The retinoblastoma gene was identified over a decade ago as the first tumor suppressor. Although the gene was initially cloned as a result of its frequent mutation in the rare pediatric eye tumor, retinoblastoma (Friend et al. 1986; Fung et al. 1987; Lee et al. 1987), it is now thought to play a fundamental role in cellular regulation and is the target of tumorigenic mutations in many cell types. The retinoblastoma gene encodes a 928–amino acid phosphoprotein, Rb, which arrests cells in the G1 phase (Weinberg 1995). Rb is phosphorylated and dephosphorylated during the cell cycle; the hyperphosphorylated (inactive) form predominates in proliferating cells, whereas the hypophosphorylated (active) form is generally more abundant in quiescent or differentiating cells (Chen et al. 1989). As a demonstration its tumor suppressor activity, Rb was reintroduced into Rb-deficient tumor cells and it blocked several features of the malignant phenotype (Huang et al. 1988). Mutations affecting the retinoblastoma gene are frequently encountered, not only in retinoblastoma but also in other cancers such as osteosarcoma, small cell lung cancer, prostate cancer, and breast cancer (Friend et al. 1986; Fung et al. 1987; Harbour et al. 1988; Lee et al. 1988; T’Ang et al. 1988; Bookstein et al. 1990). Indeed, children with hereditary retinoblastoma have 30-fold increased risk of developing a second, nonocular malignancy, especially bone and soft tissue sarcomas in adolescence and cutaneous melanomas in adulthood (Eng et al. 1993; Moll et al. 1997). These second neoplasms occur almost exclusively in patients who have germ-line mutations in the retinoblastoma gene. As a further indication of its fundamental role in tumor suppression, Rb can be functionally inactivated by constitutive hyperphosphorylation in tumors that do not have mutations in the retinoblastoma gene (Sherr 1996). In addition, DNA tumor viruses express oncoproteins, such as adenovirus E1A, SV40 large tumor antigen, and human papillomavirus (HPV) E7, that bind and inactivate Rb; these proteins are required for the viruses to transform cells (DeCaprio et al. 1988; Whyte et al. 1988; Dyson et al. 1989). Rb function depends, at least in part, on interactions with the E2F family of DNA-binding transcription factors (E2F) (Chellappan et al. 1991; Dyson 1998; Nevins 1998). E2F sites are found in the promoters of many genes that are important for cell cycle progression, and Rb appears to repress transcription of these genes through its interaction with E2F (Blake and Azizkhan 1989; Thalmeier et al. 1989; Dalton 1992; Ohtani et al. 1995). Recent findings in the Rb/E2F field are clarifying how this pathway regulates the transition from G1 to S phase at the molecular level. Other emerging results show that this pathway also regulates other parts of the cell cycle and that it may even have roles beyond cell cycle control. In this article, we review the current understanding of the mechanism of action of Rb and its roles in cell cycle regulation, apoptosis, and development. We refer readers to recent reviews by Bartek et al. (1997), Dyson (1998), Lipinski and Jacks (1999), and Sherr and Roberts (1999) for additional in-depth analysis of the field and for historical perspectives.

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The Rb/E2F pathway: expanding roles and emerging paradigms

Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

https://www.embopress.org/doi/pdf/10.1038/msb4100134

Large‐scale mapping of human protein–protein interactions by mass spectrometry

Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.

DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters

The DNA damage response pathway is a cellular surveillance system that senses the presence of damaged DNA and elicits an appropriate and effective response to that damage. First identified as a regulator of cell cycle transitions, the DNA damage response pathway has since been shown to regulate a number of other cellular processes, which include DNA repair, apoptosis, and replication fork stabilization (Zhou and Elledge 2000; Cimprich 2003). The importance of this pathway is demonstrated by the conservation of this response from yeast to humans (Zhou and Elledge 2000; Melo and Toczyski 2002) and by several studies that have shown that loss of checkpoint proteins predisposes affected individuals to cancer (Sherr 2004).

One critical component of the DNA damage response pathway is the ATR–ATRIP complex. ATR is a phosphatidylinositol kinase-related protein kinase that is thought to function as both a sensor and transducer in the DNA damage response. ATR, and its associated protein ATRIP, respond to a broad spectrum of genotoxic agents that includes ultraviolet light (UV), topoisomerase inhibitors, alkylating agents, and cis-platinum, as well as chemicals that disrupt replication, such as aphidicolin and hydroxyurea (HU) (Zhou and Elledge 2000; Cortez et al. 2001; Melo and Toczyski 2002). Following DNA damage, ATR phosphorylates and activates the checkpoint kinase Chk1 (Melo and Toczyski 2002). In higher eukaryotes, the phosphorylation of Chk1 also requires the activities of the Rad9–Rad1–Hus1 (RHR, aka 9–1–1) complex and Claspin (Melo and Toczyski 2002). The RHR complex is a PCNA-related complex that is loaded on to primed DNA in vitro (Ellison and Stillman 2003; Zou et al. 2003) and is recruited to sites of DNA damage in vivo (Kondo et al. 2001; Melo et al. 2001). Claspin was initially identified as a protein that bound the activated form of Chk1, and it has been shown to bind chromatin throughout S phase (Kumagai and Dunphy 2000; Lee et al. 2003).

We and others previously showed that recruitment of ATR and Rad1 to chromatin (Lupardus et al. 2002) and activation of Chk1 (Lupardus et al. 2002; Stokes et al. 2002) requires DNA replication in Xenopus egg extracts following several types of DNA damage. Studies in mammalian cells also indicate that ATR binds UV-damaged chromatin in S phase but not G1 phase (Ward et al. 2004). In addition, other studies show that a replication fork must be established in Saccharomyces cerevisiae for checkpoint activation induced by methylmethane sulfonate (MMS) (Tercero et al. 2003). Taken together, these observations suggest that one or more replication-dependent events are needed to generate the signal that ATR recognizes for many types of DNA damage.

Although the exact nature of the biochemical signal(s) responsible for activating the ATR pathway and the replication-dependent steps necessary for its formation are still unclear, evidence from a number of different systems supports a central role for replication protein A (RPA)-coated single-stranded DNA (ssDNA) in the response. In yeast, certain RPA mutants exhibit a checkpoint defect and also adapt more rapidly to DNA damage (Longhese et al. 1996; Pellicioli et al. 1999). In addition, knock-down of the ssDNA-binding protein RPA results in a significant loss of both Chk1 phosphorylation and ATR foci formation following DNA damage in mammalian cells (Zou and Elledge 2003). In Xenopus egg extracts, RPA is also required for the recruitment of ATR to chromatin following treatment with aphidicolin (You et al. 2002) or etoposide (Costanzo et al. 2003) and for the recruitment of ATR to poly(dA)70 ssDNA (Lee et al. 2003). Importantly, in vitro experiments have shown that RPA is sufficient for the binding of ATRIP to ssDNA (Zou and Elledge 2003) and that RPA also facilitates the association of the RHR complex with DNA (Ellison and Stillman 2003; Zou et al. 2003).

Interestingly, the amount of ssDNA appears to increase following genotoxic stress, as RPA accumulates on chromatin in Xenopus extracts and mammalian cells treated with UV, MMS, HU, or aphidicolin (Michael et al. 2000; Mimura et al. 2000; Walter 2000; Lupardus et al. 2002; Zou and Elledge 2003). Moreover, in budding yeast, increased amounts of ssDNA have been observed by electron microscopy following HU treatment (Sogo et al. 2002). In the case of DNA damage, the mechanism by which this ssDNA accumulates is not known, nor is it clear if ssDNA accumulation is required for checkpoint activation. In principle, a number of DNA repair (e.g., nucleotide excision repair, base excision repair) and recombination processes could lead to the generation of ssDNA following DNA damage at several points in the cell cycle. Alternatively, during DNA replication, ssDNA could be formed if DNA polymerases are slowed by lesions and the replicative helicase continues to unwind DNA. Indeed, uncoupling of helicase and polymerase activities has been previously observed in the presence of aphidicolin (Walter and Newport 2000), and recent studies have shown that this aphidicolin-induced uncoupling is dependent on the MCM helicase (Pacek and Walter 2004).

In this study, we used a cell-free extract system derived from Xenopus eggs (Walter et al. 1998) to examine the mechanism by which ssDNA accumulates following DNA damage. We demonstrate that the appearance and disappearance of a highly unwound form of plasmid DNA that accumulates following aphidicolin treatment (Walter and Newport 2000) correlates with the phosphorylation of Chk1 on Ser 344 (S344). Importantly, this hyperunwound form of DNA was also observed upon replication of plasmid DNA damaged with either UV or cis-platinum. This suggests that DNA damage induces uncoupling of helicase and polymerase activities and that these lesions, as well as aphidicolin, may generate a common checkpoint-activating DNA structure. Moreover, while stalling the replication fork with aphidicolin results in a robust checkpoint response, we find that aphidicolin elicits no checkpoint when the MCM DNA helicase is inactivated. Using plasmids of varying sizes, we also show that functional uncoupling of DNA unwinding and DNA synthesis during S phase may serve to amplify the level of Chk1 phosphorylation that can be achieved at each individual replication fork. Finally, we demonstrate that although DNA unwinding is necessary for checkpoint activation, it is not sufficient and that additional DNA synthesis by Polα is needed. Taken together, these results suggest that functional uncoupling of helicase and polymerase activities is necessary to convert DNA lesions and chemical inhibitors of DNA replication into the signal(s) that activate the ATR-dependent checkpoint.

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Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint

The MCM proteins are essential replication initiation factors originally identified as proteins required for minichromosome maintenance in Saccharomyces cerevisiae. The best known among them are a family of six structurally related proteins, MCM2-7, which are evolutionally conserved in all eukaryotes. The MCM2-7 proteins form a hexameric complex. This complex is a key component of the prereplication complex that assembles at replication origins during early G1 phase. New evidence suggests that the MCM2-7 proteins may be involved not only in the initiation but also in the elongation of DNA replication. Orchestration of the functional interactions between the MCM2-7 proteins and other components of the prereplication complex by cell cycle-dependent protein kinases results in initiation of DNA synthesis once every cell cycle.

MCM Proteins in DNA Replication

A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

Detection of a novel cell cycle-regulated kinase activity that associates with the amino terminus of the retinoblastoma protein in G2/M phases.

We have shown previouslythat a novel cell cycleregulated histone HI kinase activity, retinoblastoma kinase (RbK), associates with and phosphorylates the amino terminus of the Rb protein in G2-M. We have shown also that the amino terminus of p107, a Rbrelated protein, does not associate with a similar kinase in vftro or in vivo. Here, we report that a RbKlike kinase associates with the amino terminus of p130, another Rb-related protein, only marginally. Moreover, the association of RbK with Rb in vitro is shown to require a discrete portion of the Rb amino terminus, amino acids 89-202. This region has been shown previously to be subject to inactivating mutations in retinoblastoma and to be required for Rb-mediated growth suppression in vitro. Taken together, these data indicate that the formation of Rb-RbK complexes may play an important role in Rb-mediated growth suppression. We have mapped two in vitro sites of Rb phosphorylation by RbK to sites that are phosphorylated in vivo and are targets of cyclindependent kinase phosphorylation in vitro. As such, at least some sites of RbK phosphorylation overlap with those of other proline-directed serine and threonine kinases. Consistent with this latter observation, we report that the trans-activation domain of c-myc is phosphorylated specifically by RbK in vitro at a site (senne 62) that is phosphorylated in vivo during G2-M, cell-cycle phases in which RbK activity is maximal.

http://cgd.aacrjournals.org/cgi/reprint/7/1/53.pdf

The amino terminus of the retinoblastoma (Rb) protein associates with a cyclin-dependent kinase-like kinase via Rb amino acids required for growth suppression

Jacqueline M. Sterner

Papers

Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

Detection of a novel cell cycle-regulated kinase activity that associates with the amino terminus of the retinoblastoma protein in G2/M phases.

The amino terminus of the retinoblastoma (Rb) protein associates with a cyclin-dependent kinase-like kinase via Rb amino acids required for growth suppression