Fig. 2. Oxidative stress–induced MTK1 activation requires both oxidation and reduction of MTK1.

(A and B) HEK293 (A) and M57 cells (B) were stimulated with various concentrations of H₂O₂ for 10 min. Cell extracts were electrophoresed under nonreducing (−2ME) or reducing (+2ME) conditions, and probed for endogenous MTK1 (A) and Myc-MTK1 (B) by immunoblotting. Ox-MTK1, oxidized MTK1. (C) M57 cells were treated with H₂O₂ (1 mM). Cell extracts were electrophoresed under nonreducing conditions and immunoblotted with anti-Myc antibody (top). Immunoprecipitated Myc-MTK1 was electrophoresed under reducing conditions and probed with anti–P-MTK1 or anti-Myc antibodies (middle and bottom). (D) M57 cells were pretreated with the catalase inhibitor aminotriazol and stimulated with H₂O₂ (0.5 mM). Oxidation (top), phosphorylation (middle), and the expression level (bottom) of Myc-MTK1 were assessed as in (C). (E) Schematic diagram of MTK1 structure. GBD, GADD45-binding domain; AID, autoinhibitory domain; CCD, coiled-coil domain; KD, kinase domain. (F to H and J) HEK293 cells expressing Myc-MTK1 or its mutant derivatives (ΔGBD, 3C/S, C163S, C193S, or C218S) were treated with H₂O₂. Oxidation (F and G) and phosphorylation (H and J) of Myc-MTK1 were monitored as in (C). (I) Cos7 cells were cotransfected with Myc-MTK1 (WT, ΔGBD, or 3C/S) and GADD45β. Immunoprecipitated Myc-MTK1 was probed with anti–P-MTK1 or anti-Myc antibodies.