Activation of protein kinase C in permeabilized human neuroblastoma SH-SY5Y cells.
The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.
Larsson
C
C
Department of Psychiatry and Neurochemistry, Lund University, Sweden.
Saermark
T
T
Mau
S
S
Simonsson
P
P
D000098:Acetophenones / Q000494:pharmacology; D000595:Amino Acid Sequence; D001095:Arachidonic Acids / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D004072:Digitonin; D004075:Diglycerides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D010758:Phosphorus / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D011796:Quinacrine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology / Q000473:pathology
D000098:Acetophenones; D001095:Arachidonic Acids; D004075:Diglycerides; D004676:Myelin Basic Protein; D010761:Phosphorus Radioisotopes; C044295:1,2-dioctanoylglycerol; D010758:Phosphorus; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D011796:Quinacrine; D004072:Digitonin; D013755:Tetradecanoylphorbol Acetate; C006821:4-bromophenacyl bromide; D002118:Calcium