Abstract LB-309: Interplay between E3 ligases modulate Per2 stability

Abstract

Abstract Circadian oscillation relies on autoregulatory loops generated by sequential phases of transcription/translation, protein modification, and degradation. Interestingly, disruption of the clock gene period2 (per2) in mice is associated with a hyperplasic phenotype and loss of apoptotic response. When irradiated, per2 knockout mice show a significantly higher frequency of tumor development most likely due to loss of normal circadian control of genes concerned with the regulation of the DNA-damage response as well as partial resistance to radiation-induced apoptosis. Although evidence suggests that the per2 gene functions in tumor suppression, no mechanism of Per2 action is known. Our work show direct evidences of crosstalk mechanisms that involve Per2 and the oncogenic E3-ligase Mdm2. Whereas we know much about the role of b-TrCP in modulating the oscillatory levels of Per2 in the cell, the functional relevance of Mdm2 targeting of Per2 has just been uncovered. Bacterial two-hybrid screenings led to the identification of Mdm2 as a new PER2 binding partner, an interaction that was later validated by immunoprecipitation studies from both ectopicaly expressed and endogenous proteins. In vitro experiment show that recombinantly expressed Per2 serves as a substrate for Mdm2 and polyubiquitination takes place shortly after interaction. These results were later confirmed when Mdm2 was overexpressed and ubiquitination of Per2 was detected in immunoprecipitated samples. Interestingly, knock down of either beta-TrCP or Mdm2 dampened PER2 oscillation, although the mechanism by either E3 ligase seems to act relies in the subcellular localization of the substrate., Lastly, there is an interplay between these two ligases as we showed that downregulation of Mdm2 influences the total levels of beta-TrCP and direct interaction between these protein was confirmed by immunoprecipitation. Moreover, experiments carried out with constitutively negative active forms of each ligase show they are each other targeted for ubiquitination and that Per2 exerts a very relevant control on stability of b-TrCP by binding to Mdm2 and inhibiting its E3-ligase activity. Citation Format: jingjing liu, Carla V. Finkielstein. Interplay between E3 ligases modulate Per2 stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-309. doi:10.1158/1538-7445.AM2014-LB-309