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PubMed1991

Phosphorylation of coagulation factor II by phospholipid/Ca(2+)-dependent protein kinase (protein kinase C).

Prothrombin is a major constituent of the blood coagulation cascade and requires phospholipid and Ca2+ for its activation. We have found that phospholipid/Ca(2+)-dependent protein kinase (Protein kinase C) phosphorylates prothrombin and the associated apparent Km value for prothrombin (0.86 microM) is comparable to the Km value reported for most known substrates of protein kinase C. A 2-dimension separation analysis revealed that serine residue was apparently phosphorylated by PKC. The phosphorylation was inhibited by such phosphatidylserine- and/or Ca2+ competitive protein kinase C inhibitors as trifluoperazine, palmitoylcarnitine and gossypol. These results suggest that protein kinase C phosphorylation was involved in the regulation of blood coagulation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1986

Synthetic myelin basic protein peptide analogs are specific inhibitors of phospholipid/calcium-dependent protein kinase (protein kinase C).

Synthetic peptide analogs of the bovine myelin basic protein (MBP) corresponding to residues 104-118 were found to specifically inhibit phospholipid/ Ca2+-dependent protein kinase (protein kinase C). The peptides [Ala107]MBP (104-118) and [Ala113]MBP (104-118) inhibited protein phosphorylation of intact MBP, histone H1 and peptide phosphorylation with MBP(104-123), MBP(104-118) or [Ala105]MBP (104-118) as substrates. The inhibitor peptides [Ala107]MBP(104-118) and [Ala113]MBP (104-118), containing alanine in place of the arginine recognition sites, apparently inhibited the enzyme noncompetitively with respect to substrates, with IC50 values ranging from 46-145 and 28-62 microM, respectively. These peptide analogs did not inhibit cyclic AMP-dependent protein kinase or myosin light chain kinase but inhibited phospholipid/Ca2+-dependent phosphorylation of endogenous proteins in the total, solubilized fraction of rat brain.

Amino Acid Sequence↗
PubMed1984

Activation of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) by new classes of tumor promoters: teleocidin and debromoaplysiatoxin.

The new potent tumor promoters teleocidin and debromoaplysiatoxin , which are structurally unrelated to phorbol esters, activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). The concentrations of 12-O-tetradecanoylphorbol-13-acetate, teleocidin and debromoaplysiatoxin for half-maximum activation of protein kinase C were found to be approximately 3 ng/ml, 40 ng/ml and 400 ng/ml, respectively. These three types of tumor promoters bind to protein kinase C, and appear to exhibit their pleiotropic actions through activation of this enzyme.

Alkaloids↗
PubMed1997

Dinucleotide receptor modulation by protein kinases (protein kinases A and C) and protein phosphatases in rat brain synaptic terminals.

The diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate (Ap5A), can activate an ionotropic dinucleotide receptor that induces Ca2+ transients into synaptosomes prepared from rat brain. This receptor, also termed the P4 purinoceptor, is sensitive only to adenine dinucleotides and is insensitive to ATP. Studies on the modulatory role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases on the response of diadenosine polyphosphate receptors were performed by measuring the changes in the intracellular Ca2+ levels with fura-2. Activation and inhibition of PKA were carried out by means of forskolin and the PKA inhibitory peptide (PKA-IP), respectively. The Ap5A response was inhibited by forksolin to 35% of control values, but PKA-IP induced an increase of 37%. The effect of PKC activation was similar to that observed for PKA. PKC stimulation with phorbol 12,13-dibutyrate produced an inhibition of 67%, whereas the PKC inhibitors staurosporine and PKC inhibitory peptide enhanced the responses elicited by Ap5A to 40% in both cases. Protein phosphatase inhibitors diminished the responses elicited by Ap5A to 17% in the case of okadaic acid, to 50% for microcystin, and to 45% in the case of cyclosporin A. Thus, the activity of dinucleotide receptors in rat brain synaptosomes appears to be modulated by phosphorylation/dephosphorylation. These processes could be of physiological significance in the control of transmitter release from neurons that are postsynaptic to nerves that release diadenosine polyphosphates.

Adenosine Triphosphate↗
PubMed1984

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.

Animals↗
PubMed1984

Evidence for the role of phosphorylase kinase, protein kinase C, and other Ca2+-sensitive protein kinases in the response of hepatocytes to angiotensin II and vasopressin.

Angiotensin II, catecholamines, and vasopressin can stimulate the phosphorylation of 10 hepatic cytosolic proteins via a Ca2+-linked, cyclic AMP-independent mechanism. To explore the role of known Ca2+-sensitive protein kinases in this response, [32P]PO4(3-)-labeled hepatocytes were stimulated with various agonists, the cytoplasmic proteins were separated on two-dimensional gels, and the resulting autoradiographs were computer analyzed. The role of phosphorylase kinase was examined using hepatocytes from gsd/gsd rats which are deficient in this enzyme. The phosphorylation state of phosphorylase was not increased by glucagon, angiotensin II, or vasopressin in hepatocytes from the gsd/gsd animals. The phosphorylation state of all other substrates was changed by glucagon or the Ca2+-linked hormones to the same extent in gsd/gsd hepatocytes as in normal Wistar controls, suggesting that phosphorylase kinase plays a restricted role in the hormone response. The role of the Ca2+- and phospholipid-sensitive protein kinase (protein kinase C) was examined by stimulating hepatocytes with phorbol esters which are thought to activate protein kinase C by substituting for diacylglycerol. Phorbol esters increased the phosphorylation state of 3 of the 10 substrates affected by angiotensin II or vasopressin, but did not stimulate Ca2+ fluxes in hepatocytes. Treatment of hepatocytes with the Ca2+ ionophore A23187 mimicked the effect of the Ca2+-linked hormones on the phosphorylation of the other 7 substrates. The results demonstrate that at least three Ca2+-sensitive protein kinases are involved in the response of hepatocytes to Ca2+-linked hormones. Since these kinases can be activated independently by phorbol esters or A23187, the results imply that hormones such as vasopressin generate two intracellular messengers, diacylglycerol and Ca2+ ion.

Angiotensin II↗
PubMed2004

Up-regulation of IL-4 production by the activated cAMP/cAMP-dependent protein kinase (protein kinase A) pathway in CD3/CD28-stimulated naive T cells.

The signal transduction of the cAMP/cAMP-dependent protein kinase [protein kinase A (PKA)] pathway through multiple receptors is critical for many processes in all cell types. In T cells, the engagement of both the TCR-CD3 complex and the CD28 co-stimulatory molecule also induces cAMP, and subsequently activates PKA. It is believed that elevation of cAMP levels in T cells is inhibitory of IL-2 production and T cell proliferation. However, the function and detailed signal transduction mechanisms of the cAMP/PKA pathway in naive T(h) cells are less well understood. In this study, we show that calcitonin gene-related peptide (CGRP) down-regulates IL-2 and IFN-gamma production and up-regulates IL-4 production to promote T(h)2 differentiation by moderate activation of the cAMP/PKA pathway via the CGRP receptor in the presence of a CD3/CD28 co-stimulation signal. The IL-4 production and transcriptional activation of T(h)2 cytokine mRNAs were also reproduced by the addition of a cAMP analogue, dibutyryl-cAMP, in CD3/CD28-stimulated naive T(h) cells. More interestingly, cAMP/PKA activation in naive T(h) cells stimulated with anti-CD3 plus anti-CD28 mAb is essential for inducing IL-4 production and promoting T(h)2 differentiation; in addition, NF-AT is a downstream effector of the cAMP/PKA signaling pathway. These findings indicate that the cAMP/PKA pathway transduces the critical activation signal to T(h)2 polarization by a CD3/CD28 co-stimulation signal and a PKA activating reagent.

Animals↗
PubMed1991

Isolation and identification of a novel cellular protein that potently activates Ca2+/phospholipid-dependent protein kinase (protein kinase C).

A novel heat-stable cellular protein that significantly stimulated Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) was identified. It is ubiquitous and has a molecular mass of over 150 kDa, and was shown to be specific for PKC. It activates PKC in the absence and presence of phospholipids; however, its maximal stimulatory potential was achieved when optimal amounts of phospholipids were also present in the reaction medium. Thus, in comparison with classical cofactors such as phospholipid, where activation of PKC mainly involves interaction with the regulatory domain of PKC, the mechanism of activation of PKC by the activator appears to involve several binding sites.

Animals↗
PubMed1991

Isolation and characterization of the calcium- and phospholipid-dependent protein kinase (protein kinase C) subtypes from bovine heart.

Protein kinase C was isolated from bovine heart by chromatography on DEAE-Sephacel, phenyl-Sepharose, poly(L-lysine) agarose, and hydroxylapatite. Estimates based upon enzyme recovery indicate 10-20 nmol/min of protein kinase C activity per gram of bovine ventricular myocardium. Hydroxylapatite column chromatography resolved the preparation into two peaks of calcium- and phospholipid-dependent protein kinase activity. By Western blot analysis, peaks 1 and 2 contained subtypes II (beta 2) and III (alpha), respectively. No cross-reactivity was observed, indicating that separation was complete. Type III, the major subtype detected, was subsequently purified to apparent homogeneity by chromatography on phosphatidylserine (PS) acrylamide. Type II activity could not be recovered following phosphatidylserine affinity chromatography. Phospho amino acid analysis showed that type III autophosphorylated at serine residues, whereas type II autophosphorylated at both serine and threonine residues. Among the various phospholipids tested for activity, PS was the most effective. Both subtypes were activated by 1-stearoyl-2-arachidonylglycerol (SAG) in the presence of phosphatidylserine and calcium. Activation of both subtypes occurred at calcium concentrations of less than 1 microM. In addition to several similarities, these two subtypes showed differences in activation and kinetic properties: type II was activated by cardiolipin, 1,2-and 1,3-dioleoylglycerol, and both cis- and trans-unsaturated fatty acids. Type III was activated to a lesser degree by cardiolipin and showed no response to 1,3-dioleoylglycerol. Type III was activated to a greater extent by 1,2-diacylglycerols and by cis-unsaturated fatty acids. In the presence of PS and SAG, type II exhibited substantial activity in the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) without added calcium. Activation of types II and III by unsaturated fatty acids was independent of phospholipid and showed a lower apparent calcium affinity than that observed for activation by phosphatidylserine. These results show that cardiac protein kinase C subtypes II and III were functionally distinguishable and may play unique roles in the regulation of cardiac function.

Amino Acids↗
PubMed1986

Phospholipid/calcium-dependent protein kinase (protein kinase C) system: a major site of bioregulation.

The substrate specificity determinants of protein kinase C are probed using synthetic peptides encompassing the major phosphorylation site serine 115 in bovine MBP. The results indicate that basic residues arginine 107 and 113 N-terminal to the phosphorylation site are essential for the substrate activity of the peptides. Substitutions of these basic residues by alanine cause a marked decrease in their substrate activity and the resulting peptide analogs become specific and rather potent inhibitors of protein kinase C. Leukemic cells are particularly abundant in protein kinase C and its substrate proteins, and the enzyme system has been shown to play a key role in cell growth. The agents that stimulate protein kinase C include tumor promoting phorbol esters (such as TPA) and mezerein, and the putative second messenger diacylglycerol. Many antineoplastic agents, on the other hand, inhibit the enzyme which include adriamycin, tamoxifen, alkyl-lysophospholipid, selenium, retinal and lipoidal amine CP-46, 665-1. Immunocytochemical studies of protein kinase C in leukemic cells indicate that it is localized in the plasma membrane, cytoplasm, nucleus and Golgi apparatus, and the subcellular distribution of the enzyme might be related to the phases of the cell cycle. TPA induces translocation of the enzyme to plasma membrane, suggesting an additional mode of action for the tumor promotor.

Animals↗
PubMed1987

Catalytic and receptor-binding properties of the calcium-sensitive phospholipid-dependent protein kinase (protein kinase C) in swine luteal cytosol.

We have evaluated the catalytic and receptor-binding properties of protein kinase C in swine luteal cytosol using two complementary approaches: assay of catalytic activity assessed as the enzymatic transfer of radiolabeled phosphate to histone III-s acceptor protein in the presence of specific phospholipid, diacylglycerol, or phorbol ester and ionic calcium; and, the high-affinity binding of [3H]phorbol-12,13-dibutyrate ([3H]PDB) to the protein kinase C receptor. Catalytic properties of pig luteal protein kinase C included: absolute dependence on calcium ions for maximal activation (approximate ka = 0.5 microM); synergistic activation by 1,2-sn-diolein, phospholipid and calcium ions; and rank order of specific phospholipid activational potency: phosphatidylserine greater than phosphatidic acid greater than phosphatidylinositol greater than phosphatidylethanolamine greater than phosphatidylcholine. The enzyme was also activated by specific phorbol esters at the following half-maximally effective (ED50) concentrations: 12-O-tetradecanoylphorbol-13-acetate (TPA) 11 nM; phorbol-12,13-dibenzoate (PDBe) 26 nM; phorbol-12,13-dibutyrate (PDBu) 33 nM; mezerein 65 nM; and phorbol-12,13-diacetate (PDA) 130 nM. Phorbol-ester receptor properties of protein kinase C included specific, high-affinity (kd congruent to 19 nM), saturable, low-capacity (congruent to 44 pmol/mg protein) [3H]PDB binding sites. Moreover, the rank order of the equilibrium binding ID50s for various phorbol compounds was similar to that of catalytic ED50s: viz. 3 nM TPA; 8 nM PDBe; 16 nM PDBu; 19 nM mezerein; and 590 nM PDA. Thus, swine luteal cytosol contains catalytically active protein kinase C with specific phospholipid sensitivity, synergistic activation by diacylglycerol, phospholipid and calcium, and a strict dependence on ionic calcium concentrations that is influenced markedly by the presence of diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Phosphorylation of the ribosomal protein S6 during agonist-induced exocytosis in exocrine glands is catalyzed by calcium-phospholipid-dependent protein kinase (protein kinase C). Experiments with guinea pig parotid glands.

The ribosomal protein S6 in exocrine cells is phosphorylated during stimulation of exocytosis by cAMP-dependent or calcium-dependent agonists. Under both conditions the same tryptic S6 phosphopeptides (termed A, B, and C) were found [Padel, Kruppa, Jahn & Söling (1983) FEBS Lett. 159, 112-118]. Studies have now been made of the phosphorylation pattern of protein S6 from purified guinea pig parotid ribosomes following in vitro phosphorylation with calmodulin-dependent, phospholipid-dependent, and cAMP-dependent protein kinases. Only the phospholipid-dependent enzyme led to the phosphorylation of peptides A, B, and C, while the cAMP-dependent enzyme phosphorylated only peptides A and C, and the calmodulin-dependent enzyme did not phosphorylate any of the phosphopeptides found in S6 from unstimulated or stimulated intact cells. Guinea pig parotid microsomes contain substantial phospholipid-dependent protein kinase activity. Stimulation of intact parotid glands with tetradecanoylphorbol acetate led to a significant phosphorylation of S6 and a similar tryptic S6 phosphopeptide pattern as seen with carbamoylcholine. It is concluded that activation of phospholipid-dependent protein kinase is responsible for the phosphorylation of protein S6 during stimulation with calcium-dependent and cAMP-dependent secretagogues.

Animals↗
PubMed1998

Rapid activation of the novel serine/threonine protein kinase, protein kinase D by phorbol esters, angiotensin II and PDGF-BB in vascular smooth muscle cells.

Protein kinase D (PKD) is a novel serine/threonine kinase structurally distinct from all protein kinase C (PKC) isoforms but which like classic and novel PKCs is activated by phorbol esters and diacylglycerol. This study investigated the regulation of PKD in vascular smooth muscle cells (VSMC) by physiological regulators of VSMC function and growth factors. Treatment of rabbit aortic VSMC with phorbol ester, angiotensin II and PDGF-BB all stimulated PKD activity in a time- and concentration-dependent manner in VSMC. The effect of angiotensin II was particularly rapid and potent (maximum stimulation within 1 min and at 0.5 nM). In contrast, the maximum effect of PDGF-BB was obtained after 5 min. Other factors, including basic FGF, IGF-I, IGF-II, endothelin-1 and endothelin-2, had no effect on PKD activity in VSMC. These results show for the first time that PKD activity is regulated in VSMC, and is activated by the vasoconstrictor angiotensin II. PKD may be an important mediator for the biological function(s) of one or more PKC isoforms in VSMC and/or may represent a component of a novel PKC-independent signalling pathway in VSMC.

Angiotensin II↗
PubMed1991

Structural and functional diversities of a family of signal transducing protein kinases, protein kinase C family; two distinct classes of PKC, conventional cPKC and novel nPKC.

Recent molecular cloning and biochemical experiments on the nature of protein kinase C (PKC) have revealed the existence of two distinct classes of phorbol ester (and diacylglycerol) receptor/protein kinase, conventional PKC (cPKC) and novel PKC (nPKC). Each of these classes contains multiple related molecules expressed in tissues and cells in a type-specific manner. Although nPKC does not show the typical PKC activity ascribable to conventional PKCs and thus was neglected in earlier studies, several lines of evidence suggest that nPKCs are involved in a variety of cell responses to physiological stimuli and phorbol esters. It is possible that in some cases nPKC is the major mediator of the so-called PKC-activators, such as phorbol esters, mezerein, and bryostatins. In addition to the clear difference between cPKC and nPKC, functional diversity among conventional PKCs has also been demonstrated; PKC gamma differs in its competence to mediate the signal toward transcriptional activation through TPA-responsive cis-acting elements from cPKC alpha and nPKC epsilon. The differences between cPKC and nPKC and among the individual members of each of these two classes, and their specific pattern of distribution in tissues and cells, provide a rationale by which to explain the specificity and diversity of cellular responses to external stimuli generating DAG and to phorbol esters. The results presented here also provide a means to dissect the complex signaling pathway in cells and to analyze the molecular basis underlying the signal transduction processes mediated by this family of protein kinases.

Animals↗
PubMed1989

Glutathione S-transferase is an in vitro substrate of Ca++-phospholipid-dependent protein kinase (protein kinase C).

Glutathione S-transferase was found to be a good substrate of Ca++-phospholipid-dependent protein kinase in vitro. Of 6 isozymes of glutathione transferase purified from rat liver cytosol (1-1, 1-2, 2-2, 3-3, 3-4, 4-4), only isozymes 1-1, 1-2 and 2-2 were significantly phosphorylated by the kinase purified from rabbit brain. Phosphorylation was more pronounced in subunit 1 than in subunit 2, and the degree of the phosphorylation was similar in all three homo- and heterodimers, where 1 mol of phosphoryl group per mol subunit was transferred to the subunit 1. The phosphorylated transferase 1-1 showed decreased affinity for bilirubin, suggesting that the phosphorylation affects the function of glutathione S-transferase in an isozyme-specific manner.

Animals↗
PubMed1986

Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils.

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose-dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose-dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the "translocation" of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.

Calcium↗