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Mol. Cells 2013; 36(4): 322-332

Published online October 31, 2013

https://doi.org/10.1007/s10059-013-0114-x

© The Korean Society for Molecular and Cellular Biology

Phosphorylation of DYNLT1 at Serine 82 Regulates Microtubule Stability and Mitochondrial Permeabilization in Hypoxia

Xue Xu, Qiong Zhang, Jiong-yu Hu, Dong-xia Zhang, Xu-pin Jiang, jie-zhi Jia, Jing-ci Zhu, and Yue-sheng Huang

1School of Nursing, The Third Military Medical University, Chongqing, China, 2Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, The Third Military Medical University, Chongqing 400038, China

Received: April 11, 2013; Revised: July 9, 2013; Accepted: August 22, 2013

Abstract

Hypoxia-induced microtubule disruption and mitochondrial permeability transition (mPT) are crucial events leading to fatal cell damage and recent studies showed that microtubules (MTs) are involved in the modulation of mitochondrial function. Dynein light chain Tctex-type 1 (DYNLT1) is thought to be associated with MTs and mitochondria. Previously we demonstrated that DYNLT1 knockdown aggravates hypoxia-induced mitochondrial permeabilization, which indicates a role of DYNLT1 in hypoxic cytoprotection. But the underlying regulatory mechanism of DYNLT1 remains illusive. Here we aimed to investigate the phosphorylation alteration of DYNLT1 at serine 82 (S82) in hypoxia (1% O2). We therefore constructed recombinant adenoviruses to generate S82E and S82A mutants, used to transfect H9c2 and HeLa cell lines. Development of hypoxia-induced mPT (MMP examining, Cyt c release and mPT pore opening assay), hypoxic energy metabolism (cellular viability and ATP quantification), and stability of MTs were examined. Our results showed that phosph-S82 (S82-P) expression was increased in early hypoxia; S82E mutation (phosphomimic) aggravated mitochondrial damage, ele-vated the free tubulin in cytoplasm and decreased the cellular viability; S82A mutation (dephosphomimic) seemed to diminish the hypoxia-induced injury. These data suggest that DYNLT1 phosphorylation at S82 is involved in MTs and mitochondria regulation, and their interaction and cooperation contribute to the cellular hypoxic tolerance. Thus, we provide new insights into a DYNLT1 mechanism in stabilizing MTs and mitochondria, and propose a potential therapeutic target for hypoxia cytoprotective studies.

Keywords DYNLT1, energy metabolism, hypoxia, microtubule, mPT, phosphorylation

Article

Research Article

Mol. Cells 2013; 36(4): 322-332

Published online October 31, 2013 https://doi.org/10.1007/s10059-013-0114-x

Copyright © The Korean Society for Molecular and Cellular Biology.

Phosphorylation of DYNLT1 at Serine 82 Regulates Microtubule Stability and Mitochondrial Permeabilization in Hypoxia

Xue Xu, Qiong Zhang, Jiong-yu Hu, Dong-xia Zhang, Xu-pin Jiang, jie-zhi Jia, Jing-ci Zhu, and Yue-sheng Huang

1School of Nursing, The Third Military Medical University, Chongqing, China, 2Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, The Third Military Medical University, Chongqing 400038, China

Received: April 11, 2013; Revised: July 9, 2013; Accepted: August 22, 2013

Abstract

Hypoxia-induced microtubule disruption and mitochondrial permeability transition (mPT) are crucial events leading to fatal cell damage and recent studies showed that microtubules (MTs) are involved in the modulation of mitochondrial function. Dynein light chain Tctex-type 1 (DYNLT1) is thought to be associated with MTs and mitochondria. Previously we demonstrated that DYNLT1 knockdown aggravates hypoxia-induced mitochondrial permeabilization, which indicates a role of DYNLT1 in hypoxic cytoprotection. But the underlying regulatory mechanism of DYNLT1 remains illusive. Here we aimed to investigate the phosphorylation alteration of DYNLT1 at serine 82 (S82) in hypoxia (1% O2). We therefore constructed recombinant adenoviruses to generate S82E and S82A mutants, used to transfect H9c2 and HeLa cell lines. Development of hypoxia-induced mPT (MMP examining, Cyt c release and mPT pore opening assay), hypoxic energy metabolism (cellular viability and ATP quantification), and stability of MTs were examined. Our results showed that phosph-S82 (S82-P) expression was increased in early hypoxia; S82E mutation (phosphomimic) aggravated mitochondrial damage, ele-vated the free tubulin in cytoplasm and decreased the cellular viability; S82A mutation (dephosphomimic) seemed to diminish the hypoxia-induced injury. These data suggest that DYNLT1 phosphorylation at S82 is involved in MTs and mitochondria regulation, and their interaction and cooperation contribute to the cellular hypoxic tolerance. Thus, we provide new insights into a DYNLT1 mechanism in stabilizing MTs and mitochondria, and propose a potential therapeutic target for hypoxia cytoprotective studies.

Keywords: DYNLT1, energy metabolism, hypoxia, microtubule, mPT, phosphorylation

Mol. Cells
Aug 31, 2022 Vol.45 No.8, pp. 513~602
COVER PICTURE
Cryo-EM structure of human porphyrin transporter ABCB6 (main figure) shows that binding of hemin (inset, magenta) in concert with two glutathione molecules (cyan) primes ABCB6 for high ATP turnover (Kim et al., pp. 575-587).

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