Sublethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppress protein phosphatases in Jurkat T cells.

Sublethal concentrations of reactive oxygen intermediates including H2O2 can alter human T cell function and inhibit proliferative responses but relatively little is known about the effects of low levels of oxidant stress on signaling pathways. In the present study, we investigated whether the exposure of Jurkat T cells to micromolar concentrations of H2O2 might influence the activity of certain serine/threonine kinases and protein phosphatases important for T cell signaling as well as initiation of nuclear events. Jurkat cells treated with 100-200 microM H2O2 exhibited rapid increases in cytosolic protein kinase C (PKC) activity without detectable translocation of PKC to the membrane/particulate compartment. The stimulation of PKC activity by H2O2 was associated with an increase in the activation of kinases phosphorylating myelin basic protein (MBP), a substrate for mitogen-activated protein (MAP) kinase and RRLSSLRA (S6 peptide; a substrate for the approximately 90-kDa ribosomal S6 kinases). Optimal activation of MAP kinase in cells treated with H2O2 was preceded by increases in protein tyrosine phosphorylations and occurred at sublethal concentrations of H2O2 which did not markedly deplete intracellular ATP. Pretreatment of cells with the PKC inhibitors sangivamycin and H7 suppressed but did not block the stimulation of MAP kinase activity in response to H2O2 or phytohemagglutinin. The activities of both protein tyrosine phosphatase (PTP) and protein phosphatase 2A (PP2A) were reduced after H2O2 treatment of intact cells. Furthermore, kinetic studies showed that H2O2 was capable of suppressing the activities of PTP and PP2A before inducing optimal increases in MAP kinase activity. These results demonstrate that the exposure of T cells to sublethal levels of oxidant stress acutely stimulates the MAP kinase cascade and suggest that this activation may involve PKC-dependent and -independent pathways as well as inhibition of certain protein phosphatases.

Regulation of protein kinase enzymatic activity in Jurkat T cells during oxidative stress uncoupled from protein tyrosine kinases: role of oxidative changes in protein kinase activation requirements and generation of second 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 and AP1 has been described in cells exposed to oxidative stress and following the direct stimulation of protein kinase C (PKC) by phorbol diesters. However, the mechanisms that may contribute to redox regulation of PKC are unknown. We studied the expression of PKC activity and several second messengers in human Jurkat T cells exposed to oxidative stress in the form of H2O2. Micromolar concentrations of H2O2 rapidly induced increased cytosolic PKC enzymatic activity in Jurkat T cells that was associated with a marked arrest of cellular proliferation. The increase in cytosolic PKC activity in cells treated with H2O2 was accompanied by elevations in intracellular free calcium ([Ca2+]i), generation of inositol phosphates, and release of arachidonic acid. Functional studies showed that H2O2 enhancement of cytosolic PKC activity required phospholipase C activity but was not primarily mediated by arachidonic acid. The response of PKC to oxidative stress displayed a lack of Ca2+ dependence and was uncoupled from the activity of protein tyrosine kinases (PTK). Furthermore, the reduced activation requirements of PKC from cells treated with H2O2 were associated with shifts in elution profiles of PKC enzymatic activity after Mono-Q chromatography. These 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 intracellular events can intersect phosphorylation events mediated by PKC through the release of second messengers as well as direct changes in PKC activation requirements. Moreover, redox regulation of PKC is distinct from T cell receptor signaling in that the activity of PKC is uncoupled from the regulatory influences of PTK.