Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein kinase C functions prominently in cell regulation via its pleiotropic role in signal transduction processes. Certain oncogene products resemble elements involved in transmembrane signaling, elevate cellular sn-1,2-diacylglycerol second messenger levels, and activate protein kinase C. Sangivamycin was unique among the nucleoside compounds tested in its ability to potently inhibit protein kinase C activity. Inhibition was competitive with respect to ATP for both protein kinase C and the catalytic fragment of protein kinase C prepared by trypsin digestion. Sangivamycin was a noncompetitive inhibitor with respect to histone and lipid cofactors (phosphatidylserine and diacylglycerol). Sangivamycin inhibited native protein kinase C and the catalytic fragment identically, with apparent Ki values of 11 and 15 microM, respectively. Sangivamycin was an effective an inhibitor of protein kinase C as H-7, an isoquinolinsulfonamide. Sangivamycin did not inhibit [3H]phorbol-12,13-dibutyrate binding to protein kinase C. Sangivamycin did not exert its action through the lipid binding/regulatory domain; inhibition was not affected by the presence of lipid or detergent. Unlike H-7, sangivamycin selectively inhibited protein kinase C compared to cAMP-dependent protein kinase. The discovery that protein kinase C is inhibited by sangivamycin and other antitumor agents suggests that protein kinase C may be a target for rational design of antitumor compounds.
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PMID:Sangivamycin, a nucleoside analogue, is a potent inhibitor of protein kinase C. 333 87

Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C) isolated from rat brain cytosol undergoes autophosphorylation in the presence of Mg2+, ATP, Ca2+, phosphatidylserine, and diolein. Approximately 2-2.5 mol of phosphate were incorporated per mol of the kinase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the phosphorylated kinase showed a single protein band of Mr = 82,000 compared to the Mr = 80,000 of the nonphosphorylated enzyme. Analysis of the 32P-labeled tryptic peptides derived from the autophosphorylated kinase by peptide mapping revealed that multiple sites were phosphorylated. Both serine and threonine residues were found to be labeled with 32P. Limited proteolysis of the autophosphorylated kinase with trypsin resulted in the conversion of the kinase into a phospholipid- and Ca2+-independent form. Two major 32P-labeled fragments, Mr = 48,000 and 38,000, were formed as a result of proteolysis, suggesting that the catalytic domain and possibly the Ca2+- and phospholipid-binding region were both phosphorylated. Protein kinase C autophosphorylation has a Km for ATP (1.5 microM) about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation appears to be an intramolecular reaction. The autophosphorylated protein kinase C, unlike the protease-degraded enzyme, still depends on Ca2+ and phospholipid for maximal activity. However, the autophosphorylated form of the kinase has a lower Ka for Ca2+ and a higher affinity for the binding of [3H]phorbol-12, 13-dibutyrate. These findings suggest that autophosphorylation of protein kinase C may be important in the regulation of the enzymic activity subsequent to signal transduction.
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PMID:Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase. 346 87

Protein kinase C isolated from retina catalyzes the stoichiometric phosphorylation of bovine rhodopsin. Enzymological studies using receptor in rod outer segment membranes stripped of peripheral proteins reveal that the phosphorylation is independent of receptor conformation or liganded state; the half-time for phosphorylation of unbleached (dark-adapted) rhodopsin, bleached (light-activated) rhodopsin, and opsin (chromophore removed) is the same. The phosphorylation by protein kinase C is Ca2+ and lipid regulated; the Km for Ca2+ decreases with increasing concentrations of membrane, consistent with known properties of Ca(2+)-regulated protein kinase Cs. The Km for ATP is 27 microM, with an optimal concentration for MgCl2 of approximately 1 mM. The phosphorylation of rhodopsin by protein kinase C is inhibited by the protein kinase C-selective inhibitor sangivamycin. Proteolysis by Asp-N reveals that all the protein kinase C phosphorylation sites are on the carboxyl terminus of the receptor. Cleavage with trypsin indicates that Ser338, the primary phosphorylation site of rhodopsin kinase, is not phosphorylated significantly; rather, the primary phosphorylation site of protein kinase C is on the membrane proximal half of the carboxyl terminus. The protein kinase C-catalyzed phosphorylation of rhodopsin is analogous to the ligand-independent phosphorylation of other G protein-coupled receptors that is catalyzed by second messenger-regulated kinases.
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PMID:Kinetics and localization of the phosphorylation of rhodopsin by protein kinase C. 789 14

PKN, a novel protein kinase with catalytic domain homologous to PKC family and unique amino terminal leucine zipper-like sequences, was purified partially from COS7 cells transfected with the cDNA construct encoding human PKN for enzymatic characterization of the enzyme. Using serine containing synthetic peptides based on PKC pseudosubstrate sites as the phosphate acceptors, kinase activities estimated from partially purified PKN were not stimulated by Ca2+/phosphatidylserine/diolein but were activated several-fold to several tens-fold by 40 microM unsaturated fatty acids, such as arachidonic acid, linoleic acid, and oleic acid. Autophosphorylation of the immunoprecipitates using anti-PKN antiserum was also stimulated by various unsaturated fatty acids. Limited proteolysis of PKN with trypsin induced an enhancement of the peptide kinase activity that was almost independent of arachidonic acid.
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PMID:Activation of PKN, a novel 120-kDa protein kinase with leucine zipper-like sequences, by unsaturated fatty acids and by limited proteolysis. 794 81

The cyclic GMP phosphodiesterase (PDE) of retinal rods plays a key role in phototransduction and consists of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Here we report that PDE alpha and PDE gamma are phosphorylated by protein kinase(s) C (PKC) from brain and rod outer segments (ROS). These same two types of PKC also phosphorylate PDE alpha in trypsin-activated PDE (without PDE gamma). In contrast, cyclic-AMP-dependent protein kinase catalytic subunit phosphorylates both PDE alpha and PDE beta, but not PDE gamma. This kinase does not phosphorylate trypsin-activated PDE. The synthetic peptides AKVISNLLGPREAAV (PDE alpha 30-44) and KQRQTRQFKSKPPKK (PDE gamma 31-45) inhibited phosphorylation of PDE by PKC from ROS. These data suggest that sites (at least one for each subunit) for phosphorylation of PDE by PKC are localized in these corresponding regions of PDE alpha and PDE gamma. Isoenzyme-specific PKC antibodies against peptides unique to the alpha, beta, gamma, delta, epsilon and zeta isoforms of protein kinase C were used to show that a major form of PKC in ROS is PKC alpha. However, other minor forms were also present.
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PMID:Phosphorylation of bovine rod photoreceptor cyclic GMP phosphodiesterase. 821 38

Ribonucleotide reductase is responsible for supplying the deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two dissimilar protein components called R1 and R2. Immunoprecipitation of R1 and R2 proteins from [32P]orthophosphate-labeled exponentially growing mouse L cells showed that the R2 protein but not the R1 protein of ribonucleotide reductase could be phosphorylated in vivo. Two-dimensional phosphopeptide mapping experiments of trypsin-digested R2 protein showed a major spot containing more than 90% of the total radioactivity and a minor spot with the remaining radioactivity. Phosphoamino acid analysis of R2 phosphorylated protein indicated that phosphorylation occurred exclusively on serine. Protein kinase C, cAMP-dependent protein kinase, p34cdc2, and CDK2 were capable of phosphorylating the R2 protein in vitro, whereas casein kinase II was not. To determine whether any of these enzymes could phosphorylate peptides observed to be phosphorylated in actively growing cells, tryptic phosphopeptide maps of R2 that had been phosphorylated in vitro were compared with maps of R2 that had been isolated from [32P]-labeled cells. Only the phosphopeptide maps obtained with p34cdc2 and CDK2 matched the pattern found in [32P]-labeled cells. Experiments in which tryptic digests from different samples were mixed prior to two-dimensional separation demonstrated comigration of phosphopeptides obtained by in vivo phosphorylation with phosphopeptides derived from p34cdc2 or CDK2 obtained by in vitro phosphorylations. These studies indicate that protein R2 phosphorylation may play an important role in the regulation of ribonucleotide reduction, DNA synthesis, and cell cycle progression, and suggest a potentially important p34cdc2 and/or CDK2 regulation point in DNA replication.
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PMID:Phosphorylation of ribonucleotide reductase R2 protein: in vivo and in vitro evidence of a role for p34cdc2 and CDK2 protein kinases. 825 5

A peptide inhibiting either corpuscolate or purified PKC has been identified from microsomes of PHA-activated human PBMC but it is not detectable in microsomes of resting PBMC. The peptide was obtained from a microsomal preparation in an oligomeric form that could be transformed into a monomeric form by beta-MSH. The active peptide (IN) was retained on a PC-11 chromatographic column and could be eluted with NaCl. IN is ineffective on PKC-dependent protamine phosphorylation of protamine and on Ca2+ and phospholipid-independent activity generated by mild hydrolysis with trypsin of PKC. Ca2+ binding is permissive for IN activity. IN inhibits particulate PKC in PHA-activated PBMC, but is ineffective after TPA activation. All these data indicate that IN acts at the regulatory domain of PKC.
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PMID:Identification of a novel protein kinase C inhibitor in microsomes from phytohaemagglutinin activated human peripheral blood mononuclear cells. 836 75

Protein kinase C-theta (PKC-eta) is a member of the protein kinase C family that is characterized by Ca2+ independence and restricted histone kinase activity (Dekker, L. V., Parker, P. J., and McIntyre, P. (1992) FEBS Lett. 312, 195-199). Here we have investigated the molecular basis of this low histone kinase activity by limited proteolysis and site-directed mutagenesis. It is shown that a 46-kDa C-terminal tryptic fragment, representing the catalytic domain of PKC-eta, can phosphorylate histone. The Km value for histone of this catalytic fragment is 25-fold lower than that of intact PKC-eta. Thus, sites in the N-terminal regulatory domain upstream of the trypsin cleavage site (near residue 320) restrict histone kinase activity of intact PKC-eta. Deletion of the "Vo domain" (residues 2-137) generates a PKC-eta mutant that shows the same cofactor dependence and substrate phosphorylation as wild-type PKC-eta, indicating that the relevant sites do not appear to lie in the Vo domain but between amino acid 137 and the start of the catalytic domain. Deletion of the pseudosubstrate region (residue 155-171) generates a cofactor-independent kinase that has high histone kinase activity. A pseudosubstrate site point mutation in which the alanine residue at position 161 is replaced with a glutamic acid residue shows the same properties as the pseudosubstrate site deletion mutant. Km values for histone for both mutants are similar to that observed for the catalytic fragment. Therefore, in addition to its role in conferring cofactor dependence, the pseudosubstrate site also mediates the low histone kinase activity of wild-type PKC-eta. The data are discussed in the light of current models for PKC activation.
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PMID:Mutagenesis of the regulatory domain of rat protein kinase C-eta. A molecular basis for restricted histone kinase activity. 839 39

The protein kinase C-alpha (PKC-alpha)-catalyzed phosphorylation of the peptide [Arg]4-Tyr-Gly-Ser-[Arg]5-Tyr is independent of Ca2+ and phospholipid. The binding of this peptide to PKC-alpha induces a conformational change in the enzyme that results in the exposure of hydrophobic groups that subsequently insert into a membrane. Induction of a conformational change in the enzyme by this peptide is demonstrated by susceptibility to trypsin cleavage. Additionally, exposure of hydrophobic sites on the enzyme is shown by the binding of the fluorescent probes PRODAN and bis-ANS and by the partitioning of the enzyme into a Triton X-114-enriched phase. In the presence of a phospholipid bilayer containing phosphatidylserine, this peptide promotes the translocation of PKC-alpha to the membrane in the absence of Ca2+ as observed by increased resonance energy transfer between Trp on the enzyme and dansyl-groups attached to the lipid, as well as by changes in the intrinsic tryptophan fluorescence of the enzyme. Also, once bound to the membrane the peptide.PKC-alpha complex undergoes further conformational change which is evident by an increased sensitivity to trypsin cleavage at the hinge region. These results demonstrate that substrate binding can also induce translocation of PKC to the membrane and suggest that the removal of the pseudosubstrate domain is coupled to a conformational change in the enzyme that results in the exposure of hydrophobic groups.
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PMID:Substrate-induced translocation of PKC-alpha to the membrane. 855 12

Phospholipase D (PLD) which was partially purified from membranes of porcine brain could be stimulated by multiple cytosolic components; these included ADP-ribosylation factor (Arf) and RhoA, which required guanine nucleotides for activity, and an unidentified factor which activated the enzyme in a nucleotide-independent manner (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Here, we report purification of the latter factor, its identification as the alpha isoform of protein kinase C (PKCalpha), and characterization of its regulation of PLD activity. Stimulation of PLD by purified PKCalpha or recombinant PKCalpha (rPKCalpha) occurred in the absence of any nucleotide and required activators such as Ca2+ or phorbol ester. This action was synergistic with stimulation of PLD evoked by either Arf or RhoA. Dephosphorylation of rPKC alpha with protein phosphatase 1 or 2A resulted in a loss of its kinase activity, but had little effect on its ability to stimulate PLD either alone or in conjunction with Arf. Staurosporine inhibited the kinase activity of PKCalpha without affecting activation of PLD. Finally, gel filtration of PKCalpha that had been cleaved with trypsin demonstrated that stimulatory activity for PLD coeluted with the regulatory domain of the enzyme. These data indicate that PKC may regulate signaling events through direct molecular interaction with downstream effectors as well as through its well characterized catalytic modification of proteins by phosphorylation.
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PMID:Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity. 862 5


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