Proteolytic regulation of the mitochondrial cAMP-dependent protein kinase

The mitochondrial cAMP-dependent protein kinase (PKA) is activatable in a cAMP-independent fashion. The regulatory (R) subunits of the PKA holoenzyme (R(2)C(2)), but not the catalytic (C) subunits, suffer proteolysis upon exposure of bovine heart mitochondria to digitonin, Ca(2+), and a myriad of electron transport inhibitors. Selective loss of both the RI- and RII-type subunits was demonstrated via Western blot analysis, and activation of the C subunit was revealed by phosphorylation of a validated PKA peptide substrate. Selective proteolysis transpires in a calpain-dependent fashion as demonstrated by exposure of the R and C subunits of PKA to calpain and by attenuation of R and C subunit proteolysis in the presence of calpain inhibitor I. By contrast, exposure of mitochondria to cAMP fails to promote R subunit degradation, although it does result in enhanced C subunit catalytic activity. Treatment of mitochondria with electron transport chain inhibitors rotenone, antimycin A, sodium azide, and oligomycin, as well as an uncoupler of oxidative phosphorylation, also elicits enhanced C subunit activity. These results are consistent with the notion that signals, originating from cAMP-independent sources, elicit enhanced mitochondrial PKA activity.     

Mechanistic studies of cAMP-dependent protein kinase action

The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzyme's catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase

The kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase has been investigated employing the heptapeptide Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as substrate. Initial velocity measurements performed over a wide range of ATP and Kemptide concentrations indicated that the reaction follows a sequential mechanistic pathway. In line with this, the results of product and substrate inhibition studies, the patterns of dead end inhibition obtained employing the nonhydrolyzable ATP analogue, AMP X PNP (5'-adenylylimidodiphosphate), and equilibrium binding determinations, taken in conjunction with the patterns of inhibition observed with the inhibitor protein of the cAMP-dependent protein kinase that are reported in the accompanying paper (Whitehouse, S., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3682-3692), are best fit by a steady state Ordered Bi-Bi kinetic mechanism. Although the inhibition patterns obtained employing the synthetic peptide analogue in which the phosphorylatable serine was replaced by alanine were apparently incompatible with this mechanism, these inconsistencies appear to be due to some element of the structure of this latter peptide such that it is not an ideal dead end inhibitor substrate analogue. The data presented both here and in the accompanying paper suggest that both this substrate, analogue and the ATP analogue, AMP X PNP, do not fully mimic the binding of Kemptide and ATP, respectively, in their mechanism of interaction with the protein kinase. It is proposed that, as with some other kinase reactions, the configuration of the terminal anhydride bond of ATP assumes a conformation once the nucleotide is bound to the protein kinase that assists in the binding of either Kemptide or the inhibitor protein but not the alanine-substituted peptide and that AMP X PNP, because of its terminal phosphorylimido bond, cannot assume this conformation which favors protein (or peptide) binding.     

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 ( 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2–3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15–20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2–5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2–3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.Abbreviations C catalytic subunit of protein kinase A - CKI protein kinase C inhibitor protein - Cx connexin protein - dbcAMP N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate - OAG 1-oleoyl-2-acetyl-sn-glycerol - protein kinase A cAMP-dependent protein kinase - protein kinase C Ca²⁺-sensitive phospholipid-dependent protein kinase - PKI protein kinase A inhibitor protein - R regulatory subunit of protein kinase A - TRA 12-O-tetradecanoylphorbol-13-acetate - 8Br-cAMP 8-bromoadenosine 3:5 cyclic monophosphate     

Redox regulation of cAMP-dependent protein kinase signaling: kinase versus phosphatase inactivation

Many components of cellular signaling pathways are sensitive to regulation by oxidation and reduction. Previously, we described the inactivation of cAMP-dependent protein kinase (PKA) by direct oxidation of a reactive cysteine in the activation loop of the kinase. In the present study, we demonstrate that in HeLa cells PKA activity follows a biphasic response to thiol oxidation. Under mild oxidizing conditions, or short exposure to oxidants, forskolin-stimulated PKA activity is enhanced. This enhancement was blocked by sulfhydryl reducing agents, demonstrating a reversible mode of activation. In contrast, forskolin-stimulated PKA activity is inhibited by more severe oxidizing conditions. Mild oxidation enhanced PKA activity stimulated by forskolin, isoproterenol, or the cell-permeable analog, 8-bromo-cAMP. When cells were lysed in the presence of serine/threonine phosphatase inhibitor, NaF, the PKA-enhancing effect of oxidation was blunted. These results suggest oxidation of a PKA-counteracting phosphatase may be inhibited, thus enhancing the apparent kinase activity. Using an in vivo PKA activity reporter, we demonstrated that mild oxidation does indeed prolong the PKA signal induced by isoproterenol by inhibiting counteracting phosphatase activity. The results of this study demonstrate in live cells a unique synergistic mechanism whereby the PKA signaling pathway is enhanced in an apparent biphasic manner.     

Adenylate cyclase of myometrium and its regulation by cAMP-dependent protein kinase

The purpose of this study was to examine activity of adenylate cyclase (AC) and its cAMP-dependent phosphorylation by cAMP-dependent protein kinase (PKA) in the membrane of rabbit myometrium. Isoproterenol (IP) significantly increased AC activity of nonpregnant rabbits myometrium plasma membranes. However, during pregnancy AC of myometrium plasma membranes did not respond to IP. Phosphorylation of the myometrium membrane by PKA was followed by significantly decreased response of AC to IP.     

Role of cAMP-dependent protein kinase in the regulation of platelet procoagulant activity

The membrane microparticle (MP) formation and phosphatidylserine (PS) exposure evoked by platelet activation provide catalytic surfaces for thrombin generation. Several reports have indicated the effects of cAMP-elevating agents on agonist-induced MP formation and PS exposure; however, the mechanism still remains unclear. Here we show that inhibition of basal cyclic AMP-dependent protein kinase (PKA) activity incurred platelet MP formation and PS exposure. Pretreatment of platelets with cAMP-elevating agent, forskolin, abolished thrombin plus collagen-induced MP formation and PS exposure, and obviously decreased calcium ionophore-evoked MP shedding. Moreover, the inhibitory effects of forskolin on agonists-induced MP formation and PS exposure were reversed by the PKA inhibitor H89. PKA inhibitor-induced MP formation was dose-dependently inhibited by calpain inhibitor MDL28170, and forskolin abrogated thrombin plus collagen-induced calpain activation. In conclusion, PKA plays key roles in the regulation of platelet MP formation and PS exposure. PKA-mediated MP shedding is dependent on calpain activation.     

Cytosolic cAMP-dependent protein kinase of Polysphondylium violaceum: developmental regulation and properties

Summary The cellular slime mould Polysphondylium violaceum contains a cAMP-dependent protein kinase resembling the mammalian type I enzyme. The appearance of this enzyme is developmentally regulated. The level of kinase activity is very low in vegetative cell and increases more than tenfold during differentiation.The catalytic subunit of this cAMP-dependent protein kinase has a native molecular weight of 60–80 kDa, an isoelectric point of 5.7 and an apparent K_m for ATP and Kemptide of 50 and 13.4 µM respectively. It is characterised by its sensitivity to a synthetic inhibitor specific for cAMP-dependent protein kinase. The regulatory subunit has a molecular weight of 50 kDa.Abbreviations HEPES N-2-Hydroxyethylpiperazine-N-2-ethane sulphonic acid - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(ßaminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulphate     

Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation

We report the results of experiments which support the hypothesis that, in mouse oocytes, a decrease in intraoocyte cyclic AMP (cAMP) initiates meiotic maturation; oocytes microinjected with cyclic nucleotide phosphodiesterase (PDE) underwent germinal vesicle breakdown (GVBD) in the presence of 3-isobutyl-1-methylxanthine (IBMX), which inhibited GVBD both in oocytes not injected with PDE and in oocytes injected with heat-inactivated PDE. Cyclic AMP-dependent protein kinase (PK) has been proposed to mediate maintenance of meiotic arrest by cAMP. In support of this hypothesis is the observation that 2'-deoxy cAMP, which does not activate PK, did not maintain meiotic arrest as did cAMP; this result was obtained both by microinjection of these compounds and by incubating oocytes in the presence of their membrane-permeable N6-monobutyryl derivatives. Furthermore, microinjection into oocytes of the heat-stable inhibitor of PK, PKI, induced GVBD in the presence of either dibutyryl cAMP (dbcAMP) or IBMX. Meiotic arrest was maintained in the absence of dbcAMP or IBMX, however, by microinjected catalytic subunit of PK, but not by catalytic subunit coinjected with PKI. In addition, specific changes in oocyte phosphoproteins that preceded resumption of meiosis were induced, in the presence of dbcAMP, by microinjected PKI; these changes were also tightly coupled with commitment of oocytes to resume meiosis. These results are discussed in terms of our model for regulation of meiotic arrest and maturation.     

Involvement of cAMP-dependent protein kinase and pH on the regulation of cardiac sarcoplasmic reticulum

Cyclic AMP-dependent autophosphorylation of canine cardiac sarcoplasmic reticulum (SR) vesicles occurred on a 22,000 M_r protein and it was maximum at pH 6.0. At pH 6.0 cAMP-dependent phosphorylation of cardiac SR resulted in stimulation of the depressed initial rate of formation and the levels of the acid stable phosphorylated intermediate of the Ca²⁺ ATPase (E～P). At pH 6.8 phosphorylation of cardiac SR had no effect on either the initial rate or levels of E～P formed. These findings indicate that cAMP-dependent phosphorylation of cardiac SR can compensate for the effect of acidosis on SR function.     

Influence of key residues on the reaction mechanism of the cAMP-dependent protein kinase

The reaction mechanism of the catalytic phosphoryl transfer of cAMP-dependent protein kinase (cAPK) was investigated by semi-empirical AM1 molecular orbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier is calculated as 20.7 kcal mol(-1) and the reaction itself to be exothermic by 12.2 kcal mol(-1). The active site residue Asp166, which was often proposed to act as a catalytic base, does not accept a proton in any of the reaction steps. Instead, the hydroxyl hydrogen of serine is shifted to the simultaneously transferred phosphate group of ATP. Although the calculated transition state geometry indicates an associative phosphoryl transfer, no concentration of negative charge is found. To study the influence of protein mutations on the reaction mechanism, we compared two-dimensional energy hypersurfaces of the protein kinase wild-type model and a corresponding mutant in which Asp166 was replaced by alanine. Surprisingly, they show similar energy profiles despite the experimentally known decrease of catalytic activity for corresponding mutants. Furthermore, a model structure was examined, where the charged NH3 group of Lys168 was replaced by a neutral methyl group. The energetic hypersurface of this hypothetical mutant shows two possible pathways for phosphoryl transfer, which both require significantly higher activation energies than the other systems investigated, while the energetic stabilization of the reaction product is similar in all systems. As the position of the amino acid side chains and the substrate peptide is virtually unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer itself, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys168, however, is necessary to stabilize the intermediate reaction states, particularly the side chain of the substrate peptide.     

The mechanism of protein kinase C regulation

Protein kinase C (PKC) is a family of serine/threonine protein kinases that plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis. Nine PKC genes have been identified in the human genome, which encode 10 proteins. Each member of this protein kinase family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations. Activation of PKC has been implicated in the regulation of cell growth and differentiation. This review summarizes works of the past years in the field of PKC biochemistry that covers regulation and activation mechanism of different PKC isoforms.     

Probes of the mitochondrial cAMP-dependent protein kinase

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca^2 +, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R₂C₂) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides.

A set of six different diastereomeric hexapeptides RRASVA, each with a D-amino acid residue successively in the six positions, was synthesized and tested as substrates of protein kinase A. It was found that the peptide with D-Ser was neither a substrate, nor an inhibitor of the enzyme. The other five peptides were active as substrates with slightly lower kcat values than that of the all-L amino acid peptide. However, the apparent Km values increased by one to two orders of magnitude, especially when the second arginine or the alanine residue preceding the serine was substituted. The results are discussed.     

Mechanism of action of cAMP-dependent protein kinase. V. Free energy spectra

The catalytic subunit of cAMP-dependent protein kinase from pig brain was shown to catalyse an isotope exchange reaction ATP in equilibrium with ADP. The kinetic parameters of the exchange were determined. The enzyme can also use GTP as the donor substrate; phosphotransferase and "GTPase" reactions were investigated. Based on the kinetic data obtained in this and in the previous paper the free energy profiles of protein kinase catalysed reactions are discussed.