent protein kinase, phosphorylase kinase, myosin light-chain kinase, casein kinase II, or protein kinase C.     

Phosphorylation of yeast phosphatidylserine synthase in vivo and in vitro by cyclic AMP-dependent protein kinase.

Evidence is presented that demonstrates that phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) from Saccharomyces cerevisiae is phosphorylated in vivo and in vitro by cAMP-dependent protein kinase. Phosphatidylserine synthase activity in cell extracts was reduced in the bcy1 mutant (which has high cAMP-dependent protein kinase activity) and elevated in the cyr1 mutant (which has low cAMP-dependent protein kinase activity) when compared with wild-type cells. The reduced phosphatidylserine synthase activity in the bcy1 mutant correlated with elevated levels of a phosphorylated form of the phosphatidylserine synthase Mr 23,000 subunit. The elevated phosphatidylserine synthase activity in the cyr1 mutant correlated with reduced levels of the phosphorylated form of the enzyme. There was negligible phosphorylation of the phosphatidylserine synthase Mr 23,000 subunit from stationary-phase cells. Pure phosphatidylserine synthase was phosphorylated by the cAMP-dependent protein kinase catalytic subunit, which resulted in a 60-70% reduction in phosphatidylserine synthase activity. The cAMP-dependent protein kinase catalytic subunit catalyzed the incorporation of 0.7 mol of phosphate per mol of phosphatidylserine synthase Mr 23,000 subunit. The specific cAMP-dependent protein kinase inhibitor prevented the phosphorylation of phosphatidylserine synthase and the inhibition of its activity by the catalytic subunit. Analysis of peptides derived from protease-treated labeled phosphatidylserine synthase showed only one labeled peptide. Phospho amino acid analysis of labeled phosphatidylserine synthase showed that the enzyme was phosphorylated at a serine residue.     

Thyroid hormone increases type I adenosine 3', 5'-monophosphate-dependent protein kinase and casein kinase activities in rat liver cytosol: analysis of protein kinases by polyacrylamide disc gel electrophoresis

We investigated the action of thyroid hormone on each protein kinase in rat liver cytosol. Kinases were analyzed by polyacrylamide disc gel electrophoresis and isoelectric focusing in polyacrylamide gel. Polyacrylamide disc gel electrophoresis separated cAMP-dependent protein kinase type I (Rf = 0.35), type II (Rf = 0.44), their catalytic subunit (Rf = 0.26), and cAMP-independent protein kinase (Rf = 0.50). Casein kinase was detected at Rf = 0.37. In addition to the catalytic subunit with Rf = 0.26, another catalytic subunit was found at Rf = 0.44 when the cytosol was preincubated with cAMP. The administration of T3 (20 micrograms/100 g BW for 3 days) to hypothyroid rats increased enzyme activities of type I holoenzyme and casein kinase by 48%. Free catalytic subunit, separated from holoenzyme, had the same level of enzyme activity in both groups, suggesting greater endogenous dissociation of type I holoenzyme in hypothyroid rats. When heat-inactivated rat liver cytosol was used as substrate in the assay of protein kinase activity, the peak enzyme active in phosphorylating the cytosol corresponded to the casein kinase peak. Our data indicate that casein kinase is the main enzyme that mediates phosphorylation of endogenous proteins in rat liver cytosol, and that T3 treatment increases the activity of casein kinase and of type I cAMP-dependent protein kinase.     

Phosphorylation of the hepatic EGF receptor with cAMP-dependent protein kinase

The 170 000 dalton hepatic epidermal growth factor (EGF) receptor is phosphorylated on serine and tyrosine residues. The evidence indicates that distinct protein kinases are involved. Since EGF and agents that elevate cAMP are believed to participate in the regulation of liver regeneration, we tested whether or not the catalytic subunit of cAMP-dependent protein kinase (catalytic subunit), a known serine kinase, would utilize the EGF receptor as a substrate. The catalytic subunit increased phosphorylation of the EGF receptor in purified rat liver plasma membranes. The serine specificity of the catalytic subunit was established by phosphoamino acid analysis of electrophoretically purified EGF receptor. The result was confirmed by catalytic subunit phosphorylation of affinity purified preparations of the EGF receptor. The rates of dephosphorylation of the membrane-associated EGF receptor phosphorylated on different residues were compared. Dephosphorylation of serine residues (after catalytic subunit phosphorylation) was considerably slower (t1/2 greater than 120 sec) than the removal of phosphotyrosine after stimulation with EGF (t1/2 less than 30 sec).     

A conserved helix motif complements the protein kinase core.

Residues 40-300 of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase define a conserved bilobal catalytic core shared by all eukaryotic protein kinases. Contiguous to the core is an extended amphipathic alpha-helix (A helix). Trp30, a prominent feature of this helix, fills a deep hydrophobic pocket between the two lobes on the surface opposite to the active site. The C subunit in Dictyostelium discoideum shows sequence conservation of residues 40-350 with the mouse enzyme but contains an N-terminal extension of 332 residues. A sequence corresponding to the A helix contiguous to the core is absent. However, we have now identified a remote A-helix motif (residues 77-98). When the core of the Dictyostelium C subunit was modeled, based on the mouse C subunit, complementarity between this putative A helix and the surface of the core was found to be conserved. Analysis of other protein kinases reveals that the A-helix motif is not restricted to cAMP-dependent protein kinase. In the Src-related family of protein kinases, for example, an A helix is very likely contiguous to the core, thus serving as a linker between the conserved catalytic core and the Src homology 2 domain. We predict that an A-helix motif complementary to the core will be a conserved feature of most eukaryotic protein kinases.     

Structural features that specify tyrosine kinase activity deduced from homology modeling of the epidermal growth factor receptor.

To identify structural features that distinguish protein-tyrosine kinases from protein-serine kinases, a molecular model of the kinase domain of epidermal growth factor receptor was constructed by substituting its amino acid sequence for the amino acid sequence of the catalytic subunit of cAMP-dependent protein kinase in a 2.7-A refined crystallographic model. General folding was conserved as was the configuration of invariant residues at the active site. Two sequence motifs that distinguish the two families correspond to loops that converge at the active site of the enzyme. A conserved arginine in the catalytic loop is proposed to interact with the gamma phosphate of ATP. The second loop provides a binding surface that positions the tyrosine of the substrate. A positively charged surface provides additional sites for substrate recognition.     

Phosphorylation and activation of cAMP-dependent protein kinase by phosphoinositide-dependent protein kinase

Although phosphorylation of Thr-197 in the activation loop of the catalytic subunit of cAMP-dependent protein kinase (PKA) is an essential step for its proper biological function, the kinase responsible for this reaction in vivo has remained elusive. Using nonphosphorylated recombinant catalytic subunit as a substrate, we have shown that the phosphoinositide-dependent protein kinase, PDK1, expressed in 293 cells, phosphorylates and activates the catalytic subunit of PKA. The phosphorylation of PKA by PDK1 is rapid and is insensitive to PKI, the highly specific heat-stable protein kinase inhibitor. A mutant form of the catalytic subunit where Thr-197 was replaced with Asp was not a substrate for PDK1. In addition, phosphorylation of the catalytic subunit can be monitored immunochemically by using antibodies that recognize Thr-197 phosphorylated enzyme but not unphosphorylated enzyme or the Thr197Asp mutant. PDK1, or one of its homologs, is thus a likely candidate for the in vivo PKA kinase that phosphorylates Thr-197. This finding opens a new dimension in our thinking about this ubiquitous protein kinase and how it is regulated in the cell.     

A putative mediator of insulin action which inhibits adenylate cyclase and adenosine 3',5'-monophosphate-dependent protein kinase: partial purification from rat liver: site and kinetic mechanism of action

A novel putative mediator of insulin action which acts to inhibit adenylate cyclase and cAMP-dependent protein kinase has been purified from livers of insulin-treated streptozotocin-diabetic rats. It was increased by short term (5-min) insulin injections in vivo and purified several thousand-fold by Sephadex and HPLC. Its mol wt was somewhat larger (2500) than previous mediators identified, and it was more hydrophobic in character. Its mechanism of action or adenylate cyclase was determined and found to be chiefly directed against the catalytic subunit. Its action on the cAMP-dependent protein kinase was found to be competitive with regard to protein substrate, but noncompetitive with regard to ATP and cAMP. Its relationship to other putative insulin mediators and the mechanism of insulin action is discussed.     

Mechanism of translational control by hemin in reticulocyte lysates.

The formation of translational inhibitor (active eIF-2 kinase) from proinhibitor (inactive eIF-2 kinase) in reticulocyte lysates, known to be controlled by hemin, can, as we recently reported, be induced by 3':5'-cyclic AMP(cAMP)-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) or its catalytic subunit. We find that in crude preparations from rabbit reticulocyte lysates, hemin inhibits the conversion of proinhibitor to inhibitor catalyzed by endogenous cAMP-dependent protein kinase upon addition of cAMP, but not that caused by the addition of free protein kinase catalytic subunit. Hemin prevents the binding of cAMP to the regulatory subunit of cAMP-dependent protein kinase and blocks the cAMP-induced dissociation of regulatory and catalytic subunits of the enzyme whereby the enzyme is inactivated. The mechanism by which hemin prevents the formation of the inhibitor and maintains protein synthesis in reticulocyte lysates is thus explained.     

Interaction of the Subunits of Adenosine 3′:5′-Cyclic Monophosphate-Dependent Protein Kinase of Muscle

Two cAMP-dependent protein kinases purified from rabbit skeletal muscle were shown to bind the same amount of cAMP per unit of enzyme activity at several concentrations of this nucleotide. A preparation containing both of these kinases was separated into catalytic (C) and regulatory (R) subunit fractions in the presence of cAMP, the regulatory subunit being obtained as an R.cAMP complex. Addition of increasing amounts of the R.cAMP complex to the holoenzyme (RC) increased the concentration of cAMP required for half-maximal activity of the enzyme. cAMP was liberated from the R.cAMP complex in the presence of added catalytic subunit in a reaction that was facilitated by Mg(2+), ATP, and warming. These findings are presented in support of a model for activation of the protein kinase by cAMP. The possibility that excess regulatory subunit may serve as a sink for intracellular cAMP is also discussed. It is shown that cAMP bound to the R subunit is not a substrate for the cAMP phosphodiesterase.     

Phosphorylation induces a decrease in the biological activity of the protein inhibitor (GABA-modulin) of gamma-aminobutyric acid binding sites.

gamma-Aminobutyric acid (GABA)-modulin is a brain protein of Mr 16,500 that down-regulates the high-affinity binding site for GABA which is located in crude synaptic membranes. This protein can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase and by a partially purified preparation of calmodulin-sensitive Ca2+-dependent protein kinase. The GABA-modulin sites that are phosphorylated by the two enzymes are different, as revealed by HPLC analysis of tryptic digests. The capacity of GABA-modulin to decrease the number of sites that bind [3H]muscimol was completely abolished by phosphorylation of this protein with the cAMP-dependent protein kinase but not with the Ca2+-dependent enzyme. GABA-modulin present in crude synaptic membranes prepared from rat cortex also was shown to be phosphorylated by endogenous protein kinases activated by cAMP, Ca2+ and calmodulin, and Ca2+ and phosphatidylserine. These results suggest a potentially important role for protein kinase and GABA-modulin in the regulation of the number of GABA recognition sites.     

High-affinity binding of the regulatory subunit (RII) of cAMP-dependent protein kinase to microtubule-associated and other cellular proteins.

Interaction of the regulatory subunit of the type II cAMP-dependent protein kinase (RII) with tissue-specific cellular binding proteins has been demonstrated by two independent methods. Complexes of RII and its binding proteins were isolated on a cAMP analog-Sepharose affinity column, eluted from the column, and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Alternatively, nitrocellulose blots made from polyacrylamide gels containing samples of tissue extracts or affinity column eluates were treated with sequential overlays of RII, monospecific antibody, and radioiodinated protein A. In bovine cerebrum, specific high-affinity interactions between RII and several binding proteins, including major proteins of 300, 80, and 68 kDa, were recognized by the two methods. The 300-kDa and 68-kDa proteins were identified as microtubule-associated protein 2 (300 kDa) and a protein of lower molecular weight (68 kDa) that copurifies with it. The additional major binding protein of 80 kDa requires further characterization. In addition, several binding proteins distinct from those observed in bovine cerebrum were found in bovine heart. Many of the RII binding proteins from brain and heart served to differing extents as substrates for the purified catalytic subunit of cAMP-dependent protein kinase. One hypothesis of the significance of the protein kinase regulatory subunit interaction with cellular binding proteins is that this may control the protein kinase holoenzyme localization and, thereby, define the substrate targets most accessible for phosphorylation by the activated protein kinase catalytic subunit. Alternatively, RII binding to a variety of cellular proteins might regulate their function--i.e., RII could be a regulator for multiple proteins in addition to the catalytic subunit of the cAMP-dependent protein kinase.     

On the question of translocation of heart cAMP-dependent protein kinase.

Rat hearts were perfused with epinephrine and/or 1-methyl-3-isobutylxanthine for 2 min. These agents raised the concentration of cAMP and increased the fraction of cAMP-dependent protein kinase (EC 2.7.1.70) in the active form. However, the content of cAMP-dependent protein kinase in the soluble fraction of homogenates of these hearts was reduced and the amount in the particulate fraction was increased. A similar redistribution was obtained by adding cAMP to homogenates of control hearts. The reduction in soluble protein kinase content was due to apparent binding of the free catalytic subunit of the enzyme to particulate material (12,000 times g pellet) in media of low ionic strength (smaller than 100 mM KCl). The amount bound was, therefore, proportional to the dissociation of the holoenzyme. The binding was not altered by prior boiling or trypsin treatment of the particulate material, but it was prevented or reversed by the addition of 150 mM KCl. The catalytic subunit of the protein kinase from heart also bound to particulate fractions from liver or Escherichia coli and to various denatured proteins. These findings suggest that the protein kinase activity of membranes and particulate fractions has frequently been overestimated, since isolation of particulate materials has usually been carried out at low ionic strength. The data also imply that intracellular translocation of the protein kinase catalytic subunit, at least in heart tissue, is of questionable physiological significance.     

cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor

Postsynaptic membranes, rich in the nicotinic acetylcholine receptor, were isolated from the electric organ of Torpedo californica and shown to contain a cAMP-dependent protein kinase and a calcium/calmodulin-dependent protein kinase. The cAMP-dependent protein kinase phosphorylated the gamma and delta subunits of the acetylcholine receptor. The phosphorylated subunits were identified after purification of the acetylcholine receptor by affinity chromatography on a choline carboxymethyl affinity gel. In contrast, the calcium/calmodulin-dependent protein kinase phosphorylated proteins that were separated from the acetylcholine receptor by affinity chromatography. Protein kinase inhibitor, a specific inhibitor of the catalytic subunit of cAMP-dependent protein kinase, abolished the basal endogenous phosphorylation of the gamma and delta subunits of the receptor. cAMP activation of the endogenous phosphorylation of the gamma and delta subunits was dose dependent with a half-maximal response at 25 nM. Studies were also carried out with acetylcholine receptor purified from T. californica and catalytic subunit of cAMP-dependent protein kinase purified from bovine heart. The purified acetylcholine receptor was rapidly and specifically phosphorylated on the gamma and delta subunits by the purified catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.0 and 0.89 mol of (32)P per mol of receptor, respectively. The initial rates of phosphorylation of the gamma and delta subunits of the receptor were comparable to those of histone f2B and synapsin I (protein I), two of the most effective substrates for the catalytic subunit. Under the conditions used, the gamma and delta subunits had K(m) values of 4.0 and 3.3 muM and V(max) values of 2.7 and 2.1 mumol/min per mg, respectively. The results are consistent with the idea that the acetylcholine receptor is phosphorylated in vivo by a cAMP-dependent protein kinase.     

Follicle-stimulating hormone-dependent phosphorylation of vimentin in cultures of rat Sertoli cells.

Endogenous protein phosphorylation was investigated in cultured rat Sertoli cells after treatment with follicle-stimulating hormone (FSH) and pharmacological agents that activate cAMP-dependent protein kinases. In intact Sertoli cells, both phosphorylation and dephosphorylation of proteins occurred in response to treatment with these agents. Studies using cell-free preparations suggest that four phosphoproteins phosphorylated by cAMP or the catalytic subunit of cAMP-dependent protein kinase were also phosphorylated in a FSH-dependent manner in intact cells. These data suggest that FSH-dependent phosphorylation in Sertoli cells occurs through activation of a cAMP-dependent protein kinase. A FSH-dependent phosphoprotein with a molecular weight of 58,000 was identified as the intermediate filament protein vimentin, based on its migration in two-dimensional gels and its peptide map. The cellular distribution of vimentin was monitored by immunofluorescence in Sertoli cells after treatment with FSH. Results of this study support a role for intermediate filaments in FSH-dependent events in Sertoli cells.     

Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the Km for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the Ki for dopamine from 4.2 microM to 47.5 microM, and produced no change in the Km for tyrosine. Little or no change in apparent Vmax was observed. These kinetic changes are similar to those seen upon activation of tyrosine hydroxylase by cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps of tyrosine hydroxylase were identical whether the phosphorylation was catalyzed by protein kinase C or by the catalytic subunit of cAMP-dependent protein kinase. Both protein kinases phosphorylated serine residues. The results suggest that protein kinase C and cAMP-dependent protein kinase phosphorylate the same site(s) on tyrosine hydroxylase and activate tyrosine hydroxylase by the same mechanism.     

A constitutively active holoenzyme form of the cAMP-dependent protein kinase.

The major function of the regulatory (R) subunit of the cAMP-dependent protein kinase is to bind tightly to the catalytic (C) subunit to form an inactive holoenzyme in the absence of cAMP. The hinge region of the R subunit resembles the substrate recognition site for the C subunit and is known to be involved in the R.C subunit interaction. Two arginine residues in this region, Arg-92 and Arg-93, are suggested to be essential for holoenzyme formation. In this study, a mutant in which Arg-92 and Arg-93 of type II regulatory subunit (RII) were replaced with alanine was constructed. Formation of the holoenzyme from mutant RII and C subunits was analyzed by gel-filtration and cation-exchange chromatography. Mutant RII in its cAMP-free form formed a stable holoenzyme with the C subunit, which dissociated in the presence of cAMP. Interestingly, the holoenzyme formed from mutant RII and C subunits retained full enzymatic activity even in the absence of cAMP. Although mutant RII could no longer be phosphorylated by the C subunit, the rate of [3H]cAMP release from mutant RII.cAMP was increased by addition of the C subunit, indicating that C-induced cAMP release is not the result of the interaction of the C subunit with the hinge region. These results demonstrate that Arg-92 and Arg-93 are not essential for holoenzyme formation but are critical for inhibiting kinase activity in the holoenzyme probably by occupying the substrate binding site. The results suggest that, in addition to the hinge region, a second site on the RII subunit may interact with the C subunit in a cAMP-dependent manner.     

Phosphorylation of the calcium antagonist receptor of the voltage-sensitive calcium channel by cAMP-dependent protein kinase.

Physiological studies indicate that voltage-sensitive calcium channels are regulated by cAMP and protein phosphorylation. The calcium antagonist receptor of the voltage-sensitive calcium channel from transverse-tubule membranes consists of three subunits, designated alpha, beta, and gamma. The catalytic subunit of cAMP-dependent protein kinase phosphorylates both the alpha and beta subunits of the purified receptor at a rate and extent that suggests they are potential physiological substrates of this enzyme. The phosphorylation of the alpha and beta subunits in transverse-tubule membranes was analyzed by two-dimensional gel electrophoresis. In intact transverse-tubule membranes, the alpha subunit is not significantly phosphorylated. However, the beta subunit, identified by its Mr, pI, and binding to wheat germ agglutinin-Sepharose, was one of the substrates selectively phosphorylated by cAMP-dependent protein kinase in transverse-tubule membranes. These results suggest that cAMP-dependent phosphorylation of the beta subunit of the calcium antagonist receptor may be an important regulatory mechanism for calcium channel function.     

Cyclic AMP-dependent ATPase activity of bovine heart protein kinase.

The adenosine 3",5"-monophosphate (cAMP)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from bovine heart is characterized. That the ATPase activity is intimately associated with the catalytic subunit of the enzyme is suggested by the following: (i) the similar dependences of ATPase and protein kinase activities on cAMP; (ii) the dissociation of ATPase activity from the holoenzyme on addition of cAMP and its co-elution with the catalytic subunit on gel filtration chromatography; (iii) the similarity of the relative effectiveness of divalent metal ions in ATPase and protein kinase catalysis; and (iv) the correspondence of kinetically determined Km(MgATP) and Ki(MgADP) values with thermodynamic dissociation constants determined by equilibrium dialysis. The hydrolysis of ATP is stimulated 10- to 20-fold by cAMP in the holoenzyme. The molar specific activity of the catalytic subunit ATPase is approximately 0.7 min-1 with Km(MgATP) = 5 muM. MgADP is a competitive inhibitor of the reaction with a Ki value of approximately muM. The order of the relative effectiveness of metal ions for both ATPase and peptide kinase activities is Mg2+ greater than Mn2+ greater than Ca2+. A possible interpretation of these observations is that the role that the metal ion plays is more directly manifested in bond-breaking than in bond-forming.