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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Protein kinase C catalyzes the incorporation of about 1.1, 0.7 and 0.4 mole of phosphate per mole of Lipocortin-I (P35), Lipocortin-II (P36) and Lipocortin-85 (P36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While Lipocortin-I is phosphorylated on threonine residues, Lipocortin-II and Lipocortin-85 are phosphorylated on serine residues. The substoichiometric phosphorylation of Lipocortin-85 appears to preclude the potential regulation of this protein by protein kinase C. The phosphorylation of Lipocortin-I on threonine residues and Lipocortin-II on serine residues suggests these proteins may be regulated by distinct phosphorylation-dephosphorylation reactions.
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PMID:Phosphorylation of lipocortins in vitro by protein kinase C. 294 7

Experimental conditions are described for simultaneous purification of three forms of lipocortin (lipocortin I, lipocortin II and lipocortin-85) from bovine lung. The procedure yields milligram quantities of all three lipocortins. Using antisera against lipocortin I and lipocortin II, purified proteins show no cross contaminations. All forms of lipocortin exhibit equal potency as in vitro bovine pancreatic phospholipase A2 inhibitors. Protein kinase C catalyzes the in vivo incorporation of about 1.0, 0.7 and 0.4 mole of phosphate per mole of lipocortin I (p35), lipocortin II (p36) and lipocortin-85 (p36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While lipocortin I is phosphorylated on threonine residues, lipocortin II and lipocortin-85 are phosphorylated on serine residues.
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PMID:Purification of three forms of lipocortin from bovine lung. 295 2

We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation.
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PMID:Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts. 310 61

Protein kinase C (PKC), a Ca2+-and phospholipid-dependent protein kinase, is now known to be regulated by sn-1,2-diacylglycerol (DAG) second messengers and is the intracellular phorbol ester receptor. Models of transmembrane signaling events that elicit DAG production include receptor-mediated G protein-dependent activation of phospholipase C. Several products of oncogenes resemble transmembrane signaling elements; critical second-messenger levels may, therefore, be altered by genetic defects in these elements. We found that normal rat kidney cells transformed with ras and sis contained elevated levels of DAG, and cells transformed with temperature-sensitive K-ras had elevated DAG levels at the permissive but not the restrictive temperature. To study the mechanism of PKC activation by phosphatidylserine (PS), DAG, and Ca2+, we used mixed micelles of Triton X-100, and analogous methods to examine PS dependence on [3H]phorbol-dibutyrate binding and activation. PKC activation occurs at physiological mole fractions of PS and DAG and does not require a bilayer. Activation by PS, which was cooperative, required four or more molecules. Activation by DAG was not cooperative and one molecule was sufficient. Monomeric PKC is the active species. Our activation model suggests that PKC binds to Ca2+ and four PS carboxyl groups to form a surface-bound, "primed" but inactive complex. DAG binds to the complex of the four PS carboxyl groups, the Ca2+, and the PKC through three bonds, two to ester carbonyls and one to the 3-hydroxyl moiety. Collectively, these may cause a conformational change and activate the enzyme.
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PMID:Mechanism of regulation of protein kinase C by lipid second messengers. 332 5

The phospholipid, sn-1,2-diacylglycerol, and calcium dependencies of rat brain protein kinase C were investigated with a mixed micellar assay (Hannun, Y., Loomis, C., and Bell, R.M. (1985) J. Biol. Chem. 260, 10039-10043). Protein kinase C activity was independent of the number of Triton X-100, phosphatidylserine (PS), and sn-1,2-dioleoylglycerol (diC18:1) mixed micelles. Activation was strongly dependent on the mole per cent of PS and diC18:1. Activity of protein kinase C was dependent on PS, diC18:1, and calcium in mixed micelles prepared from detergents other than Triton X-100. This is consistent with the micelle providing an inert surface into which the lipid cofactors partition. Molecular sieve chromatography provided direct evidence for the homogeneity of Triton X-100, PS, and diC18:1 mixed micelles. Mixing studies and surface dilution studies indicated that PS and diC18:1 rapidly equilibrate among the mixed micelles. At saturating calcium, the diC18:1 dependence was strongly dependent on the mole per cent PS present. At 10 mol % PS, 0.25 mol % diC18:1 gave maximal activity whereas 6 mol % PS and 6 mol % diC18:1 did not give maximal activity. diC18:1 dependencies were hyperbolic at all PS levels tested. The data support the conclusion that a single molecule of diC18:1/micelle is sufficient to activate monomeric protein kinase C. The mole per cent PS required for maximal activation was reduced markedly as the mole per cent diC18:1 increased. Under all conditions tested, the PS dependence of protein kinase C activation lagged until greater than 3 mol % PS was present. Then activation occurred in a cooperative manner with Hill numbers near 4. These data indicate that 4 or more molecules of PS are required to activate monomeric protein kinase C. PS was the most effective of all the phospholipids tested in the mixed micelle assay. diC18:1 was found to modulate the amount of calcium required for maximal activity. As the level of Ca2+ increased, the mole per cent PS required reached a limiting value of 3 mol %. A number of sn-1,2-diacylglycerols containing short chain fatty acids activated protein kinase C in a saturable manner in mixed micelles. The data are discussed in relation to a model for protein kinase activation.
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PMID:Protein kinase C activation in mixed micelles. Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies. 371 Oct 83

Protein kinase C has been previously shown both to phosphorylate and to desensitize the ability of the human 5-HT1A receptor to inhibit adenylyl cyclase [Raymond, J. R. (1991) J. Biol. Chem. 266, 14747-14753]. In this study, we examined the effects of short-term treatment with protein kinase A activators on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the human serotonin 5-HT1A receptor in CHO cells that stably express 1200 fmol of receptor/mg of protein. Forskolin induced a concentration- and time-dependent phosphorylation of the receptor that was detectable at 5 min and maximal at 15-30 min with a half-maximal concentration of 10-20 microM. Phosphorylation was also induced by Sp-cAMPS or dibutyryl-cAMP, and blocked by Rp-cAMPS and a pseudosubstrate inhibitor of PKA, but not by heparin (inhibitor of receptor kinase) or sphingosine (inhibitor of PKC). The stoichiometry of phosphorylation induced by forskolin was 1 mol of phosphate per mole of receptor. PKA activators did not induce a measurable desensitization of 5-HT1A receptor-inhibited adenylyl cyclase activity. However, forskolin augmented the desensitization caused by a submaximal concentration of phorbol 12-myristate 13-acetate (300 nM PMA) as evidenced by a rightward shift of the concentration-response curve for 5-HT, and approximately doubled the amount of phosphate incorporated into the receptor by PMA. Forskolin did not augment desensitization or increase the degree of phosphorylation induced by a maximal concentration of PMA (5 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein kinase A induces phosphorylation of the human 5-HT1A receptor and augments its desensitization by protein kinase C in CHO-K1 cells. 772 77

Protein kinase C plays an important role in regulation of the cytoskeleton. We found that protein kinase C activity is associated with the retinal cytoskeleton, and that it catalyses the stoichiometric phosphorylation of two cytoskeletal proteins, with apparent molecular masses of 56 kDa and 100 kDa. The 56-kDa substrate was identified as vimentin on the basis of its apparent molecular mass, pI, solubility, immunoreactivity, pattern of proteolysis by Lys-C and a partial amino acid sequence. Immunomicroscopy was consistent with previous reports that in the retina vimentin has the unusual property of being present in neuronal cells--horizontal cells--as well as non-neuronal cells. The characteristics of protein kinase C phosphorylation of vimentin that was enriched in neuronal vimentin were determined. Hyperactivation of protein kinase C by treatment of retinas with phorbol myristate acetate resulted in the phosphorylation of vimentin in situ, indicating that the phosphorylation is physiologically relevant. In vitro, purified retinal protein kinase C catalysed the incorporation of nearly 2 mol phosphate per mole of monomeric vimentin. The phosphorylation was highly dependent on the presence of phosphatidylserine. Thus, protein kinase C functions in the retinal cytoskeleton, where a major role is in the modification of vimentin. The characterization of the phosphorylation of outer retinal vimentin by protein kinase C provides a basis for further studies on the regulation and function of this cytoskeletal element.
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PMID:Protein kinase C association with the retinal cytoskeleton and phosphorylation of vimentin. 792 14

Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP. cAMP stimulation of Dictyostelium cells leads to translocation of MHC-PKC from the cytosol to the membrane fraction, as well as causing an increase in both MHC-PKC phosphorylation and its kinase activity. MHC-PKC undergoes autophosphorylation with each mole of kinase incorporating about 20 mol of phosphate. The MHC-PKC autophosphorylation sites are thought to be located within a domain at the COOH-terminal region of MHC-PKC that contains a cluster of 21 serine and threonine residues. Here we report that deletion of this domain abolished the ability of the enzyme to undergo autophosphorylation in vitro. Furthermore, after this deletion, cAMP-dependent autophosphorylation of MHC-PKC as well as cAMP-dependent increases in kinase activity and subcellular localization were also abolished. These results provide evidence for the role of autophosphorylation in the regulation of MHC-PKC and indicate that this MHC-PKC autophosphorylation is required for the kinase activation in response to cAMP and for subcellular localization.
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PMID:Autophosphorylation of Dictyostelium myosin II heavy chain-specific protein kinase C is required for its activation and membrane dissociation. 899 70

Protein kinase C displays high apparent cooperativity in its activation by phosphatidylserine. This contribution uses a novel approach to address the physical basis for this apparent cooperativity. We examine the binding of protein kinase C betaII to large unilamellar vesicles as a function of increasing mole fraction phosphatidylserine and as a function of increasing total lipid concentrations. Binding data are subjected to an analysis, described in the Appendix, that allows calculation of the fractional saturation of phosphatidylserine binding sites with this ligand. This analysis reveals that (1) protein kinase C betaII binds approximately eight phosphatidylserine molecules and (2) the binding of each lipid is not cooperative. Rather, the apparent cooperativity observed in protein kinase C's interaction with multiple phosphatidylserine molecules arises from effects specific to the interaction of a multivalent macromolecule with multiple membrane-associated ligands. Nor does diacylglycerol, which has been previously shown to dramatically increase protein kinase C's affinity for phosphatidylserine-containing membranes, induce cooperativity. Thus, protein kinase C binds multiple phosphatidylserine molecules in the absence of interaction between potential binding sites. The method presented for determining the stoichiometry and cooperativity in the interaction of protein kinase C with phosphatidylserine is applicable to any multivalent molecule binding to monovalent ligands incorporated into lipid membranes.
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PMID:Mechanism of the apparent cooperativity in the interaction of protein kinase C with phosphatidylserine. 986 Aug 41


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