Mitochondria are highly motile organelles that constantly undergo fission and fusion. Impairment of mitochondrial dynamics is associated with mitochondrial dysfunction and is frequently linked to the pathogenesis of neurodegenerative diseases and cancer. We have previously shown that biallelic inactivation of the suppressor of cytokine signaling 6 (SOCS6) is a frequent event in human gastric cancer, and that overexpression of SOCS6 inhibits cell growth as well as colony formation in soft agar, suggesting a potential role of SOCS6 as a tumor suppressor. We also showed that SOCS6 is targeted to mitochondria, and induces mitochondrial fragmentation accompanied with intrinsic apoptosis. SOCS6 induces mitochondrial fragmentation is in part mediated through its interaction with DRP1 and regulation of DRP1 fission activity. In this study, we further showed that SOCS6 interacts with the Elongin B/C and Cullin 5 forming an ECS E3 ubiquitin ligase complex, and that formation of an intact ECS E3 ligase complex is important for SOCS6-mediated mitochondrial fragmentation. First, the interaction of SOCS6 with Elongin B/C and/or Cullin 5 prolonged SOCS6 stability. Second, mutations of the conserved Leu500 and Cys504 residues in SOCS6 BC-box which are critical for Elongin B/C binding yielded a SOCS6 mutant failed to induce mitochondrial fragmentation and apoptosis. Most importantly, ablation of Elongin C activity protected cells from SOCS6-mediated mitochondrial fission. Taken together, our data suggest an important role of SOCS6 in modulating mitochondrial dynamics that is dependent on ECS E3 ubiquitin ligase complex activity. Citation Format: Huan-Yu Lin, Shiu-Ting Lin, Mei-June Wang, Jeou-Yuan Chen. Suppressor of cytokine signaling 6 (SOCS6) promotes mitochondrial fission through E3 ubiquitin ligase complex activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1699. doi:10.1158/1538-7445.AM2013-1699

Mitochondria are highly motile organelles that constantly undergo fission and fusion. Impairment of mitochondrial dynamics is associated with mitochondrial dysfunction and frequently linked to the pathogenesis of neurodegenerative diseases and cancer. We have previously shown that loss-of-function of suppressor of cytokine signaling 6 (SOCS6) is a frequent event in human gastric cancer. In this study, we recapitulated the event of loss of SOCS6 in cancer-derived cell lines where endogenous SOCS6 was expressed, and we showed that elimination of SOCS6 expression delays UV-induced apoptosis process. By contrast, overexpression of SOCS6 in cancer cell lines that express endogenous SOCS6 at minimal levels promotes apoptosis. SOCS6 induces intrinsic apoptotic process and facilitates cytochrome c release by enhancing Bax conformational change, mitochondrial targeting and oligomerization. We further demonstrated that SOCS6 is a novel mitochondrial protein that regulates mitochondrial dynamics. By confocal microscopy, we showed that ectopically expressed SOCS6 is predominantly localized in mitochondria, and the carboxyl-terminal SOCS box is important for its mitochondrial localization. Localization of SOCS6 in mitochondria is accompanied with concomitant mitochondrial fragmentation, both of which can be reversed by coexpression of mitochondrial fusion proteins (Mfn1 and Mfn2) or a dominant-negative form of Drp1. Ectopic expression of SOCS6 severely impairs the dynamics and function of mitochondria. Taken together, our data suggest that SOCS6 is a novel mitochondrial protein that induces intrinsic apoptosis pathway through perturbing mitochondrial dynamics and facilitating Bax activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 180. doi:10.1158/1538-7445.AM2011-180

Mitochondria are highly motile organelles that constantly undergo fission and fusion. Impairment of mitochondrial dynamics is associated with mitochondrial dysfunction and frequently linked to the pathogenesis of neurodegenerative diseases and cancer. We have previously shown that loss-of-function of suppressor of cytokine signaling (SOCS) 6 is a frequent event in human gastric cancer and ectopic expression of SOCS6 sensitized cells to UV-induced apoptosis. In this study, we have identified SOCS6 as a novel mitochondrial protein that regulates mitochondrial dynamics. By confocal microscopy, we showed that ectopically expressed SOCS6 is predominantly localized in mitochondria, and that the carboxyl-terminal SOCS box is important for its mitochondrial localization. We further showed that localization of SOCS6 in mitochondria is accompanied with increased mitochondrial fragmentation with attenuated membrane potential, both of which can be reversed by coexpression of mitochondrial fusion proteins Mfn1 or Mfn2. Our data further suggested that SOCS6 interferes with mitochondrial dynamics by interrupting the fusion process. Firstly, SOCS6 expression severely impaired the dynamics and mobility of mitochondria in live cells as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Secondly, SOCS6 expression significantly hampered intermitochondrial transfer and fusion in the somatic hybrid produced by PEG-induced fusion. Furthermore, elimination of the expression of endogenous SOCS6 by RNA interference technique led to elongated mitochondria, and conferred cells increased resistance to UV-induced apoptosis. Taken together, our data suggest that SOCS6 is a mitochondrial targeting protein involved in the regulation of mitochondrial dynamics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1208.

SOCS proteins were initially identified as the suppressors of cytokine signaling. There are eight members in the SOCS family, each containing a central SH2 domain and a carboxyl-terminal SOCS box motif. Among the SOCS family proteins, CIS and SOCS1-3 have been shown to be expressed at elevated levels upon cytokine treatment, and the up-regulated proteins can then turn around to suppress the cytokine signaling through recruiting the phosphorylated signaling intermediates for ubiquitylation-mediated protein degradation, leading to the blockade of the signaling pathway. However, the involvement of other SOCS family proteins, including SOCS6, in protein ubiquitylation remains to be addressed. We have previously shown that biallelic inactivation of SOCS6 is a frequent event in human gastric cancer, and that overexpression of SOCS6 inhibits cell growth as well as colony formation, suggesting a potential role of SOCS6 as a tumor suppressor. We further demonstrated that SOCS6 is targeted to the mitochondria, and induces mitochondrial fragmentation accompanied with intrinsic apoptosis. In this study, we explored the possible linkage of SOCS6 to the formation of an E3 ubiquitin ligase complex. By GST-pulldown and immunoprecipitation assays, we showed that SOCS6 interacted with Elongin B/C and Cullin 5. At steady state, SOCS6 is expressed at relatively low level, but its level is increased in the presence of proteasome inhibitor MG132. In addition, ectopic expression of Elongin B/C and/or Cullin 5 also led to the stabilization of SOCS6 protein. We also showed that formation of an intact Elongin B/C-Cullin 5-SOCS (ECS) E3 ligase complex is important for SOCS6-mediated mitochondrial fragmentation. Mutations of the conserved Leu500 and Cys504 residues in the BC-box located in SOCS box motif yielded a SOCS6 mutant that not only lost the ability to interact with Elongin B/C but also failed to induce mitochondrial fragmentation and apoptosis. Most importantly, ablation of Elongin C activity protected cells from SOCS6-mediated mitochondrial fission. These data suggest an important role of SOCS6 in modulating mitochondrial dynamics mediated through ECS E3 ubiquitin ligase complex activity. We have also searched for substrates of SOCS6-mediated ECS E3 ubiquitin ligase complex by immunoprecipitation using an anti-K-ϵ-GG antibody followed by proteome study. The characterization of the ubiquitylated candidate proteins will be further discussed. Note: This abstract was not presented at the meeting. Citation Format: Shu-Chuan Chen, Huan-Yu Lin, Shiu-Ting Lin, Mei-Jung Wang, Jeou-Yuan Chen. The involvement of suppressor of cytokine signaling 6 (SOCS6) in the ECS E3 ubiquitin ligase complex. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4432. doi:10.1158/1538-7445.AM2014-4432