The retinoblastoma (RB) family proteins are pivotal transcriptional corepressors involved in diverse cellular events. As a potent tumor suppressor, RB plays a variety of important roles including regulating cell cycle, promoting cellular differentiation, inducing cell senescence and balancing apoptotic death, many of which are controlled by specific post-translational modifications. We identified an evolutionarily conserved instability element (IE) in the C-terminus of Drosophila RB-related protein Rbf1 that simultaneously regulates degradation and repression activity. Paradoxically, when the IE is deleted, increased protein levels do not cause enhanced repression activity. Rather, these mutations diminish repression activity of Rbf1, indicating a linkage between Rbf1 activity and instability. I found that the IE is an independent module which may serve as an interaction domain for multiple cofactors linking protein turnover and transcriptional repression. I showed that loss of the IE promotes cell growth, a disastrous consequence favorable to cancer progression. To better understand the control of this unique bifunctional element of Rbf1, I investigated the effects of phosphorylation by Cyclin-Cdk complexes. I show that protein stability and activity governed by the IE are cleanly separable, possibly by coordinated interactions with E2F transcription factors and E3 ubiquitin ligases. A highly conserved lysine residue K774 in the IE, frequently mutated in RB family protein p130 in lung cancers, is critical for Cyc-Cdk-mediated phosphorylation. My data suggest that the IE governs distinct functional outputs of Rbf1 through post-translational modifications and potentially cofactor binding. The similarities with mammalian systems suggest that parallel processes may regulate RB family proteins in human cancers.
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