Differential regulation of retinoblastoma protein function by specific Cdk phosphorylation sites

The retinoblastoma tumor suppressor protein, RE, contains at least three distinct protein binding domains. The A/B pocket binds proteins with the LXCXE motif, the C pocket binds the nuclear c-Abl tyrosine kinase, and the large A/B pocket binds the transcription factor E2F. Dissociation of RE from its targets is observed as RE becomes phosphorylated during G(1)/S progression. There are 16 Cdk consensus phosphorylation sites in RE. It was previously unknown whether the many phosphorylation sites had redundant or distinct functions in the regulation of RE. Using RE mutant proteins lacking specific phosphorylation sites, we show that each of the binding domains is inhibited by different sites. Thr-821/826 phosphorylation is required to inhibit the binding to LXCXE containing proteins. Mutation of these two sites does not interfere with the hyperphosphorylation of RE. However, this phosphorylated mutant retains the ability to bind T-Ag, E7, and Elf-1, all of which contain the LXCXE motif. In contrast, Ser-807/811 phosphorylation is required to disrupt c-Abl binding. Mutation of Ser-807/811 and Thr-821/826 does not abolish the regulation of E2F binding. Taken together, these results show that the protein binding domains of RE are each regulated by distinct Cdk phosphorylation sites.