E6AP ubiquitin ligase mediates ubiquitin-dependent degradation of peroxiredoxin 1.

E6-associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of tumor suppressor p53 in conjunction with the high-risk human papillomavirus E6 protein. We previously reported that E6AP targets annexin A1 protein for ubiquitin-dependent proteasomal degradation. To gain a better understanding of the physiological function of E6AP, we have been seeking to identify novel substrates of E6AP. Here, we identified peroxiredoxin 1 (Prx1) as a novel E6AP-binding protein using a tandem affinity purification procedure coupled with mass spectrometry. Prx1 is a 25-kDa member of the Prx family, a ubiquitous family of antioxidant peroxidases that regulate many cellular processes through intracellular oxidative signal transduction pathways. Immunoprecipitation analysis showed that E6AP binds Prx1 in vivo. Pull-down experiments showed that E6AP binds Prx1 in vitro. Ectopic expression of E6AP enhanced the degradation of Prx1 in vivo. In vivo and in vitro ubiquitylation assays revealed that E6AP promoted polyubiquitylation of Prx1. RNAi-mediated downregulation of endogenous E6AP increased the level of endogenous Prx1 protein. Taken together, our data suggest that E6AP mediates the ubiquitin-dependent proteasomal degradation of Prx1. Our findings raise a possibility that E6AP may play a role in regulating Prx1-dependent intracellular oxidative signal transduction pathways.

Identification of annexin A1 as a novel substrate for E6AP-mediated ubiquitylation.

E6-associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high-risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull-down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP-binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi-mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca(2+)-dependent manner. GST pull-down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP-mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin-proteasome pathway.