MLK3 localizes mainly to the cytoplasm and promotes oxidative stress injury via a positive feedback loop

Activation of mixed lineage kinase 3 (MLK3) by phosphorylation at Thr277/Ser281 stimulates downstream apoptotic pathways and ultimately leads to cell injury. MLK3 is reported to localize to both the cytoplasm and nucleus in human ovarian cancer cells and immortalized ovarian epithelial cells (T80 and T90 cells), and phosphorylation at Thr477 is required for the cytoplasmic retention of MLK3 in T80 cells. However, the subcellular distribution of MLK3 in other cell types has rarely been reported, and whether phosphorylation of MLK3 at Thr277/Ser281 affects its subcellular distribution is unknown. Here, our bioinformatics analysis predicted that MLK3 was mainly distributed in the cytoplasm and nucleus. In the human HEK293T embryonic kidney cell line and murine HT22 hippocampal neuronal cell line, endogenous MLK3 was more abundant in the cytoplasm and less abundant in the nucleus. In addition, overexpressed Myc-tagged MLK3 and EGFP-tagged MLK3 were also observed to localize mainly to the cytoplasm. MLK3 that was activated by phosphorylation at Thr277/Ser281 was mainly distributed in the cytoplasm, and phosphorylation deficient (T277A/S281A) and mimic (T277E/S281E) mutants both showed distributions similar to that of wild type (wt) MLK3, further proving that phosphorylation at Thr277/Ser281 was not involved in regulating MLK3 subcellular localization. In HEK293T cells, H 2 O 2 stimulation accelerated MLK3 phosphorylation (activation), and this phosphorylation was reduced by the antioxidant N-acetylcysteine in a dose-dependent manner. Overexpressing wt MLK3 promoted the production of intracellular reactive oxygen species and increased cell apoptosis, both of which were enhanced by the phosphorylation-mimic (T277E/S281E) MLK3 variant but not by the phosphorylation-deficient (T277A/S281A) MLK3 variant. These findings provided additional evidence for the cytoplasmic and nuclear distribution of MLK3 in HEK293T cells or HT22 cells and revealed the pivotal role of MLK3 in the positive feedback loop of oxidative stress injury.