Ubiquitin Ligase RNF20/40 Facilitates Spindle Assembly And Promotes Breast Carcinogenesis Through Stabilizing Motor Protein Eg5

Objective:1.To explore the molecular detail involved in the interaction between Eg5 and RNF20/40 and investigate whether the RNF20/40 complex catalyzes the ubiquitination of Eg5.2.To investigate the role of RNF20/40 in mitotic spindle assembly.3.To explore the role of the RNF20/40-Eg5 axis in breast carcinogenesis.Methods and results:This project was divided into four part:Part one: In an effort to further explore the biological activity of RNF20/40 complex,affinity purification and mass spectrometry were applied to identify proteins that are potentially associated with RNF20/40 in vivo.We found that Eg5,a cellular motor protein,was co-purified with RNF20,suggesting a physical association of Eg5 with the RNF20/40 complex in cells.And we validated affinity purification results by co-immunoprecipitation and GST pull-down assay.In order to gain a functional insight into the physical association between the RNF20/40 complex and Eg5,we examined the cellular localization of RNF20/40 and Eg5.And we found that RNF20/40 was co-localized with Eg5 throughout every stage of mitosis and was especially enriched at spindle poles.Collectively,these results indicate that the ubiquitin ligase RNF20/40 complex is physically associated with the motor protein Eg5 at spindle poles during mitosis.Part two: The physical association between the RNF20/40 complex and Eg5 prompted us to investigate whether the RNF20/40 complex catalyzes the ubiquitination of Eg5.We analyzed the protein using liquid chromatography-tandem mass spectrometry(LCMS/MS)and detected a +114-Dalton mass shift for K745 of the Eg5 protein retrieved from the Ub-HA-Eg5 band,indicating that Eg5 is monoubiquitinated at K745 in cells.To support RNF20/40-mediated Eg5 monoubiquitination,an in vitro ubiquitination assay was performed.Cycloheximide(CHX)chase assays showed a reduced Eg5 half-life during mitosis in RNF20,or RNF40,depleted cells.To further investigate the hypothesis that the RNF20/40complex-mediated Eg5 monoubiquitination protects Eg5 from proteasome-dependent degradation,MCF-7 cells were transfected with RNF20 or RNF40 specific si RNAs and treated with or without MG132.Taken together,these results indicate that the RNF20/40-mediated monoubiquitination promotes Eg5 stability.Part three: The observations that the RNF20/40 complex is co-localized with Eg5 at spindle poles during mitosis and regulates Eg5 stability prompted us to investigate the role of RNF20/40 in mitotic spindle assembly.We demostrated that the RNF20/40 complex is functionally implemented in mitotic spindle assembly through regulating Eg5 stability.MTT assays and colony formation assays showed that knockdown of either RNF20/40 or Eg5 resulted in a significant decrease in the viability of MCF-7cells.In addition,flow cytometry analysis indicated that either RNF20/40 or Eg5 depletion,or Eg5 activity inhibition by monastrol led to a remarkable increase in G2/M cell percentage and apoptosis of MCF-7 cells.These data indicate that the RNF20/40-Eg5 pathway is essential for the growth and survival of breast cancer cells.Part four: To further explore the role of the RNF20/40-Eg5 axis in breast carcinogenesis,breast tumors developed from MCF-7 cells were transplanted into nude mice.The results indicated that tumor growth was significantly suppressed in athymic mice which had received tumors with either RNF20,RNF40,Eg5 knockdown,or monastrol treatment.Immunohistochemistry(IHC)staining of RNF20,RNF40,and Eg5 suggests a concurrent up-regulation of RNF20,RNF40,and Eg5 in breast cancer.Conclusion:This study report that the RNF20/40 complex interacts with the motor protein Eg5 during mitosis and participates in spindle assembly.We show that the RNF20/40 complex monoubiquitinates and stabilizes Eg5.Loss of RNF20/40 results in spindle assembly defects,cell cycle arrest and apoptosis.Consistently,depletion of either RNF20/40 or Eg5 suppresses breast cancer in vivo.Significantly,RNF20/40 and Eg5 are concurrently upregulated in human breast carcinomas and high Eg5 expression is associated with poorer overall survival of patients with luminal A,or B,breast cancer.Our study uncovers an important spindle assembly role of the RNF20/40 complex,and implicates the RNF20/40-Eg5 axis in breast carcinogenesis,supporting the pursuit of these proteins as potential targets for breast cancer therapeutic interventions.