Abstract

The retinoblastoma susceptibility gene, Rb, was the first tumour suppressor gene to be identified and characterized. Rb belongs to the family of so-called pocket proteins, which also includes p107 and p130. Inactivation of Rb may contribute to many human malignancies including familial retinoblastoma, small-cell lung carcinomas, cervical carcinomas, prostate carcinomas, breast carcinomas, and some forms of leukemias. The most studied function of Rb protein is in the regulation of cell cycle progression at the G1/S boundary (Giacinti and Gordano, 2006). However, Rb is also considered to be involved in chromatin remodeling, development, differentiation and apoptosis. Due to the important position of Rb as a regulator of cell cycle progression at the G1/S phase boundary, Rb is highly regulated. Hypophosphorylated Rb binds E2F and recruits histone deacetylases and methytransferases to repress the expression of E2F controlled gene expression. Phosphorylation by cyclin/CDKs over the course of G1-phase leads to hyperphosphorylation, disassociation of Rb from E2F and the expression of E2F controlled S-phase inducing genes (Trimarchi et al, 2002).

The multiple roles of Rb are facilitated by its interaction with different protein partners, dependent on the cell type, and on the developmental and cell cycle stages. The interactions of Rb with its binding partners are conserved throughout a wide variety of taxa, from plants to invertebrates and mammals (Van den Heuvel and Dyson, 2008). The Rb protein is commonly represented as consisting of three modules, the N-domain, pocket domain and the C-domain (Morris and Dyson, 2001). The pocket domain is further separated into the A and B domains which each possess the helical cyclin fold. The pocket domain acts as a binding region for numerous cellular proteins, including the E2F transcription factors, histone deacetylases and cell cycle regulators as well as viral oncoproteins (Fattaey et al.1992).

To date, two pocket domain binding motifs have been identified. An LxCxE motif mediates binding to the conserved "pocket region" in the B-domain and an LxxLFD motif that binds a deep conserved groove between the pocket A and B domains. The LxCxE motif is found in numerous kinases, histone deacetylases and methytransferases (e.g. 11226179, 12502741, 10958676). Recruitment of histone deacetylases and methytransferases via the LxCxE binding pocket mediates repression of E2F controlled genes. The LxxLFD motif in the transactivation (TA) domain of E2F transcription factors is partially responsible for the recruitment of Rb (Rb also contacts E2F through an additional larger disordered region that binds across the E2F/DP1 interface (2AZE )) thereby repressing E2F mediated transcription. Deregulation of Rb-E2F interaction or the LxCxE mediated binding results in hyperproliferation, lack of differentiation, apoptosis and can lead to cancer.

Rb is a common target of viral oncoproteins, predominantly of DNA viruses, most often via the LxCxE motif (first identified in the adenovirus E1A and papilloma virus E7 proteins (2198278)). Convergently evolved mimics are known in multiple viruses including both plant (RepA in wheat dwarf virus and Clink in faba bean necrotic yellows virus) and mammalian (EBNAC in Epstein-Barr Virus (EBV), pp71 in Human cytomegalovirus, Ta in SV40, E7 in HPV, E4 and E1A in Adenovirus, NSP90 in Rubella, Tax in HTLV) viral proteins. Adenovirus E1A has both an LxCxE and an LxxLFD motif (SV40 Lt and HPV E7 are also proposed to contain both motifs however possess insufficient evidence for high confidence in the LxxLFD motif, see below). These viral proteins use their Rb targeting motifs to deregulate E2F binding to Rb, alleviating the Rb-mediated repression and forcing the cell into S-Phase thereby activating the replication machinery necessary for completion of the DNA viral life cycle. For example, the LxxLFD motif contained in the CR1 region of Adenovirus E1A and the TA domain of E2F use analogous residues to directly compete for the AB pocket of Rb (Liu and Marmorstein, 2007). E1A has also been shown to recruit Rb to anti-viral promoters leading to their repression.

Cellular C/EPBs, SV40 Lt and HPV E7 have been proposed to possess the pocket groove-binding motif yet they do not match the motif conservation of the canonical E2F and E1A motifs (these both have structural evidence for pocket groove binding (Cho et al., 2002; Liu and Marmorstein, 2007)). There might be a possibility of additional binding sites as the surface of the pocket domain contains extensive grooves outside the LxxLFD and LxCxE binding sites. Furthermore, the structure of the extended E2F peptide revealed an interface adjacent to the LxxLFD binding site.