Abstract 53: Interplay among E3 ubiquitin ligases regulate timely degradation of the circadian factor Period 2

Circadian rhythms are mechanisms that measure time on a scale of about 24 h and that adjusts our body to external environmental signals. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Oscillatory rhythms are generated as result of the activation of positive and negative transcriptional feedback loops in which post-translational modifications, shuttling, and degradation are common themes. In mammals, the positive feedback loop involves regulation of bmal1 transcription. BMAL1/CLOCK selectively binds to E-box enhancers and drives the expression of per, cry and rev-erbα genes. REV-ERBa protein then represses bmal1 transcription through Rev-Erba/ROR response elements in its promoter. Then, the level of bmal1 RNA falls, as per and cry RNA levels peak. Period proteins (i.e., PER1, 2, and 3) accumulate in the cytoplasm, becomes phosphorylated by CK1e/d, ubiquitylated and degraded. Later in the day, cryptochrome accumulates (i.e., CRY2), associates with PER2/CK1e/d and this complex translocates to the nucleus where CRY disrupts the CLOCK/BMAL1-associated transcriptional complex, resulting in the inhibition of cry, per and rev-erba and de-repression of bmal1 transcription. In addition to the transcriptional loops, ubiquitin E3 ligases-mediated protein degradation is critical to sustain physiological rhythms. The F-box proteins b-TrCP1/2 target PER1 and PER2 for degradation via ubiquitination. Whereas knocking down β-trcp results in PER stabilization and period lengthening in synchronized culture cells; knock-out b-trcp1 mice do not display an abnormal circadian rhythmicity. Our findings indicate that PER2 ubiquitination engages two different types of E3 ligases, a process that directly influences the timely control of PER2 accumulation in the nucleus. Our data show that the mouse double-minute 2 homolog (Mdm2) RING finger E3 ligase binds to PER2 in multiple regions including an overlapping site with b-TrCP. We determined that PER2 is a specific substrate for Mdm2 and that PER29s polyubiquitination occurs both in vitro and in cells. Accordingly, overexpression Mdm2 results in the decrease of PER2 half-life, whereas, down-regulation of Mdm2 by siRNA increases PER2 stability. Furthermore, we found Mdm2-dependent post-translational modification of PER2 directly influences the amplitude of the circadian oscillatory response in synchronized cells and that both, b-TrCP and Mdm2 target PER2 at different circadian times. Overall, our findings support a model in which, the rate of nuclear degradation of PER2 leads to slow accumulation of the protein in the nucleus and, later in the day, to a temporal switch in which b-TrCP takes control over PER2 degradation. Thus, the interplaying between these two E3 ligases is indispensible for sustaining robust circadian oscillation. Citation Format: Carla V. Finkielstein, jingjing liu. Interplay among E3 ubiquitin ligases regulate timely degradation of the circadian factor Period 2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2015-53