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Distinct intra-mitochondrial localizations of pro-survival kinases and regulation of their functions by DUSP5 and PHLPP-1.
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease ( IF 6.2 ) Pub Date : 2020-05-30 , DOI: 10.1016/j.bbadis.2020.165851
Wataru Ohwada 1 , Masaya Tanno 1 , Toshiyuki Yano 1 , Sang-Bing Ong 2 , Koki Abe 1 , Tatsuya Sato 3 , Atsushi Kuno 4 , Takayuki Miki 1 , Hirohito Sugawara 1 , Yusuke Igaki 1 , Tetsuji Miura 1
Affiliation  

ERK and Akt have been shown to regulate cell sensitivity to death-inducing stress by phosphorylating GSK-3β, 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-3β 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-3β 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-3β 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-3β. 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-3β regulation and that recruitment of DUSP5 as well as PHLPP-1 to mitochondria contributes to ROS-induced termination of the protective signaling.



中文翻译:

生存前激酶的不同线粒体内定位及其通过DUSP5和PHLPP-1对其功能的调节。

ERK和Akt已显示可通过磷酸化GSK-3β来调节细胞对死亡诱导应激的敏感性,GSK-3β是线粒体通透性转变阈值的主要调节剂。在这里,我们检查了促生存激酶的线粒体内定位及其受磷酸酶的调节。从HEK293或H9c2细胞分离的线粒体进行了胰蛋白酶消化,免疫印迹显示GSK-3β和ERK主要位于外膜(OM)中,而Akt在OM,内膜(IM)和内膜中处于相当水平。矩阵。IGF-1处理可增加基质中Akt的蛋白水平,而OM中ERK和GSK-3β的蛋白水平会增加。同时,IGF-1处理可提高IM和基质中的Thr202 / Tyr204-ERK水平以及OM,IM和基质中的Ser473-Akt和Ser9-GSK-3β水平。通过使用抗霉素A将细胞暴露于活性氧(ROS),可增加OM中DUSP5和PHLPP-1的水平,并诱导Akt,ERK和GSK-3β的去磷酸化。通过用Percoll梯度离心法纯化的线粒体以及用GFP标记的DUSP5转染细胞,实验证实了DUSP5的线粒体定位。敲低DUSP5或PHLPP-1均可增加线粒体中Thr202 / Tyr204-磷酸化ERK和Ser473-磷酸化Akt的水平。siRNA介导的DUSP5敲低可抑制抗霉素A诱导的细胞死亡。

更新日期:2020-05-30
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