REGULATION OF PROTEIN-KINASE ENZYMATIC-ACTIVITY IN JURKAT T-CELLS DURING OXIDATIVE STRESS UNCOUPLED FROM PROTEIN-TYROSINE KINASES - ROLE OFOXIDATIVE CHANGES IN PROTEIN-KINASE ACTIVATION REQUIREMENTS AND GENERATION OF 2ND-MESSENGERS

Prior studies have suggested that intracellular phosphorylation events and cellular redox mechanisms may interact in regulating a variety of cellular functions, including the transcriptional activation of gene expression. Increased activity of transcriptional factors NF kappa B a nd AP1 has been described in cells exposed to oxidative stress and fol lowing the direct stimulation of protein kinase C (PKC) by phorbol die sters. However, the mechanisms that may contribute to redox regulation of PKC are unknown. We studied the expression of PKC activity and sev eral second messengers in human Jurkat T cells exposed to oxidative st ress in the form of H2O2. Micromolar concentrations of H2O2 rapidly in duced increased cytosolic PKC enzymatic activity in Jurkat T cells tha t was associated with a marked arrest of cellular proliferation. The i ncrease in cytosolic PKC activity in cells treated with H2O2 was accom panied by elevations in intracellular free calcium ([Ca2+](i)), genera tion of inositol phosphates, and release of arachidonic acid. Function al studies showed that H2O2 enhancement of cytosolic PKC activity requ ired phospholipase C activity but was not primarily mediated by arachi donic acid. The response of PKC to oxidative stress displayed a lack o f Ca2+ dependence and was uncoupled from the activity of protein tyros ine kinases (PTK). Furthermore, the reduced activation requirements of PKC from cells treated with H2O2 were associated with shifts in eluti on profiles of PKC enzymatic activity after Mono-Q chromatography. The se shifts appeared to represent intrinsic changes in the conformation of PKC induced by oxidative stress because western blotting failed to reveal any PKC cleavage products or reductions in native PKC alpha or beta. These findings indicate that oxidative regulation of intracellul ar events can intersect phosphorylation events mediated by PKC through the release of second messengers as well as direct changes in PKC act ivation requirements. Moreover, redox regulation of PKC is distinct fr om T cell receptor signaling in that the activity of PKC is uncoupled from the regulatory influences of PTK.