Oncogene (2006) 25, 5220–5227 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc REVIEW RB and cell cycle progression C Giacinti1,2 and A Giordano1,2 1Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA and 2Department of Human Pathology and Oncology, University of Siena, Siena, Italy The Rb protein is a tumor suppressor, which plays a of a tumor suppressor. Several human tumors show pivotal role in the negative control of the cell cycle and in mutations and deletions of the Rb gene, and inherited tumor progression. It has been shown that Rb protein allelic loss of Rb confers increased susceptibility to (pRb)is responsible for a major G1 checkpoint, blocking cancer formation (Dunn et al., 1988). The Rb gene is S-phase entry and cell growth. The retinoblastoma family functionally inactivated in most human neoplasms includes three members, Rb/p105, p107 and Rb2/p130, either by direct mutation/deletion, such as in retino- collectively referred to as ‘pocket proteins’. The pRb blastoma, osteosarcoma and small-cell lung carcinoma, protein represses gene transcription, required for transi- or indirectly through altered expression/activity of tion from G1 to S phase, by directly binding to the upstream regulators (Liu et al., 2004). Nevertheless, transactivation domain of E2F and by binding to the the Rb protein plays a pivotal role in the negative promoter of these genes as a complex with E2F. pRb control of the cell cycle and in tumor progression. represses transcription also by remodeling chromatin It has been shown that Rb protein (pRb) is structure through interaction with proteins such as responsible for a major G1 checkpoint (restriction hBRM, BRG1, HDAC1 and SUV39H1, which are point) blocking S-phase entry and cell growth, promot- involved in nucleosome remodeling, histone acetylation/ ing terminal differentiation by inducing both cell cycle deacetylation and methylation, respectively. Loss of pRb exit and tissue-specific gene expression (Weinberg, functions may induce cell cycle deregulation and so lead to 1995). a malignant phenotype. Gene inactivation of pRB through The Rb gene family includes three members, Rb/p105, chromosomal mutations is one of the principal reasons p107 and Rb2/p130, collectively referred to as ‘pocket for retinoblastoma tumor development. Functional inacti- proteins’. The term ‘pocket proteins’ derives from the vation of pRb by viral oncoprotein binding is also shown in conserved binding pocket region through which pRb, many neoplasias such as cervical cancer, mesothelioma p107 and Rb2/p130 bind viral oncoproteins and cellular and AIDS-related Burkitt’s lymphoma. factors such as the E2F family of transcription factors. Oncogene (2006) 25, 5220–5227. doi:10.1038/sj.onc.1209615 Further studies have shown that overexpression of all three pocket proteins in cells can induce growth arrest in Keywords: Rb;cell cycle;E2F;cancer;Rb2/p130;tumor the G1 phase of the cell cycle (Dyson, 1998). These suppressor studies helped define the role of the Rb gene family in regulating transition between cell proliferation and terminal differentiation. How pRb family members control cell cycle proliferation is not completely under- stood. Moreover, the interaction between the pRb Introduction family proteins and the E2F family transcription factors plays a central role in governing cell cycle progression The Rb gene is an archetypal tumor suppressor gene and DNA replication by controlling the expression of that was first identified in a malignant tumor of the cell cycle E2F-dependent genes (Macaluso et al., 2005). retina known as retinoblastoma. In addition, pRb recruits chromatin remodeling factors Retinoblastoma is a sporadic or hereditary pediatric such as histone deacetylase 1 (HDAC1) (Luo et al., neoplasm arising from retinal cells, and Knudson (1971) 1998), SWI/SNF factors (Harbour and Dean, 2000; hypothesized that the tumor phenotype is not apparent De Luca et al., 1997), Polycomb group proteins unless both copies of the gene are damaged. The cloning (Dahiya et al., 2001) or methyltransferase (Nielsen of the retinoblastoma Rb gene and the identification of et al., 2001) that act on the nearby surrounding biallelic Rb mutations in retinoblastoma tumors confirm nucleosome structure. the hypothesis that such gene product exerts the action The pRb protein has also a central role in various differentiation processes, including eye, lens, brain, peri- pheral nervous system, epidermis, melanocytes, hair cells, Correspondence: Dr A Giordano, Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, muscle and liver. For example, pRb is necessary for the PA 19122, USA. completion of the muscle differentiation program and E-mail: [email protected] for myogenic helix–loop–helix-dependent transcription RB and cell cycle progression C Giacinti and A Giordano 5221 (reviewed by De Falco, in this issue). The most from Rb family proteins. Rb proteins can be phos- convincing evidence of the importance of pRb in cellular phorylated both on A/B domains of the pocket region differentiation comes from studies of Rb knockout mice, and on the C-terminal domain. The main hypothesis is where the disruptions of the Rb gene cause death by day that the phosphorylation at the carboxy terminus of 14 of gestation, associated with defects in the develop- pRb by cyclin D/cdk4 (or ckd6) causes a conformational ment of the hematopoietic system and central nervous modification, which displaces the HDAC bound to pRb system (Lee et al., 1992). through its LXCXE motif. This facilitates the phos- phorylation of the A/B pocket region by cyclin E (A)/ cdk2, leading to the release of E2F from pRb/p105. Pocket protein phosphorylation is often reversed by Rb family proteins dephosphorylation, which causes transient inactivation. Pocket protein dephosphorylation is known to take The Rb/p105 and Rb2/p130 map to human chromo- place in the period from anaphase to G1 and to depend somal areas 13q14 and 16p12.2, respectively (Baldi upon protein phosphatase 1 (Ludlow et al., 1993); et al., 1996), in which mutations have been found in dephosphorylation occurs in response to growth in- several human neoplasias. The p107 maps to chromo- hibitory signals. In some situations, phosphorylation somal area 20q11.2, which is not frequently found to be can cause permanent inactivation, for example, by altered in human neoplasia, although there is a recent binding skp2 (Tedesco et al., 2002) and leading to report (Ichimura et al., 2000) of deletion or functional degradation. High levels of Cdk activity may result in inactivation for p107 in human tumors. The Rb family phosphorylation of pRb-Ser567, which exposes the Rb proteins share large regions of homology, especially in a protein to a proteolytic cleavage site and degradation by bipartite region, the so-called ‘pocket domain’ (Claudio an unidentified protease (Ma et al., 2003). et al., 2002). Rb2/p130 and p107 are more related to The C-terminal region of pRb proteins also plays an each other, with about 50% amino-acid identity, than important role in orchestrating the activity of these they are to Rb/p105 (30–35% identity). The Rb family proteins. The carboxy-terminal region differs in length proteins show a peculiar steric conformation owing to between Rb/p105 and the other two proteins Rb2/p130 the pocket region, which is responsible for many of the and p170, which are instead very similar in the amino- protein–protein interactions in the homeostasis of the acid sequence. As described above, analysis of structural cell cycle (Paggi et al., 1996). The pocket region is similarities among the Rb family members discloses that characterized structurally by two conserved functional p107 and Rb2/p130 are more strictly related to each domains (A and B) separated by a spacer (S), which other than to Rb. The carboxy-terminal region is quite substantially differs among the three Rb proteins, Rb/ important as it contains the nuclear localization signal p105, Rb2/p130 and p107. Members of the E2F family (NLS) and domains responsible for HADC1 and cyclin/ of transcription factors, which are cellular Rb-binding cdk complex binding. The NLS controls the transport of proteins, interact with the A and B pocket domains Rb proteins from the cytoplasm to the nucleus; (Puri et al., 1999), as well as proteins that possess the it consists of two cluster basic residues separated LXCXE peptide motif, such as oncoproteins from by a stretch of amino acids including proline residues several DNA tumor viruses (Lee et al., 1998), the (Dingwall et al., 1991). Nuclear localization signal D-type cyclins (Weinberg, 1995) and HDAC. Rb proteins works as a carrier also for E2F4 and E2F5 proteins, can bind E2F and several other LXCXE-containing both of which do not present the NLS. proteins at the same time because the two binding sites It has been demonstrated that HDAC1 needs both the for E2F peptide and LXCXE peptide are close but LXCXE-binding motif of the pocket region and the distinct from each other (Lee et al., 1998;Gallo and C-terminus region in order to form a stable complex Giordano, 2005). (Stiegler et al., 1998) with Rb proteins. The binding sites Rb family members are post-translation regulated for E2F and HDAC1 are distinct from each other (Lee proteins and their gradual phosphorylation leads to et al., 1998), so we can hypothesize that Rb works as a their functional inactivation;Rb protein phosphoryla- link between HDAC1 and E2F proteins when associated tion is a cell cycle-dependent phenomenon and cyclin/ to E2F-responsive promoters. cyclin-dependent kinase (cdk) complexes are responsible for this regulation. In the early G1 phase, D-type cyclins interact with the A/B pocket of the Rb gene products via their LXCXE motif;in particular, they couple with the Rb and cell cycle kinase cdk4 or cdk6 and phosphorylate pRb/105 and Rb2/p130 (Dowdy et al., 1993).