Question

Describe the following: 1) how the function of pRb protein is regulated by post-translational modifications during the normal mitotic cell cycle and 2) how the modifications control the normal cell cycle progression through the R-point and initiation of the S-phase of the cell cycle.

Answer 1

A.

RB, p107, and p130 together are known as the ‘pocket protein’ family. Approximately, RB shares 25% sequence identity with both the homologs, whereas p107 and p130 share ~54% identity with each other. Structural data characterizing p107 and p130 are limited; however, sequence analysis suggests domains comparable to those in RB are present (see the figure). Both the proteins contain predicted amino-terminal domains (107N and 130N), pocket domains, and carboxy-terminal domains (107C and 130C), with analogous secondary and tertiary structural elements. Consistent with these parallel structural features, a number of common molecular functions have been identified. All three proteins negatively regulate the cell cycle, and for each protein this effect has been tied to its ability to associate with E2F transcription factor family members and influence E2F-mediated gene expression. The pocket domains of all homologs are predicted to contain L-X-C-X-E-binding clefts, which bind viral proteins and probably partially overlapping sets of cellular proteins. These pocket proteins are inactivated by CDKs, and the sequence analysis suggests several common structural effects of phosphorylation.

Genetic and cellular investigations have revealed various key functional differences between pocket proteins. RB knockout is embryonic lethal in mice, whereas knockout of p107 or p130 doesn't have a phenotype in a mixed genetic background. Examination of mice lacking different combinations of pocket protein genes suggests that p107 and p130 have an overlapping role in development that is distinct from that of RB. Importantly, the tumor suppressor properties of the RB gene are significantly stronger than those of p107 and p130, and only RB mutations are commonly found in human cancer. Consistent with these genetic differences, pocket proteins have been observed to control distinct E2F target genes and arrest cells in different cell cycle phases. In a recent striking example, a genome-wide screen of gene repression in fibroblast cells revealed a unique role for RB in promoting senescence. Further structural and biochemical analysis is needed to understand the molecular basis for these functional differences in pocket proteins, although distinct protein interactions have already been identified. For example, pocket proteins show preferences for different E2F family members, and p107 and p130 bind and inhibit CDKs, whereas RB exclusively forms a stable complex with protein phosphatase 1 (PP1).

Post-translational modifications have an important role in the regulation of RB function. With a few exceptions, RB phosphorylation (P) results in inactivation, transcriptional derepression, and cell cycle progression. RB is phosphorylated by several different kinases, including cyclin-dependent kinases (CDKs) and checkpoint kinase 2 (CHK2). Phosphorylation controls RB interactions with other proteins. This modification typically occurs outside structured domains (see the figure) and promotes conformational transitions from disordered to ordered RB structures that mask protein-binding surfaces. Different kinases show preferences for particular phosphorylation sites, and discrete phosphorylation events induce specific structural changes. However, it remains uncertain whether, and in what context, differentially phosphorylated isoforms of RB exist in the cell.

Acetylation (Ac) and methylation (Me) sites have been identified in disordered sequences towards the RB carboxy-terminal domain (RBC). In contrast to phosphorylation, these modifications occur in response to signals, such as DNA damage and differentiation, which correlate with RB activation and repression of gene expression. Acetylation occurs on Lys873 and Lys874, which are located within the cyclin-docking sequence, and results in reduced phosphorylation, probably through kinase inhibition. Methylation on Lys873 and Lys810 by SET-domain methyltransferases similarly results in RB hypophosphorylation. SET and MYND domain-containing 2 (SMYD2) methylates Lys860, which results in the recruitment of the transcriptional repressor lethal(3)malignant brain tumor-like 1 (L3MBTL1). The reader is referred to other reviews for more details on these and other emerging post-translational modifications on RB and their roles in the regulation of function.

B.

a | RB associates with E2F transcription factors and differentiation-related polypeptide (DP) heterodimers in G1 to repress transcription of cell cycle genes. RB can also form a unique interaction with E2F1 involving sequences in the RB carboxy-terminal domain (RBC). b | In S phase, RB is phosphorylated (P) and unable to bind to E2Fs that are bound the promoters of cell cycle genes, allowing the cell cycle to advance. Phosphorylated RB can still interact with E2F1, and this complex can repress the expression of apoptotic target genes. c | In response to DNA damage, RB is dephosphorylated and regains the ability to repress E2F transcription at cell cycle promoters. Simultaneously, phosphorylated RB remains in contact with E2F1 transcription factors, and the recruitment of p300/CBP-associated factor (PCAF) through unknown signals leads to histone acetylation (not shown) and activation of pro-apoptotic target genes. RBN, RB amino-terminal domain.