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

Yu Jiang1, Bai-Xue Wang1, Yi Xie1

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

Cell Biochemistry and Biophysics
|August 7, 2023
PubMed

Insights

Mixed lineage kinase 3 (MLK3) is primarily found in the cytoplasm and nucleus. Phosphorylation at Thr277/Ser281 activates MLK3, promoting oxidative stress and apoptosis, but does not affect its subcellular localization.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Mixed lineage kinase 3 (MLK3) activation by phosphorylation at Thr277/Ser281 triggers apoptotic pathways and cell injury.
  • Previous studies indicated MLK3 localization in the cytoplasm and nucleus of ovarian cells, with Thr477 phosphorylation involved in cytoplasmic retention.
  • The subcellular distribution of MLK3 in other cell types and the role of Thr277/Ser281 phosphorylation in its localization remained largely unknown.

Purpose of the Study:

  • To investigate the subcellular distribution of MLK3 in human embryonic kidney (HEK293T) and murine hippocampal neuronal (HT22) cell lines.
  • To determine if MLK3 phosphorylation at Thr277/Ser281 influences its subcellular localization.
  • To elucidate the role of MLK3 in oxidative stress and apoptosis.

Main Methods:

  • Bioinformatics analysis to predict MLK3 distribution.
  • Western blot analysis to detect endogenous and overexpressed MLK3 in HEK293T and HT22 cells.
  • Use of phosphorylation-deficient (T277A/S281A) and phosphorylation-mimic (T277E/S281E) MLK3 mutants.
  • Hydrogen peroxide (H2O2) stimulation and N-acetylcysteine treatment to induce and inhibit oxidative stress.
  • Measurement of intracellular reactive oxygen species (ROS) and cell apoptosis.

Main Results:

  • Bioinformatics analysis predicted, and experiments confirmed, that MLK3 is predominantly localized in the cytoplasm and nucleus of HEK293T and HT22 cells.
  • Phosphorylation of MLK3 at Thr277/Ser281 did not alter its subcellular distribution, as wild-type, phosphorylation-deficient, and phosphorylation-mimic mutants showed similar localization patterns.
  • H2O2-induced MLK3 phosphorylation was attenuated by N-acetylcysteine.
  • Overexpression of wild-type MLK3 increased ROS production and apoptosis, effects that were enhanced by the T277E/S281E mutant but not the T277A/S281A mutant.

Conclusions:

  • MLK3 is distributed in both the cytoplasm and nucleus in HEK293T and HT22 cells.
  • Phosphorylation at Thr277/Ser281 is not involved in regulating MLK3 subcellular localization.
  • Activated MLK3 plays a critical role in the positive feedback loop of oxidative stress-induced injury and apoptosis.
Keywords:
CytoplasmMLK3NucleusPhosphorylationReactive oxygen species.Subcellular distribution

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