No abstract

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

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