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

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Abstract

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, H2O2 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.

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Fig. 1: MLK3 is mostly localized to the cytoplasm with a small amount of protein in the nucleus in HEK293T cells and HT22 cells.
Fig. 2: Overexpressed MLK3-EGFP is mainly located in the cytoplasm of HEK293T cells.
Fig. 3: Phosphorylated MLK3 (Thr277/Ser281) is mainly located in the cytoplasm of HEK293T cells.
Fig. 4: H2O2 promotes MLK3 phosphorylation at T277/S281, which in turn leads to intracellular ROS release.
Fig. 5: MLK3 phosphorylation at T277/S281 promotes cell apoptosis.

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Abbreviations

MLK3:

mixed lineage kinase 3

MAPKKKs:

mitogen-activated protein kinase kinase kinases

JNK:

c-Jun N-terminal kinase

ERK:

extracellular signal-regulated kinase

Thr:

threonine

Ser:

serine

ROS:

reactive oxygen species

ANOVA:

one-way analysis of variance

AAs:

amino acids

Myc-MLK3:

Myc-tagged MLK3

MLK3-EGFP:

EGFP-fused MLK3

T277/S281:

Thr277/Ser281.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (32100769 to M.L. and 81100852 to C.-P.D.), the Natural Science Foundation of the Jiangsu Higher Education Institutions (20KJA310010 to C.-P.D.), and Xuzhou Medical University (D2020054 to M.L.)

Author contributions

Y.J., M.L., and C.P.D. designed research; Y.J., B.X.W., Y.X., and L.M. performed biochemical research and analyzed data; M.L. and C.P.D. wrote the paper and provided supervision and funding.

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  1. These authors contributed equally: Yu Jiang, Bai-Xue Wang

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  1. Research Center for Biochemistry and Molecular Biology, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou, Jiangsu, 221004, China

    Yu Jiang, Bai-Xue Wang, Yi Xie, Li Meng, Meng Li & Cai-Ping Du

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Jiang, Y., Wang, BX., Xie, Y. et al. MLK3 localizes mainly to the cytoplasm and promotes oxidative stress injury via a positive feedback loop. Cell Biochem Biophys 81, 469–479 (2023). https://doi.org/10.1007/s12013-023-01159-8

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