Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW The Rb/E2F pathway: expanding roles and emerging paradigms J. William Harbour1,2 and Douglas C. Dean1,3 1Division of Molecular Oncology and 2Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri 63110,USA Received April 21, 2000; revised version accepted July 17, 2000. The retinoblastoma gene was identified over a decade Rb; these proteins are required for the viruses to trans- ago as the first tumor suppressor. Although the gene was form cells (DeCaprio et al. 1988; Whyte et al. 1988; Dy- initially cloned as a result of its frequent mutation in the son et al. 1989). rare pediatric eye tumor, retinoblastoma (Friend et al. Rb function depends, at least in part, on interactions 1986; Fung et al. 1987; Lee et al. 1987), it is now thought with the E2F family of DNA-binding transcription fac- to play a fundamental role in cellular regulation and is tors (E2F) (Chellappan et al. 1991; Dyson 1998; Nevins the target of tumorigenic mutations in many cell types. 1998). E2F sites are found in the promoters of many The retinoblastoma gene encodes a 928–amino acid genes that are important for cell cycle progression, and phosphoprotein, Rb, which arrests cells in the G1 phase Rb appears to repress transcription of these genes (Weinberg 1995). Rb is phosphorylated and dephosphory- through its interaction with E2F (Blake and Azizkhan lated during the cell cycle; the hyperphosphorylated (in- 1989; Thalmeier et al. 1989; Dalton 1992; Ohtani et al. active) form predominates in proliferating cells, whereas 1995). Recent findings in the Rb/E2F field are clarifying the hypophosphorylated (active) form is generally more how this pathway regulates the transition from G1 to S abundant in quiescent or differentiating cells (Chen et al. phase at the molecular level. Other emerging results 1989). As a demonstration its tumor suppressor activity, show that this pathway also regulates other parts of the Rb was reintroduced into Rb-deficient tumor cells and it cell cycle and that it may even have roles beyond cell blocked several features of the malignant phenotype cycle control. In this article, we review the current un- (Huang et al. 1988). Mutations affecting the retinoblas- derstanding of the mechanism of action of Rb and its toma gene are frequently encountered, not only in reti- roles in cell cycle regulation, apoptosis, and develop- noblastoma but also in other cancers such as osteosar- ment. We refer readers to recent reviews by Bartek et al. coma, small cell lung cancer, prostate cancer, and breast (1997), Dyson (1998), Lipinski and Jacks (1999), and Sherr cancer (Friend et al. 1986; Fung et al. 1987; Harbour et al. and Roberts (1999) for additional in-depth analysis of the 1988; Lee et al. 1988; T’Ang et al. 1988; Bookstein et al. field and for historical perspectives. 1990). Indeed, children with hereditary retinoblastoma have Ն30-fold increased risk of developing a second, nonocular malignancy, especially bone and soft tissue Rb structure sarcomas in adolescence and cutaneous melanomas in Rb contains several functional domains. Domains A and adulthood (Eng et al. 1993; Moll et al. 1997). These sec- B are highly conserved from humans to plants, and they ond neoplasms occur almost exclusively in patients who interact with each other along an extended interdomain have germ-line mutations in the retinoblastoma gene. As interface to form the central “pocket” (Chow and Dean a further indication of its fundamental role in tumor sup- 1996; Lee et al. 1998), which is critical to the tumor- pression, Rb can be functionally inactivated by consti- suppressor function of Rb (Qin et al. 1992). The pocket is tutive hyperphosphorylation in tumors that do not have disrupted by most naturally occurring germ-line muta- mutations in the retinoblastoma gene (Sherr 1996). In tions in hereditary retinoblastoma patients (Harbour addition, DNA tumor viruses express oncoproteins, such 1998) and by most tumor-derived mutations (Horowitz as adenovirus E1A, SV40 large tumor antigen, and hu- et al. 1990). Viral oncoproteins and a number of endog- man papillomavirus (HPV) E7, that bind and inactivate enous Rb-binding proteins contain an LXCXE motif that allows them to bind Rb (Whyte et al. 1988; Dyson et al. 1989; Ludlow et al. 1989; Lee et al. 1998). The crystal 3Corresponding author. structure of the pocket in complex with an LXCXE pep- E-MAIL [email protected]; FAX (314) 747-2797. Article and publication are at www.genesdev.org/cgi/doi/10.1101/ tide revealed that the binding site for LXCXE is in do- gad.813200. main B (Lee et al. 1998). However, domain A is required GENES & DEVELOPMENT 14:2393–2409 © 2000 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/00 $5.00; www.genesdev.org 2393 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Harbour and Dean for domain B to assume an active conformation, thus thereby block the antiapoptotic activity of MDM2 by explaining the conservation of both domains (Kim and preventing the degradation of p53 (Hsieh et al. 1999). Cho 1997; Lee et al. 1998). A number of endogenous Further work is needed to elucidate the role of these proteins that interact with Rb also contain an LXCXE- protein interactions involving the carboxy-terminal re- like sequence, including histone deacetylase (HDAC)-1 gion of Rb in vivo. and HDAC2, and the ATPase, BRG1, from the SWI/SNF The function of the amino-terminal region of Rb re- nucleosome remodeling complex (Dunaief et al. 1994; mains controversial. This region contains consensus cdk Brehm et al. 1998; Luo et al. 1998; Magnaghi et al. 1998). phosphorylation sites, which may regulate Rb activity The LXCXE binding site is the best characterized but when they are phosphorylated during the cell cycle. In not the only binding site in the pocket. E2Fs do not con- addition, the amino-terminal region interacts with sev- tain an LXCXE and thus bind Rb at a distinct site that eral proteins, including MCM7 (a replication licensing appears to involve points of contact in both the pocket factor; Sterner et al. 1998), a novel G2/M cycle-regulated and in the carboxy-terminal region (Huang et al. 1992; kinase (Sterner et al. 1995), and other proteins (Durfee et Lee et al. 1998). This allows E2F to recruit complexes al. 1994), but the function of these interactions is still containing Rb and other proteins, such as those with the unclear. In experiments with genetically engineered LXCXE motif, to a promoter. Still other binding sites mice lacking Rb, introduction of an Rb transgene with appear to be in the pocket. Whereas HDAC1 and HDAC2 an amino-terminal truncation mutation delayed the em- contain LXCXE-like motifs through which they can in- bryonic lethality caused by homozygous loss of Rb but teract with Rb (Magnaghi et al. 1998), HDAC3 does not did not prevent it, suggesting that this region may be contain this sequence, and mutations in the LXCXE important for complete Rb function during development binding site of Rb do not affect its binding to Rb (Chen (Riley et al. 1997). Similarly, this Rb mutant did not and Wang 2000; Dahiya et al. 2000). Although BRG1 con- prevent the development of pituitary tumors in Rb+/− tains an LXCXE, it does not require this sequence to bind mice, although the onset of tumors was delayed, suggest- Rb, which allows Rb to recruit BRG1 and HDAC1 into a ing that this region may play some role in Rb-mediated single complex (Zhang et al. 2000). Three recent studies tumor suppression (Riley et al. 1997). However, these in which the LXCXE-binding site was mutated have pro- experiments relied on expression of an Rb transgene that vided additional, albeit somewhat conflicting, insights may not entirely recapitulate the expression pattern and into its role. In each of these studies, the mutations in- protein level of endogenous Rb. In other experiments, hibited binding to E1A. In two of the studies the muta- tumor suppression by Rb was actually enhanced when tions also inhibited binding to HDAC1 and HDAC2 but the amino-terminal region was removed (Qin et al. 1992; did not affect binding to HDAC3, BRG1, and E2F1 (Chen Xu et al. 1994; Chow et al. 1996). Thus, a physiologic and Wang 2000; Dahiya et al. 2000). In the third study role of the amino-terminal region remains unclear and mutations did not affect binding to HDAC1 (N. Dyson, may only be settled once this region of the Rb gene is pers. comm.). Because mutation of the LXCXE-binding deleted in mice. site had variable effects on Rb function in these studies, further work is needed to clarify its role and to charac- Rb-mediated inhibition of E2F terize other binding sites on Rb. Another functional domain of Rb is located within the Rb can repress transcription by at least two different carboxy-terminal region. This region contains binding mechanisms. First, it can bind transcription factors such sites for the c-abl tyrosine kinase and MDM2, which as E2F and block their ability to activate transcription appear to be distinct from the E2F site in the carboxy- (Flemington et al. 1993; Helin et al. 1993). Second, the terminal region (Welch and Wang 1993; Xiao et al. 1995). Rb–E2F repressor complex that forms at promoters can The tyrosine kinase activity of c-abl is blocked when it is actively repress transcription (Bremner et al. 1995; Sell- complexed with Rb (Welch and Wang 1993). This inter- ers et al. 1995; Weintraub et al. 1995). The balance be- action appears to be important for Rb-mediated growth tween these two activities in vivo is still in question.