In this study, we demonstrated that SOCS6 encodes a mitochondria targeting protein regulating mitochondrial morphology and apoptosis. Ectopic expression of SOCS6 induced extensive mitochondrial fragmentation, whereas silencing SOCS6 led to elongated mitochondria. SOCS6 promoted mitochondrial fragmentation in part through forming complex with DRP1 and PGAM5, attenuating DRP1 phosphorylation, and promoting DRP1 mitochondrial translocation. Concomitant with mitochondrial fragmentation, ectopic SOCS6 elicited Bax activation and apoptosis. In accordance with our previous findings that SOCS6 was frequently downregulated in gastric adenocarcinoma and ectopic expression of SOCS6 inhibited colony formation, we showed in this study that loss of SOCS6 delayed cytochrome c release and suppressed apoptosis.
SOCS6-mediated mitochondrial fragmentation could be due to reduced fusion, enhanced fission, or both. Mitochondrial fusion is initiated by mitofusin-mediated mitochondrial tethering. MFN1 and MFN2 form both homotypic and heterotypic complexes.9
SOCS6, MFN1, and MFN2 are all OMM proteins. However, SOCS6 does not interfere with mitofusin complex formation (data not shown) or affect mitofusin-mediated fusion (). In contrast, inhibition of DRP1 activity by a dominant-negative DRP1 or by shRNA-KD approach conferred cells increased resistance to SOCS6-mediated mitochondrial fragmentation (). These data suggest that SOCS6-induced mitochondrial fission is dependent on DRP1 activity. In support, we showed that ectopic SOCS6 promoted DRP1 mitochondrial localization by forming complexes with DRP1 and mitochondrial phosphatase PGAM5, leading to decreased DRP1-Ser637
phosphorylation ( and ). However, KD of PGAM5 significantly suppressed but not completely abrogated SOCS6-mediated mitochondrial fragmentation (), suggesting that activities other than that of PGAM5 are involved in SOCS6-mediated fragmentation.
Shifting the mitochondrial dynamic equilibrium toward fission or fusion by up- or down-regulation of proteins controlling fission and fusion processes confers to cells an increased sensitivity or resistance toward apoptosis.30
The mitochondrial targeting proteins endophilin B131
and multiple indicator cluster survey 1 (MICS1)32
regulate mitochondrial morphology and affect apoptosis. Ectopic expression of MICS1 induced mitochondrial elongation, and delayed cytochrome c
release and the progression of apoptosis.32
Endophilin B1, which is required for the modulation of normal mitochondrial outer membrane morphological dynamics, promoted Bax activation.31
In this study, we demonstrate that SOCS6 is a novel mitochondrial targeting protein that promotes mitochondrial fission. In addition, we showed that ectopic SOCS6 facilitated UV-induced apoptosis (), suggesting that cells expressing ectopic SOCS6 exhibited a lower threshold against apoptotic insults because of the reduced Δψm. Furthermore, ectopic SOCS6 induced Bax activation () and cell death (data not shown) in a dose-dependent manner, supporting the notion that mitochondrial fragmentation actively participates in apoptosis.
By immunofluorescence and biochemical analyses, we showed that SOCS6 is tightly associated with mitochondria exposed to the cytosol (). However, a putative mitochondrial targeting sequence was not detected in the N-terminus of SOCS6. The fact that the SOCS6-C mutant, which contains only the C-terminal half of the SOCS6 protein, mimicked the full-length protein to be localized to mitochondria () suggested the possibility of an internal mitochondrial targeting signal. Alternatively, posttranslational modification of proteins with glycosylphosphatidylinositol (GPI) moiety allows the anchorage of proteins to lipid bilayers. Using big-PI predictor for GPI modification site prediction, we identified a potential GPI modification site at the C-terminus of SOCS6 at Asn524. Mutagenesis was performed to generate a SOCS6-N524L mutant allele. Our data showed that the SOCS6-N524L mutant was no longer localized to mitochondria (data not shown). To our knowledge, SOCS6 is the first SOCS family protein reported to be located in the mitochondria. Further investigation is warranted to study the targeting of SOCS6 to mitochondria.
Like all SOCS family proteins, SOCS6 contains a central SH2 domain and a C-terminal SOCS box. The SOCS box was reported to act as a common binding domain for a large family of cullin-RING-based E3 ubiquitin protein ligase complexes.33
By functioning as an adaptor protein, SOCS protein links substrates to the Elongin BC-Cul2/Cul5-SOCS box protein (ECS) E3 ligase complexes for ubiquitylation and degradation.34
Recently, several E3 ligases were reported to regulate mitochondrial dynamics through ubiquitylation and proteasomal degradation.35
MARCH-V, membrane-associated ring finger (C3HC4) 5 (also called MITOL), bound to MFN2 and DRP1 and promoted ubiquitylation of DRP1.36
Parkin, an IBR-domain E3 ligase, induced the ubiquitylation and degradation of MFN1 and MFN2, and interacted with and ubiquitylated DRP1, thus promoting its degradation.37
In our study, we showed that an intact SOCS box is a prerequisite for SOCS6 mitochondrial localization and SOCS6-mediated mitochondrial function. According to X-ray crystallographic data,34, 35, 36
the SOCS box is a conserved structure fold with three core helices that mediate the interaction with other adaptor proteins of the ECS E3 ligase complexes. We showed that deletion of the H3 helix, which is believed to disturb the stability of SOCS box folding, abolished SOCS6-mediated mitochondrial fission and apoptosis (). SOCS6 was shown to act as a component of the ECS E3 ligase complex, interacting with c-KIT and regulating its ubiquitylation and turnover.38
Whether ECS-mediated protein ubiquitylation and degradation plays a role in SOCS6-mediated mitochondrial fragmentation and apoptosis remains to be determined.
In summary, we showed that SOCS6 is a novel mitochondrial morphogenic factor and an important component of the mitochondrial pathway for apoptosis. Our data showed that loss of SOCS6 confers cells increased antiapoptotic activity to cells, corroborating the important role of mitochondrial dynamics in malignant transformation and cancer formation. These results will allow us to investigate the role of SOCS6-mediated mitochondrial morphogenesis in other pathologies, including neurodegenerative and cardiovascular diseases and diabetes.