Hattori T, Kishino T, Stephen S, Eberspaecher H, Maki S, Takigawa M, de Crombrugghe B, Yasuda H

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 on the identification of ...

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a SOX9-binding protein E6-AP/UBE3A which may act as a ubiquitin ligase towards Sox9. E6-AP bound SOX9 through the region consisting mostly of its HMG domain in vitro. In nuclear lysates flag-tagged E6-AP coprecipitated with Sox9 and its HMG domain. This finding was estimated using nuclear lysates from chondrocytic cell line which 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 brain. These findings support the concept that E6-AP regulates SOX9 levels in developing cartilage by acting as a ubiquitin ligase.

Date: Oct. 23, 2013

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