期刊
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE
卷 1866, 期 10, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.bbadis.2020.165851
关键词
Akt; DUSP5; ERK; GSK-3 beta; Mitochondria; PHLPP-1
资金
- Japan Society for the Promotion of Science, Tokyo, Japan [26461132, 17K09584]
- Uehara Memorial Foundation, Tokyo, Japan
- Grants-in-Aid for Scientific Research [17K09584, 26461132] Funding Source: KAKEN
ERK and Akt have been shown to regulate cell sensitivity to death-inducing stress by phosphorylating GSK-3 beta, a major modulator of the threshold for mitochondrial permeability transition. Here we examined intra-mitochondrial localization of the pro-survival kinases and their regulation by phosphatases. Stepwise trypsin digestion of mitochondria isolated from HEK293 or H9c2 cells was performed, and immunoblotting revealed that GSK-3 beta and ERK localized dominantly in the outer membrane (OM), while Akt resided at comparable levels in OM, the inner membrane (IM) and the matrix. Treatment with IGF-1 increased the protein level of Akt in the matrix, while ERK and GSK-3 beta protein levels were increased in OM. Simultaneously, IGF-1 treatment elevated the level of Thr202/Tyr204-phospho-ERK in IM and matrix and levels of Ser473-phospho-Akt and Ser9-phospho-GSK-3 beta in OM, IM and matrix. Exposing cells to reactive oxygen species (ROS) by using antimycin A increased the levels of DUSP5 and PHLPP-1 mainly in OM and induced dephosphorylation of Akt, ERK and GSK-3 beta. The mitochondrial localization of DUSP5 was confirmed by experiments with mitochondria purified by Percoll gradient centrifugation and by transfection of cells with GFP-tagged DUSP5. Knockdown of either DUSP5 or PHLPP-1 increased the levels of both Thr202/Tyr204-phospho-ERK and Ser473-phospho-Akt in mitochondria. Cell death induced by antimycin A was suppressed by siRNA-mediated knockdown of DUSP5. The results suggest that Akt and ERK in mitochondria show distinct intra-mitochondrial localization and crosstalk in GSK-3 beta regulation and that recruitment of DUSP5 as well as PHLPP-1 to mitochondria contributes to ROS-induced termination of the protective signaling.
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