The Retinoblastoma (RB) family of proteins play critical roles in normal development through their governance of genes involved in cell fate. During normal growth RB family activity is tightly regulated through Cdk-dependent phosphorylation, resulting in their dissociation from E2F family transcription factors. In addition, the RB pathway is also governed through the ubiquitin-proteasome system, with deregulated degradation of RB proteins frequently associated with human cancer. Recent studies from our labs have shown in Drosophila that the Retinoblastoma family (Rbf) proteins are subjected to proteasome mediated turnover during embryonic development and this process enhances Rbf engagement in transcriptional repression. This positive link between Rbf1 activity and its destruction indicates that repressor function is governed in a manner similar to that described by the degron theory of transcriptional activation. To understand the relationship between RB family stability and their repressor function during early mammalian development, we initiated studies in mouse embryonic stem (ES) cells. Our studies suggest that differentiation of mouse ES cells is associated with the establishment of a functional RB pathway and simultaneous changes in stability of RB family members. As pluripotent ES cells are characterized by unrestrained cdk activity which plummets at the onset of differentiation, we speculated that the observed changes in protein stability upon ES cell differentiation reflects an intimate relationship between RB phosphorylation and stability. Indeed, we show that phosphorylation dependent turnover of RB, p107 and p130 is mediated by an evolutionarily conserved and autonomous instability element (IE) located in their C-terminal regulatory domain. Moreover, stabilizing mutations within the IE elements also debilitate them for transcriptional repression. We conclude that the overlap of degron sequences and repression modules is a conserved feature shared among the RB homologues, and represents a novel mode of transcriptional repression. Together, these findings implicate the Retinoblastoma family IE region as a regulatory nexus linking repressor potency to the ubiquitin-proteasome system in development and disease.
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