Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The perfused rat heart was treated with the tumour-promoter and protein kinase C activator, phorbol 12-myristate 13-acetate and the distribution of protein kinase C activity between cytosolic and particulate fractions determined. 2. Phorbol ester treatment led to a rapid loss of protein kinase C activity from the cytosol (t0.5 = 2 min) with a corresponding translocation into the particulate fraction. Translocated protein kinase C activity was tightly bound to the particulate fraction, could only be extracted with buffers containing 2% Triton X-100 and could therefore be misinterpreted as being down-regulated. 3. Claims of rapid down-regulation of protein kinase C activity by phorbol esters need to be supported by rigorous procedures for extraction of the particulate material.
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PMID:Effect of phorbol esters on the distribution and total activity of protein kinase C in the perfused rat heart. 261 27

Protein kinase C (PKC), a calcium and phospholipid dependent protein kinase C, has emerged as a key element in signal transduction and cell regulation. It is activated by sn-1,2-diacylglycerol (DAG) second messengers and it serves as the receptor for phorbol esters, potent tumor promoters. PKC is now known to occur as a family of isoenzymes sharing similar structural features that allow regulation of activity by calcium, phospholipid, and DAG. In vitro mechanisms of PKC regulation by phospholipid, DAG, and phorbol esters have been studied using mixed micelles of Triton X-100/lipids. PKC activation occurs at physiologic mole fractions of phospholipid and DAG, does not require a bilayer, and appears to occur by a two-step mechanism whereby PKC initially interacts with a phospholipid surface and is then activated by the addition of DAG. Similar methodology has been used to explore the inhibition of PKC by different inhibitors that interact with its regulatory domain. Sphingosine and lysosphingolipids are potent inhibitors of PKC that prevent its interaction with DAG/phorbol esters. These naturally occurring metabolites have been shown to affect PKC activity in different cell systems. Disturbances in sphingolipid metabolism may lead to accumulation of lysosphingolipids with consequent inhibition of PKC. Additionally, these naturally occurring metabolites may have physiologic functions in regulating PKC activity by counteracting the action of DAG. The mechanism of action of sphingosine/lysosphingolipids and their possible physiologic function will be discussed.
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PMID:Regulation of protein kinase C by sphingosine and lysosphingolipids. 269 75

The specificity of the phospholipid cofactor requirement of rat brain protein kinase C was investigated using Triton X-100 mixed micellar methods. Sixteen analogues of phosphatidylserine were prepared and tested for their ability to support protein kinase C activity, [3H]phorbol 12,13-dibutyrate binding, and protein kinase C binding to mixed micelles. Phosphatidylserinol, -L-serine methyl ester, -N-acetyl-L-serine, -2-hydroxyacetate, -3-hydroxypropionate, and -4-hydroxybutyrate did not activate protein kinase C in mixed micelles containing 2 mol % of sn-1,2-dioleoylglycerol. This indicates that both the carboxyl and amino moieties are important for activation. Phosphatidyl-D-serine and -L-homoserine were incapable of supporting full activation; this demonstrates stereospecificity and the importance of the distance between the phosphate and carboxyl and amino moieties. Since 1,2-rac-phosphatidyl-L-serine and 1,3-phosphatidyl-L-serine fully supported protein kinase C activity, the stereochemistry within the glycerol backbone at the interface was not necessary for maximal activation. Neither lysophosphatidyl-L-serine nor 1-oleoyl-2-acetyl-sn-glycero-3-phospho-L-serine supported protein kinase C activity implying that the interfacial conformation is critical to the activation process. The phospholipid dependencies of [3H]phorbol 12,13-dibutyrate binding and of protein kinase C binding to mixed micelles containing sn-1,2-dioleoylglycerol did not mirror those for activation. The data demonstrate that protein kinase C possesses a high degree of specificity with respect to phospholipid activation and implicate several functional groups within the phospho-L-serine polar head group in binding and activation.
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PMID:Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding, and binding to mixed micelles. 276 40

Most commonly used surfactants were found to be inhibitors of partially purified rat brain protein kinase C at or above their critical micellar concentrations (CMC). These include sodium lauryl sulfate, deoxycholate, octyl glucoside, dodecyl trimethylammonium bromide, linear alkylbenzene sulfonate and Triton X-100. Several detergents, including the nonionic surfactants digitonin and Neodol-12 (ethoxylated alcohol), did not inhibit protein kinase C activity, even at concentrations greater than their CMC, while the anionic surfactant, AEOS-12 (ethoxylated alcohol sulfate), inhibited enzyme activity only slightly (less than 8%). Since these latter surfactants have little or no inhibitory effect on protein kinase C, they may be of value in solubilizing cells and tissues for the determination of enzyme activity in crude extracts. Among the detergents tested, sodium lauryl sulfate and linear alkylbenzene sulfonate significantly stimulated protein kinase C activity in the absence of phosphatidylserine and calcium. This was found to be dependent on the presence of histone in the protein kinase C assay. These detergents failed to stimulate protein kinase C activity when endogenous proteins in the partially purified rat brain extracts were used as the substrate. Our results indicate that activity of protein kinase C can be modified by the conditions of the assay and by the detergents used to extract the enzyme.
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PMID:Ethoxylated alcohol (Neodol-12) and other surfactants in the assay of protein kinase C. 277 91

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca2+. It has a Ki of about 30 microns. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca2+-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.
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PMID:Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: cholesterylphosphoryldimethylethanolamine. 277 61

The phosphorylation of an Mr 82,000 protein (p82) in the Triton X-100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)-catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82-kinase system is down-modulated after TPA treatment of mouse skin, with a half-life of around 5 h. Down-modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down-modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the particulate fraction.
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PMID:A phorbol ester and phospholipid-activated, calcium-unresponsive protein kinase in mouse epidermis: characterization and separation from protein kinase C. 277 8

Three forms of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) were extensively purified from rat liver homogenate. Subcellular fractionation analysis indicated that the majority (approximately 85%) of the activity was associated with particulate fractions of the liver. Among these, the microsomal and nuclear fractions accounted for approximately 63% and approximately 10% of total activity. The remaining 15% of protein kinase C was recovered in the soluble fraction following differential centrifugation. It was also found that most of the membrane-associated protein kinase C was latent, with 4-6-fold stimulation with detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, octyl beta-glucoside, or Triton X-100. The activity of both the bound form and the soluble enzyme was enhanced by the addition of Ca2+ and phosphatidylserine, when histone H1 was used as substrate. The bound protein kinase C activity was dissociated by homogenization of liver in buffer containing ethylene glycol bis(beta-aminoethyl ether)-N,-N,N',N'-tetraacetic acid, ethylenediaminetetraacetic acid, and various proteolytic inhibitors, and the solubilized extract was used to purify multiple forms of the enzyme. The purification procedure sequentially utilized (NH4)2SO4 fractionation, ion-exchange chromatography on DEAE-cellulose, gel permeation chromatography on Fractogel TSK HW-55 (F), ion-exchange chromatography on hydroxylapatite, gel permeation chromatography on Ultrogel AcA34, and affinity chromatography on polyacrylamide-immobilized phosphatidylserine. On hydroxylapatite columns, protein kinase C activity was resolved into three isoenzymic forms designated C-I, C-II, and C-III. The molecular weights of the three isoenzymic forms were in the range of 208,000-225,000 as shown by chromatography on calibrated Ultrogel AcA34 columns and sucrose density gradient centrifugation. Furthermore, all three isoenzymes demonstrated a single peak with a sedimentation coefficient (s20.w) in the range of 9.0-9.2. However, with polyacrylamide gel electrophoresis, all the forms showed a single protein component with average molecular weight of 64K, suggesting that the native isoenzymes may be composed by subunits. Finally, all three isoenzymes exhibited nearly identical enzymatic properties.
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PMID:Calcium-activated, phospholipid-dependent protein kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms. 282 43

Rabbit peritoneal neutrophils, permeabilized with Triton X-100, contain inositol phosphate 5-phosphomonoesterase activity capable of converting [3H]inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to [3H]inositol 1,4-bisphosphate. This activity is found predominantly associated with the soluble component of fractionated neutrophils. It is comprised of specific and nonspecific activities toward Ins-1,4,5-P3 which can be separated by cation exchange chromatography. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) prior to permeabilization does not affect the rate of Ins-1,4,5-P3 breakdown by these cells. In addition, activation of endogenous protein kinase C in a soluble fraction prepared from neutrophils does not affect the specific inositol phosphate 5-phosphomonoesterase activity of this fraction. Taken together, these results provide evidence that activation of protein kinase C in the neutrophil does not affect its 5-phosphomonoesterase activity. Unlike platelets, the phosphorylation of a 5-phosphomonoesterase, if it occurs, may not play a role in the inhibitory effects of PMA on neutrophil responsiveness.
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PMID:Demonstration of inositol phosphate 5-phosphomonoesterase activity in rabbit neutrophils: absence of a role for protein kinase C. 284 77

Rabbit peritoneal neutrophils were stimulated with either the chemotactic factor, fMet-Leu-Phe (10(-8) M, 10 s) or the protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), (0.1 microgram/ml, 3 min) at 37 degrees C, lysed with Triton X-100 at the indicated times and the histone H4 kinase activity of the lysate measured. The histone H4 protein kinase activity was increased severalfold by fMet-Leu-Phe but not PMA. The inclusion of the potent protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (50 microM) inhibited little if any of the histone H4 protein kinase activity. The effect of fMet-Leu-Phe was transient, maximum stimulation occurring within 10 s and decaying thereafter. The soluble fraction (extract) of the Triton X-100 lysates from control and fMet-Leu-Phe-treated cells was found to contain both histone H4 protein kinase and calcium-phospholipid-activated protein kinase (protein kinase C) activities. The histone H4 protein kinase activity obtained after fMet-Leu-Phe treatment was very little affected by calcium, phospholipid, and PMA and preferred histone H4 but not H1 or H2A as its substrate. In contrast, the calcium-phospholipid-activated protein kinase activity of the extract preferred histones H1 or H2A as substrates and was strongly inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine. The histone H4 protein kinase was partially separated from kinase C by DEAE-cellulose and phenyl-Sepharose 4B chromatography. It phosphorylated mostly serine in histone H4. The results indicate that the chemotactic factor, fMet-Leu-Phe, stimulates a protein kinase with substrate specificity and biochemical properties distinct from calcium-phospholipid-activated protein kinase C.
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PMID:Stimulation of a histone H4 protein kinase in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with chemotactic factors. Effect of fMet-Leu-Phe and partial characterization of the protein kinase. 284 11

The rate of energy-dependent nucleoside triphosphatase (NTPase)-mediated nucleocytoplasmic translocation of poly(A)-containing mRNA [poly(A)+mRNA] across the nuclear envelope is thought to be regulated by poly(A)-sensitive phosphorylation and dephosphorylation of nuclear-envelope protein. Studying the phosphorylation-related inhibition of the NTPase, we found that phosphorylation of one polypeptide of rat liver envelopes by endogenous NI- and NII-like protein kinase was particularly sensitive to poly(A). This polypeptide (106 kDa) was also phosphorylated by nuclear-envelope-bound Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C). Activation of kinase C by tumour-promoting phorbol esters resulted in inhibition of nuclear-envelope NTPase activity and in a concomitant decrease of mRNA (actin) efflux rate from isolated rat liver nuclei. Protein kinase C, but not nuclear envelope NI-like or NII-like protein kinase, was found to be solubilized from the envelope by Triton X-100, whereas the presumable poly(A)-binding site [the 106 kDa polypeptide, representing the putative carrier for poly(A)+mRNA transport] remained bound to this structure. RNA efflux from detergent-treated nuclei lost its susceptibility to phorbol esters. Addition of purified protein kinase C to these nuclei restored the effect of the tumour promoters. Protein kinase C was found to bind also to isolated rat liver nuclear matrices in the absence but not in the presence of ATP. The NII-like nuclear-envelope protein kinase co-purified together with the 106 kDa polypeptide which specifically binds to poly(A) in an ATP-labile linkage.
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PMID:Studies on protein kinases involved in regulation of nucleocytoplasmic mRNA transport. 284 56


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