title: E6-AP/UBE3A protein acts as a ubiquitin ligase toward SOX9 protein.
abstract: SOX9 is a transcription factor that acts as a key regulator at various stages of cartilage differentiation. There is ample evidence that intracellular SOX9 protein levels are tightly regulated both by sumoylation and by degradation through the ubiquitin-proteasome pathway. Using a proteomics approach, here we report the identification of a SOX9-binding protein, E6-AP/UBE3A, that may act as a ubiquitin ligase toward Sox9. E6-AP bound SOX9 through the region consisting mostly of its high mobility group domain in vitro. In nuclear lysates, FLAG-tagged E6-AP coprecipitated with Sox9 and its high mobility group domain. This finding was estimated using nuclear lysates from a chondrocytic cell line that endogenously expresses E6-AP and SOX9. Accordingly, ectopically expressed E6-AP and SOX9 colocalized in the nucleus. We show that E6-AP ubiquitinates SOX9 in vitro and in vivo and that SOX9 levels are enhanced after addition of the proteasome inhibitor bortezomib. Similar, siRNA knockdown of E6-AP and the E2 ligase Ubc9 increased cellular SOX9 amounts, supporting the notion that SOX9 may be ubiquitinated in hypertrophic chondrocytes by E6-AP and degraded by proteasomes. This is in accordance with the distribution of SOX9 levels, which are high in proliferating and prehypertrophic chondrocytes but low in hypertrophic chondrocytes, whereas E6-AP levels are high in hypertrophic chondrocytes and low in prehypertrophic chondrocytes. Furthermore, E6-AP-deficient mice showed SOX9 accumulation in chondrocytes and the brain. These findings support the concept that E6-AP regulates SOX9 levels in developing cartilage by acting as a ubiquitin ligase.
authors.affiliation: From the Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku Okayama 700-8525, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Hattori
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kishino
authors.forename: Shelley
authors.initials: S
authors.lastname: Stephen
authors.forename: Heidi
authors.initials: H
authors.lastname: Eberspaecher
authors.forename: Sayumi
authors.initials: S
authors.lastname: Maki
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takigawa
authors.forename: Benoit
authors.initials: B
authors.lastname: de Crombrugghe
authors.forename: Hideyo
authors.initials: H
authors.lastname: Yasuda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D019902:Chondrocytes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D011485:Protein Binding; D040901:Proteomics; D034741:RNA, Small Interfering / Q000235:genetics; D055758:SOX9 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D034741:RNA, Small Interfering; D055758:SOX9 Transcription Factor; D044763:Ubiquitin-Conjugating Enzymes; C502429:Ube3a protein, mouse; D044767:Ubiquitin-Protein Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9
keywords: Chondrocytes; E3 Ubiquitin Ligase; E6-AP; Protein Degradation; Proteomics; SOX9; UBE3A; Ubiquitin; Ubiquitination