Molecular mechanisms underlying RB protein function

Abstract

Inactivation of the RB protein is one of the most fundamental events in cancer. Coming to a molecular understanding of its function in normal cells and how it impedes cancer development has been challenging. Historically, the ability of RB to regulate the cell cycle placed it in a central role in proliferative control, and research focused on RB regulation of the E2F family of transcription factors. Remarkably, several recent studies have found additional tumour-suppressor functions of RB, including alternative roles in the cell cycle, maintenance of genome stability and apoptosis. These advances and new structural studies are combining to define the multifunctionality of RB.

Figure 1. RB is a multidomain protein with several distinct protein-binding surfaces

a | Model of active and complexes with E2F and an ‘L-X-C-X-E’ peptide (Protein Data Bank (PDB) codes: 2QDJ, 1GUX, 1N4M and 2AZE). The dashed lines indicate flexible interdomain linkers. b | Schematic diagram of the domain structure and location of known binding sites for protein partners. c | Schematic structure of RB in its inactivated, phosphorylated conformation. Thr373 phosphorylation drives interdomain docking of the RB amino-terminal domain (RBN) and the ‘pocket’, whereas Ser608 and Ser612 and Thr821 and Thr826 phosphorylation induce binding of the pocket loop and the RB carboxy-terminal domain (RBC), respectively, to the pocket domain. These different conformational changes inhibit specific RB–protein interactions. CDH1, CDC20 homologue 1; CDK, cyclin-dependent kinase; DP^MB, differentiation-related polypeptide marked box; E2F^TD, E2F transactivation domain; EID1, E1A-like inhibitor of differentiation 1; PP1, protein phosphatase 1.

Figure 2. Regulation of E2Fs by RB

In the G1 phase of the cell cycle, RB is phosphorylated at low levels and associates with E2F transcription factors, which are dimeric proteins containing E2F and differentiation-related polypeptide (DP) subunits. RB also recruits enzymes that regulate chromatin structure to these complexes. Transcription of these genes is repressed until cyclin-dependent kinases (CDKs) phosphorylate (P) RB and prevent binding of E2Fs and chromatin regulators. E2F transcription factors then transcribe genes necessary for S phase and the cell cycle advances. RBC, RB carboxy-terminal domain; RBN, RB amino-terminal domain.

Figure 3. Transcription-independent regulation of cyclin-dependent kinases by RB

a | Schematic depiction of the F box protein S phase kinase-associated protein 2 (SKP2) recognizing phosphorylated p27. Binding results in ubiquitylation and degradation of p27 and the activation of cyclin-dependent kinases (CDKs). b | SKP2 is bound by the RB carboxy-terminal domain (RBC) and the ‘pocket’ domain, which competes with SKP2 for interaction with phosphorylated p27. RB thereby prevents p27 ubiquitylation, resulting in inhibition of CDK activity. c | The RBC region also binds SKP2 to recruit it to the APC/C (anaphase-promoting complex; also known as the cyclosome) E3 ubiquitin ligase complex. Ubiquitylated SKP2 (not shown) is targeted for degradation, leading to the increased expression of p27 and inhibition of CDKs. RBN, RB amino-terminal domain.

Figure 4. Regulation of pericentromeric heterochromatin by RB

The condensin II complex is enriched at pericentromeric and centromeric regions of mitotic chromosomes. Loading this complex onto chromatin is dependent on L-X-C-X-E-type interactions with RB. How this directs the condensin II complex to this genomic location is not known (indicated by a question mark), but it is suggested that additional proteins are involved. Similarly, the localization of cohesins to the centromere and pericentromere remains to be elucidated. Given the prominence of genome instability in mice deficient for L-X-C-X-E-type interactions, RB function in chromosome structure is key for the well-conserved L-X-C-X-E binding site. RBC, RB carboxy-terminal domain; RBN, RB amino-terminal domain.

Figure 5. Differential regulation of E2F1 in apoptosis

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.

RBN, RB amino-terminal domain.

RBC, RB carboxy-terminal domain; RBN, RB amino-terminal domain.