Irreversible oxidative inactivation of protein kinase C by photosensitive inhibitor calphostin C

• Published in: FEBS letters 1992-12, Vol.314 (2), p.149-154
• Main Authors: Gopalakrishna, Rayudu, Chen, Zhen Hai, Gundimeda, Usha
• Format: Article
• Language: English
• Subjects: 12-O-tetradecanoyl-phorbol 13-acetate; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Ca 2+-induced hydrophobic region; Ca2+-induced hydrophobic region; Calcium - pharmacology; Calphostin C; EGTA; Enzymes and enzyme inhibitors; ethylene glycol bis(β-aminoethylether)-N,N′-tetraacetic acid; Free Radical Scavengers; Free Radicals; Fundamental and applied biological sciences. Psychology; Naphthalenes; Oxidation-Reduction; Oxidative modification; Oxygen - pharmacology; PDBu; phorbol 12,13-dibutyrate; PKC; Polycyclic Compounds - pharmacology; Polycyclic Compounds - radiation effects; Protein kinase C; Protein Kinase C - antagonists & inhibitors; Reactive oxygen species; Singlet oxygen; TPA; Transferases; Tumor Cells, Cultured
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(92)80962-G

Isolated protein kinase C (PKC) was irreversibly inactivated by a brief (min) incubation with calphostin C in the presence of light. This inactivation required Ca 2+ either in a millimolar range in the absence of lipid activators or in a submicromolar range in the presence of lipid activators. In addition, an oxygen atmosphere was required suggesting the involvement of oxidation(s) in this inactivation process. Furthermore, PKC inactivation might involve a site-specific oxidative modification of the enzyme at the Ca 2+-induced hydrophobic region. Physical quenchers of singlet oxygen such as lycopene, β-carotene, and α-tocopherol all reduced the calphostin C-induced inactivation of PKC. In intact cells treated with calphostin C, the inactivation of PKC was rapid in the membrane fraction compared to cytosol. This intracellular PKC inactivation was also found to be irreversible. Therefore, calphostin C can bring prolonged effects for several hours in cells treated for a short time. Taken together these results suggest that the calphostin C-mediated inactivation of PKC involves a site-specific and a ‘cage’ type oxidative modification of PKC.