The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila

Abstract

In the Drosophila circadian clock, the CLOCK/CYCLE complex activates the period and timeless genes that negatively feedback on CLOCK/CYCLE activity. The 24-h pace of this cycle depends on the stability of the clock proteins. RING-domain E3 ubiquitin ligases have been shown to destabilize PERIOD or TIMELESS. Here we identify a clock function for the circadian trip (ctrip) gene, which encodes a HECT-domain E3 ubiquitin ligase. ctrip expression in the brain is mostly restricted to clock neurons and its downregulation leads to long-period activity rhythms in constant darkness. This altered behaviour is associated with high CLOCK levels and persistence of phosphorylated PERIOD during the subjective day. The control of CLOCK protein levels does not require PERIOD. Thus, CTRIP seems to regulate the pace of the oscillator by controlling the stability of both the activator and the repressor of the feedback loop.

The molecular ticks of the Drosophila circadian clock

“…Since its mRNA is itself under circadian control [40–43], USP8 regulates its own transcription, as well as per, tim and that of other CCGs by inhibiting CLK transcriptional activity through deubiquitination. CLK stability is also regulated by the HECT-domain containing E3 UB-ligase CIRCADIAN-TRIP (CTRIP) [44] (Figure 1). CTRIP downregulation results in higher CLK levels and long period without affecting Clk mRNA, which suggests that CLK stability is increased.…”

Circadian timekeeping and output mechanisms in animals

“…Once PER is degraded, CLK–CYC transcription is reactivated coincident with an increase in ubiquitylated CLK and a reduction in phosphorylated CLK, but the extent to which these processes contribute to delays that set the ~24 hours circadian period is not known. The increase in ubiquitylated CLK could be mediated by the Circadian TRIP (CTRIP) E3 ubiquitin ligase [43], ortholog of TRIP12 in mammals, whereas the reduction in phosphorylated CLK could result from CLK dephosphorylation or new CLK synthesis.…”

The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator

“…Similarly, CLK immunoreactivity in head extracts or brain tissue seems to oscillate in phase with its phosphorylation [21]–[23], although harsh extraction liberates chromatin-bound CLK, which results in relatively constant CLK levels [20],[24],[25]. Whether oscillations of CLK immunoreactivity in neurons reflect rhythmic changes of total CLK protein amount is still unclear [23],[26].…”