Figure 4. Oxidative stress inhibits MEKK1 by inducing a DTT-reversible modification.

(A) Wild-type or C1238V catalytic domain MEKK1 (Δ1172) (left) or full-length ASK1 (right) were expressed in cells that were then treated with hydrogen peroxide (H₂O₂, 10 mM), NEM (1 mM) or menadione (250 μM) as indicated. Kinase was purified and activity measured using an in vitro kinase assay. Stimuli that induce oxidative stress inhibited wild-type MEKK1, but not the C1238V (‘CV’) mutant. ASK1 is not inhibited, but instead activated, by menadione. (B) Wild-type MEKK1 was isolated from cells treated as in (A) and then incubated in vitro with 10 mM DTT. The activity of the kinase was measured by an in vitro kinase assay. DTT treatment restored the activity of H₂O₂- or menadione-inhibited MEKK1. (C) Endogenous MEKK1 was immunoprecipitated from either untreated or menadione-treated LNCaP cells. The protein was left untreated or treated with 10 mM DTT in vitro, and the activity measured by an in vitro kinase assay. Endogenous MEKK1 was inhibited by menadione treatment and was partially re-activated by reduction with DTT. Abbreviations: autoP, autophosphorylation; WT, wild-type; CV, C1238V mutant.