Sci. Signal., 4 September 2012
Apoptosis Linking Stress and Apoptosis to Impaired Mitochondrial Fusion
Science Signaling, AAAS, Washington, DC 20005, USA
Mechanistic links between cell stress and mitochondrial integrity remain elusive, despite both being implicated in apoptosis. Leboucher et al. found that the genotoxic chemotherapeutic agent doxorubicin caused fragmentation of mitochondria in U2OS cells. Doxorubicin caused a decrease in the abundance of the mitochondrial fusion protein mitofusin 2 (Mfn2) and an increase in amounts of ubiquitylated Mfn2. In cells exposed to doxorubicin, knockdown of Mfn2 increased the abundance of cleaved caspase-3, a marker of apoptosis. Pulse-chase metabolic labeling experiments showed that Mfn2 degraded faster in cells exposed to doxorubicin, and this increase in rate was abrogated by knockdown of c-Jun N-terminal kinase (JNK), which was activated by exposure of the cells to doxorubicin. Tandem mass spectrometry analysis of Mfn2 purified from U2OS uncovered a phosphorylation site at Ser27. Coimmunoprecipitation studies with an antibody specific to this site and in vitro kinase assays demonstrated that JNK phosphorylated Mfn2 at this site. In the presence of proteasome inhibitor MG132 and doxorubicin, the abundance of phosphorylated Mfn2 was increased, and knockdown of JNK abrogated the doxorubicin-induced increase in phosphorylation. A phosphomimetic S27D mutant of Mfn2 displayed an increased degradation rate in pulse-chase experiments and increased abundance of ubiquitylated species compared with wild-type or an unphosphorylatable S27A mutant. Exposure of cells to doxorubicin shifted the wild-type Mfn2 degradation rate to that of the S27D mutant. Overexpression of wild-type Mfn2 or either mutant decreased the amount of doxorubicin-induced mitochondrial fragmentation, caspase-3 activation, or cell death (cells positive for annexin V), with the most stable S27A mutant most effective and the least stable S27D mutant least effective. The S27D, but not the S27A, mutant coimmunoprecipitated with a catalytically inactive mutant of Huwe1, an E3 ubiquitin ligase. Knockdown of Huwe1 inhibited doxorubicin-induced Mfn2 degradation, mitochondrial fragmentation, and cell death, but cell death occurred when Mfn2 was also knocked down, suggesting that Mfn2 was the relevant target of Huwe1 in this process. These studies identify a signaling axis whereby genotoxic stress prevents mitochondrial fusion by inducing JNK phosphorylation of Mfn2, which associates with Huwe1, leading to Mfn2 ubiquitylation and proteasomal degradation, thus precipitating mitochondrial fragmentation and apoptosis.
G. P. Leboucher, Y. C. Tsai, M. Yang, K. C. Shaw, M. Zhou, T. D. Veenstra, M. H. Glickman, A. M. Weissman, Stress-induced phosphorylation and proteasomal degradation of mitofusin 2 facilitates mitochondrial fragmentation and apoptosis. Mol. Cell. 47, 547–557 (2012). [PubMed]
Citation: E. Andrianantoandro, Linking Stress and Apoptosis to Impaired Mitochondrial Fusion. Sci. Signal. 5, ec234 (2012).
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