Comparison of phosphorylation sites in protamines between protein kinase C and cAMP-dependent protein kinase

Phosphorylation sites of protamines by protein kinase C and cAMP-dependent protein kinase (protein kinase A) were studied. Using clupeine Y1 as a substrate, protein kinase C phosphorylates both Ser and Thr residues, whereas protein kinase A phosphorylates only Ser residue(s). Protein kinase C phosphorylates all Ser and Thr residues of clupeine Y2 and Z, however protein kinase A phosphorylates mainly Ser9 and slightly Thr5 in clupeine Y2 and Ser6 and Ser10 in clupeine Z. These results suggest that protein kinase C recognizes more sites than those of protein kinase A and may participate in protamine phosphorylation in vivo.     

Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.     

Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.     

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.     

Neuromodulation of Na+ channel slow inactivation via cAMP-dependent protein kinase and protein kinase C

Neurotransmitters modulate sodium channel availability through activation of G protein-coupled receptors, cAMP-dependent protein kinase (PKA), and protein kinase C (PKC). Voltage-dependent slow inactivation also controls sodium channel availability, synaptic integration, and neuronal firing. Here we show by analysis of sodium channel mutants that neuromodulation via PKA and PKC enhances intrinsic slow inactivation of sodium channels, making them unavailable for activation. Mutations in the S6 segment in domain III (N1466A,D) either enhance or block slow inactivation, implicating S6 segments in the molecular pathway for slow inactivation. Modulation of N1466A channels by PKC or PKA is increased, whereas modulation of N1466D is nearly completely blocked. These results demonstrate that neuromodulation by PKA and PKC is caused by their enhancement of intrinsic slow inactivation gating. Modulation of slow inactivation by neurotransmitters acting through G protein-coupled receptors, PKA, and PKC is a flexible mechanism of cellular plasticity controlling the firing behavior of central neurons.     

A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase

As an important new reagent for studying the cAMP-dependent protein kinase, a 20-residue peptide has been synthesized that corresponds to the active site of the skeletal muscle inhibitor protein. This synthetic peptide inhibits the protein kinase competitively with a Ki = 2.3 nM; its sequence, Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr- Gly-Arg-Arg-Asn-Ala-Ile-His-Asp, is that of a peptide previously reported by us which was derived from the native inhibitor protein by V8 protease digestion (Cheng, H. C., Van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem. J. 231, 655-661). Studies with analogues of this peptide show that its high affinity binding to the protein kinase (as also of the inhibitor protein) appears to be due to it mimicking the protein substrate by binding to the catalytic site via the arginine-cluster basic subsite (Formula: see text), and also to a critical contribution from one or more of the 6 N-terminal residues (Formula: see text). The availability of this high affinity synthetic peptide should open up a variety of avenues to probe the cellular actions of cAMP.     

A potent fluorescent ATP-like inhibitor of cAMP-dependent protein kinase

The fluorescent ATP analogue 8-azido-2'-O-[14C]dansyl-ATP ([ 14C]AD-ATP) was used to probe the ATP-binding site in the catalytic (C) subunit of cAMP-dependent protein kinase. AD-ATP was found to inhibit the phosphotransferase activity of C subunit with extremely high specificity. Complete inhibition was observed when each mol of C subunit was covalently labeled with 1 mol of this fluorescent ATP analogue. The labeling can be accelerated by the presence of Mg2+ or Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), whereas high concentrations of ATP can almost completely protect the enzyme from AD-ATP. Detailed studies indicated that AD-ATP competes with ATP for binding to C subunit. Analysis of the kinetic data gave dissociation constants of 2.9 and 13 microM for AD-ATP and ATP bound to C subunit, respectively. AD-ATP has a fluorescence emission peak at 510 nm in pH 7.0 aqueous buffer containing 25% glycerol. After covalent binding to C subunit this emission peak shifts to 455 nm, which suggests that the label at ATP site is in an endogenous hydrophobic environment. Upon the binding of Mg2+ or Kemptide, the fluorescence of AD-ATP-labeled C subunit can be enhanced by 50 and 45%, respectively. This enhancement suggests that the binding of either the peptide substrate or Mg2+ induces conformational change at the active site of C subunit. Analysis of the fluorescence data shows that the values of Kd for Mg2+ and Kemptide bound to AD-ATP-labeled C subunit are 0.2 mM and 2.1 microM, respectively. The normal procedure for the preparation of the C subunit from the bovine heart muscle has been simplified to require only one-fifth of the usual working time to obtain the homogeneous enzyme with 70% yield from the crude extract.     

Metabolite-controlled phosphorylation of hepatic phosphofructokinase proceeds by cAMP-dependent protein kinase

In hepatocytes 32P-incorporation into rat liver phosphofructokinase is stimulated by glucose as well as by glucagon, the effects of both stimuli being prevented by L-alanine [Eur. J. Biochem. (1982) 122, 175]. The phosphopeptides of the enzyme derived from limited proteolysis by subtilisin and from exhaustive tryptic digestion were analyzed either by one-dimensional mapping on sodium dodecyl sulphate-polyacrylamide slab gels and by fingerprint mapping, respectively. It is shown that in vivo stimulation of 32P-incorporation by glucose or by glucose plus glucagon results in identical phosphopeptide maps, and that these maps were identical with those obtained from phosphofructokinase phosphorylated in vitro with catalytic subunit of cAMP-dependent protein kinase. It is concluded that in the intact liver cell phosphofructokinase is phosphorylated by cAMP-dependent protein kinase but that the state of phosphorylation is modified by metabolite control.     

Tyrosine phosphorylation modifies protein kinase C delta-dependent phosphorylation of cardiac troponin I

Our study identifies tyrosine phosphorylation as a novel protein kinase Cdelta (PKCdelta) activation mechanism that modifies PKCdelta-dependent phosphorylation of cardiac troponin I (cTnI), a myofilament regulatory protein. PKCdelta phosphorylates cTnI at Ser23/Ser24 when activated by lipid cofactors; Src phosphorylates PKCdelta at Tyr311 and Tyr332 leading to enhanced PKCdelta autophosphorylation at Thr505 (its activation loop) and PKCdelta-dependent cTnI phosphorylation at both Ser23/Ser24 and Thr144. The Src-dependent acquisition of cTnI-Thr144 kinase activity is abrogated by Y311F or T505A substitutions. Treatment of detergent-extracted single cardiomyocytes with lipid-activated PKCdelta induces depressed tension at submaximum but not maximum [Ca2+] as expected for cTnI-Ser23/Ser24 phosphorylation. Treatment of myocytes with Src-activated PKCdelta leads to depressed maximum tension and cross-bridge kinetics, attributable to a dominant effect of cTnI-Thr144 phosphorylation. Our data implicate PKCdelta-Tyr311/Thr505 phosphorylation as dynamically regulated modifications that alter PKCdelta enzymology and allow for stimulus-specific control of cardiac mechanics during growth factor stimulation and oxidative stress.     

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.     

Tyrosine phosphorylation of phosphatase inhibitor 2

Inhibitor 2 is a heat-stable protein that complexes with the catalytic subunit of type-1 protein phosphatase. The reversible phosphorylation of Thr 72 of the inhibitor in this complex has been shown to regulate phosphatase activity. Here we show that inhibitor 2 can also be phosphorylated on tyrosine residues. Inhibitor 2 was ³²P-labeled by the insulin receptor kinase in vitro, in the presence of polylysine. Phosphorylation of inhibitor 2 was accompanied by decreased electrophoretic mobility. Dephosphorylation of inhibitor 2 by tyrosine phosphatase 1B, restored normal electrophoretic mobility. Phosphotyrosine in inhibitor 2 was detected by immunoblotting with antiphosphotyrosine antibodies and phosphoamino acid analysis. In addition, following tryptic digestion, one predominant phosphopeptide was recovered at the anode. The ability of inhibitor 2 to inhibit type-1 phosphatase activity was diminished with increasing phosphorylation up to a stoichiometry of 1 mole phosphate incorporated/mole of inhibitor 2, where inhibitory activity was completely lost. These data demonstrate that inhibitor 2 can be phosphorylated on tyrosine residues by the insulin receptor kinase, resulting in a molecule with decreased ability to inhibit type-1 phosphatase activity.     

Modulation of red cell band 4.1 function by cAMP-dependent kinase and protein kinase C phosphorylation

Human erythrocyte protein 4.1 is phosphorylated in vivo by several protein kinases including protein kinase C and cAMP-dependent kinase. We have used cAMP-dependent kinase purified from red cells and protein kinase C purified from brain to test the effects of phosphorylation on band 4.1 function. In solution, each kinase catalyzed the incorporation of 1-4 mol of PO4/mol of band 4.1. Phosphorylation of band 4.1 by each kinase resulted in a significant (50-80%) reduction in the ability of band 4.1 to promote spectrin binding to F-actin. Direct measurement of spectrin-band 4.1 binding showed that phosphorylation by each kinase also caused dramatic reduction in this association. Phosphorylation of band 4.1 by each kinase for increasing time periods enabled us to demonstrate an approximately linear inverse relationship between PO4 incorporation into band 4.1 and spectrin binding. These results show that phosphorylation of band 4.1 by cAMP-dependent kinase and protein kinase C may be central to the regulation of red cell cytoskeletal organization and membrane mechanical properties.     

Regulatory subunit of the type I cAMP-dependent protein kinase as an inhibitor and substrate of the cGMP-dependent protein kinase

The regulatory subunit of the type I cAMP-dependent protein kinase (Rt) serves as a substrate for the phosphotransferase reaction catalyzed by cGMP-dependent protein kinase (Km = 2.2 microM). The reaction is stimulated by cGMP when RI . cAMP is the substrate, but not when nucleotide-free RI is used. The cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of RI dimer in the presence of cAMP and a self-phosphorylation reaction to the extent of 4 mol of phosphate/mol of enzyme dimer. In the absence of cAMP, RI is a competitive inhibitor of the phosphorylation of histone H2B (Ki = 0.25 microM) and of the synthetic peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ki = 0.15 microM) by the cGMP-dependent enzyme. Nucleotide-free RI also inhibits the intramolecular self-phosphorylation of cGMP-dependent protein kinase. The inhibition of the phosphorylation reactions are reversed by cAMP. The catalytic subunit of cAMP-dependent protein kinase does not catalyze the phosphorylation of RIand does not significantly alter the ability of RI to serve as a substrate or an inhibitor of cGMP-dependent protein kinase. These observations are consistent with the concept that the cGMP- and cAMP-dependent protein kinases are closely related proteins whose functional domains may interact.     

Erythrocyte adducin. Comparison of the alpha- and beta-subunits and multiple-site phosphorylation by protein kinase C and cAMP-dependent protein kinase

Two major substrates for human erythrocyte protein kinase C (PK-C) of Mr 120,000 and 110,000, previously named PKC-1 and PKC-2 [Palfrey, H. C. & Waseem, A. (1985) J. Biol. Chem. 260, 16021-16029] have been found to be identical to CaM-BP 103/97 or 'adducin', recently described by K. Gardner and V. Bennett [(1986) J. Biol. Chem. 261, 1339-1348; (1987) Nature (Lond.) 328, 359-362]. These proteins have been purified from the membrane skeleton by high-salt extraction, ion-exchange and gel filtration chromatography. The two proteins co-fractionate in a ratio of approximately 1:1 under a number of conditions suggesting that they exist as a complex. Physicochemical data indicate that the native adducin complex is probably an asymmetric heterodimer of alpha and beta subunits. Adducin binds to a calmodulin (CaM) affinity matrix in a Ca2+-dependent manner and is specifically eluted with EGTA. Fingerprinting of the iodinated peptides derived from the alpha and beta subunits using three different proteases yields 16-37% overlapping peptides, indicating limited similarity between the two polypeptides. Affinity-purified polyclonal antibodies against each protein show little or no cross-reactivity with the other, indicating that the beta subunit is not derived from the alpha subunit or vice versa. Proteins reactive with both anti-(alpha-adducin) and anti-(beta-adducin) antibodies are found in erythrocytes from rat, rabbit, pig, ferret and duck. Immunoblots of adducin after non-ionic detergent extraction of ghosts reveal that a significant fraction of the protein may associate with non-skeleton membrane components. The phosphorylation of adducin is stimulated by both phorbol esters and cAMP analogues in intact erythrocytes. Fingerprinting suggests that protein kinase C preferentially phosphorylates four distinct sites on the two proteins. Phosphopeptide maps of alpha-adducin are virtually identical to those of beta-adducin after phorbol ester stimulation of intact cells, or after PK-C-catalyzed phosphorylation of the purified protein, indicating strong local similarities in the two proteins. Such maps also suggest that cAMP-dependent protein kinase (cAMP-PK) modifies adducin at some similar and some distinct sites as those modified by PK-C. In vitro phosphorylation of isolated adducin by purified PK-C results in rapid incorporation of phosphate to a final level of approximately 1.5 mol/mol in both alpha and beta subunits.(ABSTRACT TRUNCATED AT 400 WORDS)     

Secretagogue-dependent phosphorylation of the insulin granule membrane protein phogrin is mediated by cAMP-dependent protein kinase

Phogrin, a 60/64-kDa integral membrane protein of dense-core granules in neuroendocrine cells, is phosphorylated in a Ca(2+)-sensitive manner in response to secretagogue stimulation of pancreatic beta-cells. Phosphorylation of the phogrin cytosolic domain by beta-cell homogenates was Ca(2+)-independent but stimulated by cAMP. Recombinant protein kinase A (PKA) could phosphorylate phogrin directly. High performance liquid chromatography analysis of tryptic phosphopeptides, combined with site-directed mutagenesis of candidate sites, revealed the presence of two phosphorylation sites at Ser-680 and Thr-699, located in the juxtamembrane region between the transmembrane span and the protein-tyrosine phosphatase homology domain of phogrin. Full-length wild-type phogrin, as well as mutant versions where Ser-680 and Thr-699 had been replaced either by alanines or by aspartic acid residues, were targeted to secretory granules in transfected AtT20 neuroendocrine cells. Stimulation of these cells with a range of secretagogues, including K(+), BaCl(2), and forskolin, demonstrated that the in vivo phosphorylation sites are the same as those identified in vitro. In MIN6 beta-cells, the PKA inhibitor H-89 prevented Ca(2+)-dependent phogrin phosphorylation in response to glucose, suggesting that Ca(2+) exerts its effect on phogrin phosphorylation through regulating the activity of PKA.     

Selective phosphorylation of the alpha subunit of the sodium channel by cAMP-dependent protein kinase

The alpha subunit of the sodium channel purified from rat brain is rapidly and selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 3 to 4 mol of 32P/mol of saxitoxin-binding activity. The rate of phosphorylation is comparable to that of the synthetic peptide analog of the phosphorylation site of pyruvate kinase, one of the best substrates for cAMP-dependent protein kinase. An endogenous cAMP-dependent protein kinase that is present in the partially purified sodium channel preparations also selectively phosphorylates the alpha subunit. The specificity and rapidity of the phosphorylation reaction are consistent with the hypothesis that the alpha subunit is phosphorylated by cAMP-dependent protein kinase in vivo.     

Effect of the thermostable protein kinase inhibitor on intracellular localization of the catalytic subunit of cAMP-dependent protein kinase.

cAMP-dependent protein kinase mediates a variety of cellular responses in most eukaryotic cells. Many of these responses are cytoplasmic, whereas others appear to require nuclear localization of the catalytic subunit. In order to understand further the molecular basis for subcellular localization of the catalytic subunit, the effect of the heat stable protein kinase inhibitor (PKI) was investigated. The subcellular localization of the catalytic (C) subunit was determined both in the presence and absence of PKI, by microinjecting fluorescently labeled C subunit into single living cells. When injected alone, a significant fraction of the dissociated C subunit localized to the nucleus. When coin-injected with an excess of PKI, little of the C subunit localized to the nucleus, suggesting that accumulation of catalytic subunit in the nucleus requires either enzymatic activity or a nuclear localization signal. Inactivation of the catalytic subunit in vitro by treatment with N-ethylmaleimide did not prevent localization in the nucleus, indicating that enzymatic activity was not a prerequisite for nuclear localization. In an effort to search for a specific signal that might mediate nuclear localization, a complex of the catalytic subunit with a 20-residue inhibitory peptide derived from PKI (PKI(5-24)) was microinjected. In contrast to intact PKI, the peptide was not sufficient to block nuclear accumulation. In the presence of PKI(5-24), the C subunit localized to the nucleus in a fashion analogous to that of dissociated, active C subunit despite evidence of no catalytic activity in situ. Thus, nuclear localization of the C subunit appears to be independent of enzymatic activity but most likely dependent upon a signal. The signal is apparently masked by both the regulatory subunit and PKI but not by the inhibitory peptide.     

Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase

Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine. Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI. Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures.