Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: Cholesterylphosphoryldimethylethanolamine

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 μm. 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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Growth inhibition and apoptosis in human neuroblastoma SK-N-SH cells induced by hypericin, a potent inhibitor of protein kinase C

Cancer Letters ◽

10.1016/0304-3835(95)03914-i ◽

1995 ◽

Vol 96 (1) ◽

pp. 31-35 ◽

Cited By ~ 29

Author(s):

Wei Zhang ◽

Ronald E. Lawa ◽

David R. Hintona ◽

Yuzhuang Su ◽

William T. Couldwell

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Growth Inhibition ◽

Potent Inhibitor ◽

Human Neuroblastoma ◽

Kinase C

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Ca2+-binding proteins from bovine brain including a potent inhibitor of protein kinase C

Biochemical Journal ◽

10.1042/bj2320559 ◽

1985 ◽

Vol 232 (2) ◽

pp. 559-567 ◽

Cited By ~ 66

Author(s):

J R McDonald ◽

M P Walsh

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Large Scale ◽

Binding Proteins ◽

Potent Inhibitor ◽

Binding Protein ◽

Sodium Dodecyl ◽

Bovine Brain ◽

Heat Stable ◽

Kinase C

We have previously described the use of Ca2+-dependent hydrophobic-interaction chromatography to isolate the Ca2+ + phospholipid-dependent protein kinase (protein kinase C) and a novel heat-stable 21 000-Mr Ca2+-binding protein from bovine brain [Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127]. The procedure described for purification of the 21 000-Mr calciprotein to electrophoretic homogeneity has been modified to permit the large-scale isolation of this Ca2+-binding protein, enabling further structural and functional characterization. The 21 000-Mr calciprotein was shown by equilibrium dialysis to bind approx. 1 mol of Ca2+/mol, with apparent Kd approx. 1 microM. The modified large-scale purification procedure revealed three additional, previously unidentified, Ca2+-binding proteins of Mr 17 000, 18 400 and 26 000. The 17 000-Mr and 18 400-Mr Ca2+-binding proteins are heat-stable, whereas the 26 000-Mr Ca2+-binding protein is heat-labile. Use of the transblot/45CaCl2 overlay technique [Maruyama, Mikawa & Ebashi (1984) J. Biochem. (Tokyo) 95, 511-519] suggests that the 18 400-Mr and 21 000-Mr Ca2+-binding proteins are high-affinity Ca2+-binding proteins, whereas the 17 000-Mr Ca2+-binding protein has a relatively low affinity for Ca2+. Consistent with this observation, the 18 400-Mr and 21 000-Mr Ca2+-binding proteins exhibit a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, whereas the 17 000-Mr Ca2+-binding protein does not. The amino acid compositions of the 17 000-Mr, 18 400-Mr and 21 000-Mr Ca2+-binding proteins show some similarities to each other and to calmodulin and other members of the calmodulin superfamily; however, they are clearly distinct and novel calciproteins. In functional terms, none of the 17 000-Mr, 18 400-Mr or 21 000-Mr Ca2+-binding proteins activates either cyclic nucleotide phosphodiesterase or myosin light-chain kinase, both calmodulin-activated enzymes. However, the 17 000-Mr Ca2+-binding protein is a potent inhibitor of protein kinase C. It may therefore serve to regulate the activity of this important enzyme at elevated cytosolic Ca2+ concentrations.

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The intrinsic ATPase activity of protein kinase C is catalyzed at the active site of the enzyme

Biochemistry ◽

10.1021/bi00140a029 ◽

1992 ◽

Vol 31 (25) ◽

pp. 5905-5911 ◽

Cited By ~ 11

Author(s):

Nancy E. Ward ◽

Catherine A. O'Brian

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Atpase Activity ◽

Active Site ◽

Kinase C

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Tu-P7:142 Expression kinetics of NOX1 and NOX4 regulated by protein kinase C in human endothelial cells

Atherosclerosis Supplements ◽

10.1016/s1567-5688(06)80848-2 ◽

2006 ◽

Vol 7 (3) ◽

pp. 216

Author(s):

H. Xu ◽

I. Brausch ◽

M. Hortmann ◽

T. Forstermann ◽

H. Li

Keyword(s):

Endothelial Cells ◽

Protein Kinase C ◽

Protein Kinase ◽

Human Endothelial Cells ◽

Kinase C ◽

Kinetics Of ◽

Expression Kinetics

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A Pseudo-Receptor Model of the Active Site of Protein Kinase C: An Example for a New Technique in Molecular Modelling

Novel Approaches in Anticancer Drug Design - Contributions to Oncology ◽

10.1159/000424063 ◽

1995 ◽

pp. 25-39

Author(s):

C. W. v. d. Lieth ◽

G. Krauter ◽

E. Hecker

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Molecular Modelling ◽

Active Site ◽

New Technique ◽

Receptor Model ◽

Kinase C ◽

A New Technique

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The kinetics of translocation and cellular quantity of protein kinase C in human leukocytes are modified during spaceflight

The FASEB Journal ◽

10.1096/fasebj.13.9001.s23 ◽

1999 ◽

Vol 13 (9001) ◽

Cited By ~ 43

Author(s):

Jason P. Hatton ◽

Francois Gaubert ◽

Marian L. Lewis ◽

Yann Darsel ◽

Philippe Ohlmann ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Human Leukocytes ◽

Kinase C ◽

Kinetics Of

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Delayed accumulation of diacylglycerol in platelets as a mechanism for regulation of onset of aggregation and secretion

Blood ◽

10.1182/blood.v78.2.435.435 ◽

1991 ◽

Vol 78 (2) ◽

pp. 435-444 ◽

Cited By ~ 12

Author(s):

MH Werner ◽

YA Hannun

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Shape Change ◽

Human Platelets ◽

Optimal Timing ◽

Delayed Effects ◽

Kinase C ◽

Irreversible Aggregation ◽

Kinetics Of ◽

Sustained Activation

Abstract An important mechanism of platelet regulation is the formation of the second messenger diacylglycerol (DAG) and the activation of protein kinase C (PKC). Our previous studies suggested that the DAG/PKC pathway plays an important role in the induction of secretion and secondary aggregation rather than the earlier events of shape change and primary aggregation. We therefore examined the hypothesis that the delayed effects of PKC on platelets may result from delayed accumulation of DAG. The kinetics of DAG formation in human platelets were determined. When platelets were stimulated with gamma-thrombin, the largest phase of DAG accumulation was delayed for 0.6 to 0.8 minutes and DAG mass levels remained elevated for at least 2 minutes. In platelets stimulated with collagen, DAG accumulation was delayed for 1.0 to 1.2 minutes and DAG mass levels remained elevated for at least 3 minutes after stimulation. Sustained DAG production was also associated with sustained activation of PKC as monitored by phosphorylation of the 40- Kd substrate of PKC. The physiologic significance of delayed DAG accumulation was investigated using the cell-permeable DAG analog, dioctanoylglycerol (diC8). In synergy with subthreshold gamma-thrombin or collagen, exogenous diC8 reconstituted platelet activation. The optimal timing of addition of diC8 was 0.5 minutes after stimulation with gamma-thrombin or collagen. These kinetics were similar to those of endogenous DAG accumulation. These studies underscore the importance of a delayed accumulative phase of DAG generation as a mechanism controlling the onset of platelet secretion and irreversible aggregation.

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A comparative study of the activation of protein kinase C α by different diacylglycerol isomers

Biochemical Journal ◽

10.1042/bj3370387 ◽

1999 ◽

Vol 337 (3) ◽

pp. 387-395 ◽

Cited By ~ 17

Author(s):

Pilar SÁNCHEZ-PIÑERA ◽

Vicente MICOL ◽

Senena CORBALÁN-GARCÍA ◽

Juan C. G. ÓMEZ-FERNÁNDEZ

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Mixed Micelles ◽

Fatty Acyl ◽

Molar Ratio ◽

Binding Studies ◽

Scanning Calorimetry ◽

Kinase C ◽

Triton X ◽

Activation Capacity

The lipid activation of protein kinase C α (PKC α) has been studied by comparing the activation capacity of different 1,2-diacylglycerols and 1,3-diacylglycerols incorporated into mixed micelles or vesicles. Unsaturated 1,2-diacylglycerols were, in general, more potent activators than saturated ones when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)/Triton X-100 mixed micelles and pure POPS vesicles were used. In contrast, these differences were not observed when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS (4:1, molar ratio) vesicles were used. Diacylglycerols bearing short fatty acyl chains showed a very high activation capacity, however, the capacity was less in mixed micelles. Furthermore, 1,2-diacylglycerols had a considerably higher activating capacity than 1,3-diacylglycerols in POPS/Triton X-100 mixed micelles and in POPC/POPS vesicles. However, the differences between the two types of diacylglycerols were smaller when pure POPS vesicles were used. Differential scanning calorimetry (DSC) showed that POPC/POPS membrane samples containing diacylglycerols had endothermic transitions in the presence of 200 µM Ca2+ and 5 mM Mg2+. Transitions were not detected when using pure POPS vesicles due to the formation of dehydrated phases as demonstrated by FTIR (Fourier-transform infrared) spectroscopy. PKC α binding studies, performed by differential centrifugation in the presence of 200 µM Ca2+ and 5 mM Mg2+, showed that 1,2-sn-dioleoylglycerol (1,2-DOG) was more effective than 1,3-dioleoylglycerol (1,3-DOG) in promoting binding to POPC/POPS vesicles. However, when pure POPS vesicles were used, PKC α was able to bind to membranes containing either 1,2-DOG or 1,3-DOG to the same extent.

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