An okadaic acid-sensitive protein phosphatase counteracts protein kinase C-induced phosphorylation in SH-SY5Y cells.
Protein phosphorylation and subsequent dephosphorylation was studied in digitonin-permeabilized neuroblastoma SH-SY5Y cells by measuring the incorporation of [32P]phosphate into myelin basic protein (MBP). 1,2-Dioctanoyl-sn-glycerol (DOG) and calcium synergistically induced phosphorylation of MBP, which was inhibited by the protein kinase C (PKC) pseudosubstrate peptide (PKC19-36). The phosphorylation increased for 10 min when a net dephosphorylation started to appear. The dephosphorylation was inhibited by okadaic acid. Regardless of calcium concentration, the presence of DOG was necessary for significant effects of okadaic acid on MBP phosphorylation. H7 and staurosporine dose-dependently inhibited the phosphorylation of MBP, induced by DOG and calcium in the presence of okadaic acid. Different PKC pseudosubstrate peptides were applied and all showed an inhibitory effect on the phosphorylation of MBP under these conditions. These results demonstrate the presence, in SH-SY5Y cells, of a protein phosphatase, possibly protein phosphatase 2A, with a high basal activity that counteracts PKC-induced phosphorylation.
Larsson
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C
Department of Psychiatry and Neurochemistry, Lund University, Sweden.
Alling
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C
Simonsson
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P
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D002463:Cell Membrane Permeability; D004075:Diglycerides / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D009447:Neuroblastoma; D019319:Okadaic Acid; D010446:Peptide Fragments / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation / Q000187:drug effects; D010879:Piperazines / Q000494:pharmacology; D011112:Polymyxin B / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054648:Protein Phosphatase 2; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects*; D019311:Staurosporine; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D004075:Diglycerides; D004988:Ethers, Cyclic; D007527:Isoenzymes; D007546:Isoquinolines; D004676:Myelin Basic Protein; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D010879:Piperazines; C044295:1,2-dioctanoylglycerol; D019319:Okadaic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D019311:Staurosporine; D011112:Polymyxin B; D002118:Calcium