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

Abstract

The conformation and activity of pRb, the product of the retinoblastoma susceptibility gene, is dependent on the phosphorylation status of one or more of its 16 potential cyclin-dependent kinase (cdk) sites. However, it is not clear whether the phosphorylation status of one or more of these sites contributes to the determination of the various conformations and activity of pRb. Moreover, whether and how the conformation of pRb may regulate the phosphorylation of the cdk sites is also unclear. In the process of analyzing the function and regulation of pRb, we uncovered the existence of an unusual structural motif, m89 (amino acids 880 -900), the mutation of which confers upon pRb a hypophosphorylated conformation. Mutation of this structural domain activates, rather than inactivates, the growth suppressor function of pRb. In order to understand the effect of the mutation of m89 on the phosphorylation of cdk sites, we identified all the cdk sites (Thr-356, Ser-807/Ser-811, and Thr821) the phosphorylation of which drastically modify the conformation of pRb. Mutation of each of these four sites alone or in combinations 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 pRb has enhanced growth suppressing activity relative to the wild type. Our data 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 enhanced growth suppressing activity, similar to a mutant with alanine substitutions at Ser-807/Ser-811. Our recent finding, that the m89 region is part of a structural domain, p5, conserved antigenically and functionally between pRb and p53, suggests that the evolutionarily conserved p5 domain may play a role in the coordinated regulation of the activity of these two tumor suppressors, under certain growth conditions.

Pre-clinical evaluation of cyclin-dependent kinase 2 and 1 inhibition in anti-estrogen-sensitive and resistant breast cancer cells

“…We first measured pRb phosphorylation at 8 and 24 h after release from serum starvation-induced G0/1 arrest ( Figure 5A). In parental MCF7 cells, at 8 h after serum addition, pRb pSer807/811 levels, indicative of CDK4/6 activity (Driscoll et al, 1999), had substantially increased, and by 24 h, pRb pSer807/811 and pRb pThr821 levels had fully recovered. Tamoxifen caused a significant decrease in pRb phosphorylation at both time points in MCF7 cells.…”

Targeting the PI3K/Akt/mTOR signaling pathway in B-precursor acute lymphoblastic leukemia and its therapeutic potential

“…56 The Ser 807/811 and Ser 780 residues are targets of cyclin E/CDK2 and cyclin D/CDK4, respectively. 57 It should be reminded here that mTORC1 controls the translation of several proteins that are of critical importance for cell cycle progression, including CDK2. 50 It is important to emphasize that Rb gene activation is an unfavorable prognostic predictor in initial and relapsed childhood ALL.…”

The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition

“…In other instances there is no stable association of the substrate with CDK2 but the integrity of the RXL (or KXL) motif has been shown to be essential for phosphorylation of target residues such as in the substrates pRb and p53 (19,21,22). Paradoxically stronger physical association between the CDK/ cyclin and the substrate does not necessarily lead to a more efficient phosphorylation than weaker interaction, as shown with studies E2F/DP1 phosphorylation (23).…”