CRL5-SOCS6 Complex Regulates MTORC2 Function By Targeting Sin1 For Degradation

The mammalian target of rapamycin(mTOR)is a serine/threonine kinase belonging to the phosphatidylinositol kinase-related kinase(PIKK)family,and it is highly conserved in all eukaryotes and regulates many major cellular processes such as cell growth and proliferation.By interacting with different proteins,mTOR forms two structurally similar but distinct complexes,namely mTORC1 and mTORC2.Composed by Sin1,Rictor,m LST8,and mTOR,mTORC2 mainly regulates cell survival and proliferation by phosphorylating hydrophobic motif of AKT and possibly others members of the AGC kinase family.Ser473 site phosphorylation induced by mTORC2 is necessary to AKT1 full activation,which plays an important role in cell proliferation and survival.As a key component of mTORC2,Sin1 is essential for mTORC2 assemble and its kinase activity.For instance,T86 and T398phosphorylation of Sin1 impair the integrity of mTORC2 complex and suppress AKT activation,and the interaction between Ptd Ins(3,4,5)P₃(PIP3)and PH domain of Sin1 results in activation of mTORC2 complex.Thus Sin1 is a key effector between cellular physiological signal and mTORC2 kinase function.However,the upstream regulatory mechanism of Sin1 stability is largely unclear.We first treated cells with MG132,Baf-A1 and MLN4924,and identified Cullin Ring Ligase(CRL)family as the possible ubiquitin E3 ligase that mediates Sin1degradation.Furthermore,we performed si RNA experiments and found that CUL5,but not other cullin proteins functioning in regulating Sin1.CRL5 complexs are composed of CUL5,ELOB,ELOC,RBX2 and SOCS-box proteins.We designed 50sh RNA targeting over 20 SOCS-box proteins to screen for the corresponding SOCS-box protein.Then Co-Immunoprecipitation screening experiments were aslo performened.And SOCS6 was identified as the key SOCS-box protein to degrade Sin1.As the substrate of mTORC2,AKT activity is increased in pancreatic cancer cells.In the mean time,SOCS6 expression is decreased.So we used pancreatic cancer cells PANC1 and Bxpc3 as the research system.After PANC1 or Bxpc3 cells were infected by sg RNA lentivirus to deplete SOCS6,we found that Sin1 protein,as well as the level of pS473-AKT1 is increased.When treated with Cisplatin or Gemcitabine,deletion of SOCS6 increased cell survival.When we further knocked down Sin1,the level of pS473-AKT1 decreased and the drug resistance was reversed.To evaluate the functional impact of SOCS6 in regulating tumorigenesis,SOCS6-depleted PANC1cells and vector treated PANC-1 cells were subcutaneously implanted into nude mice to perform xenograft tumorigenesis assay.Strikingly,the mice transplanted with SOCS6-depleted PANC1 cells developed much bigger tumors than the mice injected with control PANC1 cells.Tumors were harvested to generate lysates for immunoblot,and the results indicated that the phospho-Ser473 AKT1 signal and the Sin1expression level in tumors from sg SOCS6 PANC-1 cells were indeed much higher than the tumors derived from control PANC-1 cells.Collectively,these results suggested a crucial function of SOCS6 in regulating cell survival possibly via the mTORC2-AKT axis through degrading Sin1.In a word,our findings revealed a crucial regulation of mTORC2 activity via Sin1 stability regelation by SOCS6,and it could expang our understanding on the mechanisms underlying cell survival and proliferation in physiological and pathological settings.