Superoxide-induced Stimulation of Protein Kinase C via Thiol Modification and Modulation of Zinc Content

Abstract

We investigated the effects of mild oxidation on protein kinase C (PKC) using the xanthine/xanthine oxidase system of generating superoxide. Exposure of various PKC preparations to superoxide stimulated the autonomous activity of PKC. Similarly, thiol oxidation increased autonomous PKC activity, consistent with the notion that superoxide stimulates PKC via thiol oxidation. The superoxide-induced stimulation of PKC activity was partially reversed by reducing agents, suggesting that disulfide bond formation contributed to the oxidative stimulation of PKC. In addition, superoxide increased the autonomous activity of the ␣, ␤ II , ⑀, and PKC isoforms, all of which contain at least one cysteinerich region. Taken together, our observations suggested that superoxide interacts with PKC at the cysteine-rich region, zinc finger motif of the enzyme. Therefore, we examined the effects of superoxide on this region by testing the hypothesis that superoxide stimulates PKC by promoting the release of zinc from PKC. We found that a zinc chelator stimulated the autonomous activity of PKC and that superoxide induced zinc release from an PKC-enriched enzyme preparation. In addition, oxidized PKC contained significantly less zinc than reduced PKC. Finally, we have isolated a persistent, autonomously active PKC by DEAE-cellulose column chromatography from hippocampal slices incubated with superoxide. Taken together, these data suggest that superoxide stimulates autonomous PKC activity via thiol oxidation and release of zinc from cysteine-rich region of PKC.

The Requirement of Reversible Cysteine Sulfenic Acid Formation for T Cell Activation and Function

“…Although cysteine oxidation inhibits the function of some proteins, in others it promotes activation. In the case of protein kinase C, oxidation by a superoxide stimulates enzymatic activity by thiol oxidation and the subsequent release of zinc from a cysteine-rich region in the amino terminus (50). Thus by modifying one amino acid in a reversible manner, cells are able to modulate signaling.…”

Superoxide as a Messenger of Endothelial Function

“…Knapp and Klann (2000) had reported an • O 2 -stimulated PKC activation via thiol oxidation. On the other hand studies on the multicomponent phagocyte 7-9 and endothelial NAD(P)H oxidase (Fontayne et al, 2002, Guzik et al 2002 Treatment of vessels from diabetic animals with SOD improved endothelium-dependent relaxations (Tesfamariam et al, 1992) and the use of vitamin C in patients with non-insulin (NIDDM) and insulin-dependent diabetes mellitus (IDDM), increased the endotheliumdependent vasodilation in the forearm circulation (Ting et al, 1996;Timimi et al, 1998).…”

A redox cycle within the cell cycle: ring in the old with the new

“…The H 2 O 2 -dependent activation of the MAPK pathway can also activate antioxidant enzymes or other detoxifying genes like GCLC (glutamate-cysteine ligase, catalytic subunit) via the antioxidant response elements found in the promoter regions of these enzymes . PKC enzyme activity can also be increased by exposure to O 2 À , inducing disulfide bond formation and release of zinc from the cysteine-rich region of the enzyme (Knapp and Klann, 2000). In addition to the redox state altering kinase activity, regulation of protein tyrosine phosphatases and low molecular weight phosphotyrosine phosphatases by ROS has been extensively studied (Chiarugi et al, 2003).…”