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Translocation and Enhancement of Phosphotransferase Activity of Protein Kinase C following Exposure in Mouse Epidermal Cells to Oxidants¹

Department of Carcinogenesis, Swiss Institute for Experimental Cancer Research, 1066 Epalinges/Lausanne, Switzerland

We have studied changes in intracellular localization and phosphorylating activity of protein kinase C (PKC) in mouse epidermal JB6 cells treated with oxidants. Exposure to hydrogen peroxide, reagent grade or generated enzymatically by glucose/glucose oxidase, at concentrations known to result in elevated intracellular free Ca²⁺ resulted in an increase in binding of [³H]phorbol dibutyrate to intact cells. Ca²⁺ chelation, either intracellularly by quin 2 or extracellularly by ethylene glycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, abolished the increase in radioligand binding. In contrast to H₂O₂, superoxide generated extracellularly by xanthine/xanthine oxidase or intracellularly by menadione was inactive. Scatchard plot analysis revealed that the enhancement in binding resulted from both increased receptor affinity and increased maximal binding capacity.

Treatment of cells with superoxide, generated extracellularly by xanthine/xanthine oxidase or intracellularly by menadione, diminished the [³H]phorbol dibutyrate-binding capacity of the cytosol fractions prepared at low Ca²⁺ concentration. This decrease was not accompanied by a compensatory increase in the binding to membrane components. In contrast to superoxide, reagent H₂O₂, H₂O₂ produced by glucose/glucose oxidase, and the Ca²⁺ ionophore A23187 had no significant effect on the [³H]phorbol dibutyrate-binding capacities of either cellular fraction.

Exposure of cells to low concentrations of extra- or intracellular superoxide resulted in an increase in the Ca²⁺- and phospholipid-dependent phosphorylating activity of cytosolic extracts towards adenosine diphosphoribose transferase which has been reported to be a specific substrate for PKC. The increase in phosphorylation could be diminished by the extracellular addition of copper-zinc-containing superoxide dismutase but not catalase suggesting that superoxide rather than H₂O₂ represents the active oxygen species in this reaction. The observation that reagent H₂O₂ or glucose/glucose oxidase failed to increase the phosphorylating activity of cytosolic preparations supports this conclusion. Treatment of cells or cytosolic extracts with the sulfhydryl reagent diamide stimulated the Ca²⁺/phospholipid-dependent phosphorylating activity toward adenosine diphosphoribose transferase. In a reconstituted system containing purified PKC, diamide induced a 25–30% increase in phospholipid-dependent phosphorylation of H1 whereas no change in activity was observed with the reducing agent dithiothreitol.

It is concluded that H₂O₂ but not superoxide induces an increase in the phorbol ester binding, presumably to PKC, of intact JB6 cells. On the other hand, treatment of cells with low concentrations of superoxide but not of H₂O₂ results in the activation of the in vitro phosphorylating capacity of cytoplasmic extracts towards acceptor proteins with specificity for PKC. While the first effect appears to be mostly due to an increase in intracellular free Ca²⁺ the second may involve the oxidation of critical sulfhydryl groups in the regulatory lipid-binding domain of PKC.

¹ This work was supported by the Swiss National Science Foundation and the Swiss Association of Cigarette Manufacturers and the Association for International Cancer Research.

Received 1/ 5/89. Revised 6/19/89. Accepted 7/21/89.

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