Lysine-independent ubiquitination and degradation of REV-ERBα involves a bi-functional degradation control sequence at its N-terminus

REV-ERB and REV-ERB{beta} proteins play crucial roles in linking the circadian system to overt daily rhythms in mammalian physiology and behavior. In most tissues, REV-ERB protein robustly cycles such that it is detected only within a tight interval of 4-6 hours each day, suggesting both its synthesis and degradation are tightly controlled. Several ubiquitin ligases are known to drive REV-ERB degradation, but how they interact with REV-ERB and which lysine residues they ubiquitinate to promote degradation are unknown. In this study, we attempted to identify both ubiquitin-ligase-binding and ubiquitination sites within REV-ERB required for its degradation. Surprisingly, mutating all lysine residues, the common sites for ubiquitin conjugation, in REV-ERB to arginines (K20R), did very little to impair its degradation in cells. K20R were degraded much faster by co-expression of two E3 ligases, SIAH2 or SPSB4, suggesting possible N-terminal ubiquitination. To explore this, we examined if small deletions at the N-terminus of REV-ERB would alter its degradation. Interestingly, deletion of amino acid (AA) residues 2 to 9 (delAA2-9) clearly resulted in a less stable REV-ERB. We found that it was the length (i.e. 8 AA), and not the specific sequence, that confers stability in this region. Simultaneously, we also mapped the interaction site of the E3 ligase SPSB4 to this same region, specifically requiring AA4-9 of REV-ERB. Thus, the first 9 AA of REV-ERB has two opposing roles in regulating REV-ERB turnover. Further, deleting eight additional AAs (delAA2-17) from the N-terminus strongly prevents REV-ERB degradation. Combined, these results suggest that complex interactions within the first 25AAs potentially act as an endogenous switch that allows REV-ERB to exist in a stabilized conformation in order to accumulate at one time of day, but then rapidly shifts to a destabilized form, to enhance its removal at the end of its daily cycle.