title: Cdc20 mediates D-box-dependent degradation of Sp100.
abstract: Cdc20 is a co-activator of the anaphase-promoting complex/cyclosome (APC/C complex), which recruits substrates at particular phases of the cell cycle and mediates their degradation. Sp100 is a PML-NB scaffold protein, which localizes to nuclear particles during interphase and disperses from them during mitosis, participates in viral resistance, transcriptional regulation, and apoptosis. However, its metabolism during the cell cycle has not yet been fully characterized. We found a putative D-box in Sp100 using the Eukaryotic Linear Motif (ELM) predictor database. The putative D-box of Sp100 was verified by mutational analysis. Overexpression of Cdc20 resulted in decreased levels of both endogenous Sp100 protein and overexpressed Sp100 mRNA in HEK 293 cells. Only an overexpressed D-box deletion mutant of Sp100 accumulated in HEK293 cells that also overexpressed Cdc20. Cdc20 knockdown by cdc20 specific siRNA resulted in increased Sp100 protein levels in cells. Furthermore, we discovered that the Cdc20 mediated degradation of Sp100 is diminished by the proteasome inhibitor MG132, which suggests that the ubiquitination pathway is involved in this process. However, unlike the other Cdc20 substrates, which display oscillating protein levels, the level of Sp100 protein remains constant throughout the cell cycle. Additionally, both overexpression and knockdown of endogenous Sp100 had no effect on the cell cycle. Our results suggested that sp100 is a novel substrate of Cdc20 and it is degraded by the ubiquitination pathway. The intact D-box of Sp100 was necessary for this process. These findings expand our knowledge of both Sp100 and Cdc20 as well as their role in ubiquitination.
authors.affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Wang
authors.forename: Ke-min
authors.initials: KM
authors.lastname: Li
authors.forename: Cai-hong
authors.initials: CH
authors.lastname: Zhou
authors.forename: Jing-lun
authors.initials: JL
authors.lastname: Xue
authors.forename: Chao-neng
authors.initials: CN
authors.lastname: Ji
authors.forename: Jin-zhong
authors.initials: JZ
authors.lastname: Chen
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D034961:Antigens, Nuclear / Q000378:metabolism*; D001324:Autoantigens / Q000378:metabolism*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D057809:HEK293 Cells; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics; D013379:Substrate Specificity; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D034961:Antigens, Nuclear; D001324:Autoantigens; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D034741:RNA, Small Interfering; C066642:SP100 protein, human; C577488:CDC20 protein, human; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome