Discovery of a regulatory motif that controls the exposure of specific upstream cyclin-dependent kinase sites that determine both conformation and growth suppressing activity of pRb

The conformation and activity of pRb, the product of the retinoblastoma sus ceptibility gene, is dependent on the phosphorylation status of one or more of its 16 potential cyclin-dependent kinase (cdk) sites. However, it is no t clear whether the phosphorylation status of one or more of these sites co ntributes to the determination of the various conformations and activity of pRb. Moreover, whether and how the conformation of pRb may regulate the ph osphorylation of the cdk sites is also unclear. In the process of analyzing the function and regulation of pRb, we uncovered the existence of an unusu al structural motif, m89 (amino acids 880-900), the mutation of which confe rs upon pRb a hypophosphorylated conformation. Mutation of this structural domain activates, rather than inactivates, the growth suppressor function o f pRb. In order to understand the effect of the mutation of m89 on the phos phorylation of cdk sites, we identified all the cdk sites (Thr-356, Ser-807 /Ser-811, and Thr821) the phosphorylation of which drastically modify the c onformation of pRb. Mutation of each of these four sites alone or in combin ations results in the different conformations of pRb, the migration pattern of which, on SDS-polyacrylamide gel electrophoresis, resembles various in vivo hypophosphorylated forms. Each of these hypophosphorylated forms of pR b has enhanced growth suppressing activity relative to the wild type. Our d ata revealed that the m89 structural motif controls the exposure of the cdk sites Ser-807/Ser-811 in vitro and in vivo. Moreover, the m89 mutant has e nhanced growth suppressing activity, similar to a mutant with alanine subst itutions at Ser-807/Ser-811, Our recent finding, that the m89 region is par t of a structural domain, p5, conserved antigenically and functionally betw een pRb and p53, suggests that the evolutionarily conserved p5 domain may p lay a role in the coordinated regulation of the activity of these two tumor suppressors, under certain growth conditions.