SMART MODE:

NORMAL GENOMIC

• Petkova V et al.
• Interaction between YY1 and the retinoblastoma protein. Regulation of cell cycle progression in differentiated cells.
• J Biol Chem. 2001; 276: 7932-6
• Display abstract

• Overexpression of the transcription factor YY1 activates DNA synthesis in differentiated primary human coronary artery smooth muscle cells. Overexpression of the retinoblastoma protein together with YY1 blocked this effect. In growth-arrested cells, YY1 resides in a complex with the retinoblastoma protein, but the complex is not detected in serum-stimulated S phase cultures, indicating that the interaction of the retinoblastoma protein and YY1 is cell cycle-regulated. Recombinant retinoblastoma protein directly interacts with YY1, destabilizing the interaction of YY1 with DNA and inhibiting its transcription initiator function in vitro. We conclude that in differentiated cells elevation of the nuclear level of YY1 protein favors progression into the S phase, and we propose that this activity is regulated by its interaction with the retinoblastoma protein.

• Kim HY, Ahn BY, Cho Y
• Structural basis for the inactivation of retinoblastoma tumor suppressor by SV40 large T antigen.
• EMBO J. 2001; 20: 295-304
• Display abstract

• Inactivation of the retinoblastoma (Rb) tumor suppressor by Simian virus 40 (SV40) large T antigen is one of the central features of tumorigenesis induced by SV40. Both the N-terminal J domain and the LxCxE motif of large T antigen are required for inactivation of Rb. The crystal structure of the N-terminal region (residues 7-117) of SV40 large T antigen bound to the pocket domain of Rb reveals that large T antigen contains a four-helix bundle, and residues from helices alpha2 and alpha4 and from a loop containing the LxCxE motif participate in the interactions with Rb. The two central helices and a connecting loop in large T antigen have structural similarities with the J domains of the molecular chaperones DnaJ and HDJ-1, suggesting that large T antigen may use a chaperone mechanism for its biological function. However, there are significant differences between large T antigen and the molecular chaperones in other regions and these differences are likely to provide the specificity needed for large T antigen to inactivate Rb.

• Roudier F et al.
• Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein.
• Plant J. 2000; 23: 73-83
• Display abstract

• In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.

• Tannoch VJ, Hinds PW, Tsai LH
• Cell cycle control.
• Adv Exp Med Biol. 2000; 465: 127-40

• Nishiwaki E et al.
• Regulation of CDK7-carboxyl-terminal domain kinase activity by the tumor suppressor p16(INK4A) contributes to cell cycle regulation.
• Mol Cell Biol. 2000; 20: 7726-34
• Display abstract

• The eukaryotic cell cycle is regulated by cyclin-dependent kinases (CDKs). CDK4 and CDK6, which are activated by D-type cyclins during the G(1) phase of the cell cycle, are thought to be responsible for phosphorylation of the retinoblastoma gene product (pRb). The tumor suppressor p16(INK4A) inhibits phosphorylation of pRb by CDK4 and CDK6 and can thereby block cell cycle progression at the G(1)/S boundary. Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II by general transcription factor TFIIH is believed to be an important regulatory event in transcription. TFIIH contains a CDK7 kinase subunit and phosphorylates the CTD. We have previously shown that p16(INK4A) inhibits phosphorylation of the CTD by TFIIH. Here we report that the ability of p16(INK4A) to inhibit CDK7-CTD kinase contributes to the capacity to induce cell cycle arrest. These results suggest that p16(INK4A) may regulate cell cycle progression by inhibiting not only CDK4-pRb kinase activity but also by modulating CDK7-CTD kinase activity. Regulation of CDK7-CTD kinase activity by p16(INK4A) thus may represent an alternative pathway for controlling cell cycle progression.

• Harbour JW, Dean DC
• The Rb/E2F pathway: expanding roles and emerging paradigms.
• Genes Dev. 2000; 14: 2393-409

• Meijer M, Murray JAH
• The role and regulation of D-type cyclins in the plant cell cycle.
• Plant Mol Biol. 2000; 43: 621-33
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• The G1 phase of the cell cycle represents a period of commitment to cell division, both for cells stimulated to resume division from a resting or quiescent state, and for cells involved in repeated cell cycles. During this period. various signals that affect the cells' ability to divide must be assessed and integrated. G1 culminates in the entry of cells into S phase, when DNA replication occurs. In addition, it is likely that several types of differentiation decision may be taken by cells in the G1 phase. In both animals and plants, it appears that D-type cyclins play an important role in the cell cycle responses to external signals, by forming the regulatory subunit of cyclin-dependent kinase complexes. The phosphorylation targets of D-cyclin kinases in mammalian cells are the retinoblastoma (Rb) protein and close relatives. Unphosphorylated Rb can associate with E2F transcription factors, preventing transcription of genes under E2F control until the G1/S boundary is reached. The conservation of Rb and E2F proteins in plants suggests that this pathway is therefore conserved in all higher eukaryotes, although it is absent in fungi and yeasts. Here we review the current understanding of the roles and regulations of D-type (CycD) cyclins in plants.

• Durfee T, Feiler HS, Gruissem W
• Retinoblastoma-related proteins in plants: homologues or orthologues of their metazoan counterparts?
• Plant Mol Biol. 2000; 43: 635-42
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• The mammalian retinoblastoma tumor suppressor protein (pRb) regulates cell division, differentiation and apoptotic pathways in specific cell types. In association with other proteins, pRb acts in part by modulating transcriptional activity. Elements of the pRb regulatory network have been identified in higher plants. Recent findings involving these proteins, which display amino acid sequence homology and biochemical binding properties analogous to their mammalian counterparts, are discussed.

• Chaubet-Gigot N
• Plant A-type cyclins.
• Plant Mol Biol. 2000; 43: 659-75
• Display abstract

• Although the basic mechanisms which control the progression through the cell cycle appear to be conserved in all higher eukaryotes, the unique features of the plant developmental programme must be somehow reflected in a plant-specific regulation of the factors which control cell division. In the past few years, considerable progress has been achieved in identifying the major components of the cell cycle machinery in plants, especially the cyclin-dependent kinases (CDKs) and their regulatory subunits, the cyclins. The question of how these components direct expression of specific genes at specific stages of the cell cycle, and how they are themselves regulated, constitutes a challenge for the present and for the years to come. This review summarizes our current knowledge of a particular class of plant cyclins, the A-type cyclins, which can be further subdivided into three structural groups. The putative functions of these A-type cyclins are discussed in relation to the presence of remarkable motifs in their amino acid sequences, and to their specific transcriptional regulation, protein amount and subcellular localization.

• Fanciulli M et al.
• Identification of a novel partner of RNA polymerase II subunit 11, Che-1, which interacts with and affects the growth suppression function of Rb.
• FASEB J. 2000; 14: 904-12
• Display abstract

• hRPB11 is a core subunit of RNA polymerase II (pol II) specifically down-regulated on doxorubicin (dox) treatment. Levels of this protein profoundly affect cell differentiation, cell proliferation, and tumorigenicity in vivo. Here we describe Che-1, a novel human protein that interacts with hRPB11. Che-1 possesses a domain of high homology with Escherichia coli RNA polymerase final sigma-factor 70 and SV40 large T antigen. In addition, we report that Che-1 interacts with the retinoblastoma susceptibility gene (Rb) by two distinct domains. Functionally, we demonstrate that Che-1 represses the growth suppression function of Rb, counteracting the inhibitory action of Rb on the trans-activation function of E2F1. These results identify a novel protein that binds Rb and the core of pol II, and suggest that Che-1 may be part of transcription regulatory complex.

• Chen XS, Garcea RL, Goldberg I, Casini G, Harrison SC
• Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16.
• Mol Cell. 2000; 5: 557-67
• Display abstract

• The papillomavirus major late protein, L1, forms the pentameric assembly unit of the viral shell. Recombinant HPV16 L1 pentamers assemble in vitro into capsid-like structures, and truncation of ten N-terminal residues leads to a homogeneous preparation of 12-pentamer, icosahedral particles. X-ray crystallographic analysis of these particles at 3.5 A resolution shows that L1 closely resembles VP1 from polyomaviruses. Surface loops contain the sites of sequence variation among HPV types and the locations of dominant neutralizing epitopes. The ease with which small virus-like particles may be obtained from L1 expressed in E. coli makes them attractive candidate components of a papillomavirus vaccine. Their crystal structure also provides a starting point for future vaccine design.

• Higashino F, Yasuda M, Shindoh M
• [The cellular target proteins of adenovirus gene products]
• Tanpakushitsu Kakusan Koso. 2000; 45: 1350-7

• Dick FA, Sailhamer E, Dyson NJ
• Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins.
• Mol Cell Biol. 2000; 20: 3715-27
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• The pocket domain of pRB is required for pRB to arrest the cell cycle. This domain was originally defined as the region of the protein that is necessary and sufficient for pRB's interaction with adenovirus E1A and simian virus s40 large T antigen. These oncoproteins, and other pRB-binding proteins that are encoded by a variety of plant and animal viruses, use a conserved LXCXE motif to interact with pRB. Similar sequences have been identified in multiple cellular pRB-binding proteins, suggesting that the viruses have evolved to target a highly conserved binding site of pRB that is critical for its function. Here we have constructed a panel of pRB mutants in which conserved amino acids that are predicted to make close contacts with an LXCXE peptide were altered. Despite the conservation of the LXCXE binding site throughout evolution, pRB mutants that lack this site are able to induce a cell cycle arrest in a pRB-deficient tumor cell line. This G(1) arrest is overcome by cyclin D-cdk4 complexes but is resistant to inactivation by E7. Consequently, mutants lacking the LXCXE binding site were able to induce a G(1) arrest in HeLa cells despite the expression of HPV-18 E7. pRB mutants lacking the LXCXE binding site are defective in binding to adenovirus E1A and human papillomavirus type 16 E7 protein but exhibit wild-type binding to E2F or DP, and they retain the ability to interact with CtIP and HDAC1, two transcriptional corepressors that contain LXCXE-like sequences. Consistent with these observations, the pRB mutants are able to actively repress transcription. These observations suggest that viral oncoproteins depend on the LXCXE-binding site of pRB for interaction to a far greater extent than cellular proteins that are critical for cell cycle arrest or transcriptional repression. Mutation of this binding site allows pRB to function as a cell cycle regulator while being resistant to inactivation by viral oncoproteins.

• Saudan P, Vlach J, Beard P
• Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb.
• EMBO J. 2000; 19: 4351-61
• Display abstract

• Adeno-associated virus (AAV) has an antiproliferative action on cells. We investigated the effect of the AAV replication proteins (Rep) on the cell division cycle using retroviral vectors. Rep78 and Rep68 inhibited the growth of primary, immortalized and transformed cells, while Rep52 and Rep40 did not. Rep68 induced cell cycle arrest in phases G(1) and G(2), with elevated CDK inhibitor p21 and reduced cyclin E-, A- and B1-associated kinase activity. Rep78-expressing cells were also impaired in S-phase progression and accumu lated almost exclusively with hypophosphorylated retinoblastoma protein (pRb). The differences between Rep78 and Rep68 were mapped to the C-terminal zinc finger domain of Rep78. Rep78-induced S-phase arrest could be bypassed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 mutants unable to bind pRb. Rb(-/-) primary mouse embryonic fibroblasts displayed a strongly reduced S-phase arrest when challenged with Rep78, compared with matched Rb(+/+) controls. These results suggest that physiological levels of active pRb can interfere with S-phase progression. We propose that the AAV Rep78 protein arrests cells within S-phase by a novel mechanism involving the ectopic accumulation of active pRb.

• Vandel L, Kouzarides T
• Residues phosphorylated by TFIIH are required for E2F-1 degradation during S-phase.
• EMBO J. 1999; 18: 4280-91
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• The transcription factor E2F-1 plays a key role in regulating cell cycle progression. Accordingly, E2F-1 activity is itself tightly controlled by a series of transcriptional and post-transcriptional events. Here we show that the E2F-1 activation domain interacts with a kinase activity which phosphorylates two sites, Ser403 and Thr433, within the activation domain. We demonstrate that TFIIH is responsible for the E2F-1 phosphorylation observed in cell extracts and that endogenous E2F-1 interacts in vivo with p62, a component of TFIIH, during S phase. When the two phosphorylation sites in E2F-1 are mutated to alanine, the stability of the E2F-1 activation domain is greatly increased. These results suggest that TFIIH-mediated phosphorylation of E2F-1 plays a role in triggering E2F-1 degradation during S phase.

• Brehm A et al.
• The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth.
• EMBO J. 1999; 18: 2449-58
• Display abstract

• E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.

• Ito N, Rubin GM
• gigas, a Drosophila homolog of tuberous sclerosis gene product-2, regulates the cell cycle.
• Cell. 1999; 96: 529-39
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• Tuberous sclerosis complex (TSC) is an autosomal dominant disorder leading to the widespread development of benign tumors that often contain giant cells. We show that the Drosophila gene gigas encodes a homolog of TSC2, a gene mutated in half of TSC patients. Clones of gigas mutant cells induced in imaginal discs differentiate normally to produce adult structures. However, the cells in these clones are enlarged and repeat S phase without entering M phase. Our results suggest that the TSC disorder may result from an underlying defect in cell cycle control. We have also identified a Drosophila homolog of TSC1.

• Lania L, Majello B, Napolitano G
• Transcriptional control by cell-cycle regulators: a review.
• J Cell Physiol. 1999; 179: 134-41
• Display abstract

• In eukaryotes, progression of the cell cycle is associated with periodic transcription activation/repression of growth-regulatory genes. We summarize here current knowledge and views on the role of critical cell-cycle regulators such as the retinoblastoma pocket family members and cyclin-dependent kinases in the regulation of gene transcription. In particular, we discuss here the role of specific cyclin-dependent kinase complexes in the regulation of basal transcription. Although the functional connections between transcription and cell-cycle regulators is far from being understood, recent progress has been made in connecting cell-cycle progression to dedicated components of the RNA polymerase II transcription apparatus complex.

• Nakagami H, Sekine M, Murakami H, Shinmyo A
• Tobacco retinoblastoma-related protein phosphorylated by a distinct cyclin-dependent kinase complex with Cdc2/cyclin D in vitro.
• Plant J. 1999; 18: 243-52
• Display abstract

• The retinoblastoma (Rb) protein was originally identified as a product of a tumour suppressor gene that plays a pivotal role in regulating both the cell cycle and differentiation in mammals. The growth-suppressive activity of Rb is regulated by phosphorylation with cyclin-dependent kinase (CDK), and inactivation of the Rb function is one of the critical steps for transition from the G1 to the S phase. We report here the cloning of a cDNA (NtRb1) from Nicotiana tabacum which encodes a Rb-related protein, and show that this gene is expressed in all the organs examined at the mRNA level. We have demonstrated that NtRb1 interacts with tobacco cyclin D by using yeast two-hybrid and in vitro binding assays. In mammals, cyclin D can assemble with CDK4 and CDK6, but not with Cdc2, to form active complexes. Surprisingly, tobacco cyclin D and Cdc2 proteins can form a complex in insect cells, which is able to phosphorylate tobacco Rb-related protein in vitro. Using immunoprecipitation with the anti-cyclin D anti-body, cyclin D can be found in a complex with Cdc2 in suspension-cultured tobacco BY-2 cells. These results suggest that the cdc2 gene modulates the cell cycle through the phosphorylation of Rb-related protein by forming an active complex with cyclin D in plants.

• Johnstone RW, Trapani JA
• Transcription and growth regulatory functions of the HIN-200 family of proteins.
• Mol Cell Biol. 1999; 19: 5833-8

• Wang MH, Doonan JH, Sastry GR
• Cloning and characterization of the unusual cyclin gene from an amphidiploid of Nicotiana glauca-Nicotiana langsdorffii hybrid.
• Biochim Biophys Acta. 1999; 1489: 399-404
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• Cyclins play an important role in the regulation of cell cycle progression in eukaryotic cells. As an aid to understanding the molecular nature of unregulated cell proliferation, a cDNA clone encoding a cyclin gene, GTcyc, was identified from genetic tumors. The clone contained 1095 bp including a 24 base poly(A) tail. GTcyc is an unusual cyclin gene, distantly related to mammalian cyclin D genes having 21-25% identity within the cyclin box. Northern blots showed that the genetic tumors express high levels of GTcyc relative to non-tumor hybrid tissues. Southern analysis suggests that GTcyc may be contained one or two families in genetic tumors.

• Suske G
• The Sp-family of transcription factors.
• Gene. 1999; 238: 291-300
• Display abstract

• GC-boxes and related motifs are frequently occurring DNA-elements present in many promoters and enhancers. In contrast to other elements it was generally thought that the transcription factor Sp1 is the only factor acting through these motifs. The cloning of paralogous genes of the Sp1 factor uncovered the existence of a small protein family consisting of Sp1, Sp2, Sp3 and Sp4. All four proteins exhibit very similar structural features. They contain a highly conserved DNA-binding domain composed of three zinc fingers close the C-terminus and serine/threonine- and glutamine-rich domains in their N-terminal regions. The high degree of structural conservation between these four proteins suggested that they do exert similar functions. Molecular, genetic and biochemical analyses, however, demonstrated that Sp2, Sp3 and Sp4 are not simply functional equivalents of Sp1. Here, I will summarize and discuss recent advances which have been made towards understanding the mode of action and biological function of individual family members.

• Foord R, Taylor IA, Sedgwick SG, Smerdon SJ
• X-ray structural analysis of the yeast cell cycle regulator Swi6 reveals variations of the ankyrin fold and has implications for Swi6 function.
• Nat Struct Biol. 1999; 6: 157-65
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• Swi6 is a 92,000 Mr protein common to two distinct transcriptional activation complexes (SBF and MBF) that coordinate gene expression at the G1-S boundary of the yeast cell cycle. The X-ray structure of a central 36,000 Mr fragment has been determined and refined at 2.1 A resolution. The structure reveals a basic framework of five ankyrin repeat modules that is elaborated through a series of helical insertions distinguishing it from structures of other ankyrin repeat proteins. A second domain contains an approximately 30-residue region of extended structure that interacts with the ankyrin repeat core over a substantial proportion of its surface. Conservation of residues buried by these interactions indicates that all members of the Swi6/Cdc10 family share a similar architecture. Several temperature-sensitive mutations within Swi6 and Cdc10 appear to disrupt these interdomain contacts rather than destabilize the ankyrin repeat core. The unusual domain arrangement may be crucial for the modulation of interactions with other co-regulatory molecules such as cyclin-CDK complexes, and has implications for the quaternary interactions within the multisubunit SBF and MBF transcription complexes.

• Markmann-Mulisch U, Reiss B, Mulisch M
• Cell type-specific gene expression in the cell cycle of the dimorphic ciliate Eufolliculina uhligi.
• Mol Gen Genet. 1999; 262: 390-9
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• The life cycle of the unicellular eukaryote Eufolliculina uhligi includes two structurally and physiologically different cell types: a motile swarmer that is arrested in the cell cycle, and a sessile cell (trophont) that feeds and reproduces. These two cell types offer an exceptionally favourable system for the isolation of genes involved in cell cycle regulation and cellular morphogenesis. Differential screening of a trophont cDNA library using a swarmer-subtracted, trophont-specific probe yielded eleven clones that represent trophont-specific transcripts and one clone that represents a swarmer-specific transcript. Sequence analysis showed that seven clones, including the only swarmer-specific one, represent unknown genes, whereas five clones could be identified by sequence comparisons. Two of the clones appear to encode proteins that are involved in the regulation of growth and metabolism. The deduced sequences of three clones resemble potential cell cycle regulators. Data are presented on a putative member of the calcium/calmodulin-dependent protein kinase family and on a TIP120-like sequence, which is the first such sequence to be described since the discovery of the rat TIP120 protein. Furthermore, a unique new sequence is presented, whose features suggest that it represents a protein that is involved in the regulation of cell division. It includes domains characteristic of two different protein families, cyclin-dependent kinases (CDKs) and cyclins, both of which are known to be cell cycle regulators. Based on our results we propose a model for cell cycle regulation in ciliated protozoa.

• Magnaghi-Jaulin L et al.
• Retinoblastoma protein represses transcription by recruiting a histone deacetylase.
• Nature. 1998; 391: 601-5
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• The retinoblastoma tumour-suppressor protein Rb inhibits cell proliferation by repressing a subset of genes that are controlled by the E2F family of transcription factors and which are involved in progression from the G1 to the S phase of the cell cycle. Rb, which is recruited to target promoters by E2F1, represses transcription by masking the E2F1 transactivation domain and by inhibiting surrounding enhancer elements, an active repression that could be crucial for the proper control of progression through the cell cycle. Some transcriptional regulators act by acetylating or deacetylating the tails protruding from the core histones, thereby modulating the local structure of chromatin: for example, some transcriptional repressors function through the recruitment of histone deacetylases. We show here that the histone deacetylase HDAC1 physically interacts and cooperates with Rb. In HDAC1, the sequence involved is an LXCXE motif, similar to that used by viral transforming proteins to contact Rb. Our results strongly suggest that the Rb/HDAC1 complex is a key element in the control of cell proliferation and differentiation and that it is a likely target for transforming viruses.

• Figarella-Branger D, Bouvier-Labit C, Civatte M, Pellissier JF
• [Cell cycle regulators and cancer]
• Ann Pathol. 1998; 18: 178-86

• Larminie CG, White RJ
• Identification of a putative BRF homologue in the genome of Caenorhabditis elegans.
• DNA Seq. 1998; 9: 49-58
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• We have identified a putative gene within the Caenorhabditis elegans genome which has the potential to encode a protein homologous to BRF, an RNA polymerase III general transcription factor. The predicted protein shares very similar overall structure with human and yeast BRF. In particular, its N-terminal half comprises a zinc-ribbon motif and a TR domain which is also present in the cyclin box. The C. elegans protein is more similar to human BRF than to the yeast BRF proteins, as would be expected from an evolutionary standpoint. Alignment of the C. elegans protein with the four known BRF proteins reveals two blocks conserved between all five sequences within the diverged C-terminal region. Profile searches using these regions suggest that they may contain evolutionarily conserved motifs. These comparisons provide insight into the structure and function of an important transcription factor.

• Nevins JR
• Toward an understanding of the functional complexity of the E2F and retinoblastoma families.
• Cell Growth Differ. 1998; 9: 585-93

• Fu YH, Nishinaka T, Yokoyama K, Chiu R
• A retinoblastoma susceptibility gene product, RB, targeting protease is regulated through the cell cycle.
• FEBS Lett. 1998; 421: 89-93
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• Degradation of cyclin B and cyclin-dependent kinase inhibitor, p27, at a specific time has been shown to play a critical role in regulating the cell cycle. SPase, a nuclear and cytosol protease with cathepsin B- and L-like proteolytic activity, has been identified in several cell lines. This proteolytic enzyme selectively degraded nuclear proteins such as retinoblastoma susceptibility gene product, RB, and transcription factor, SP-1. High levels of SPase activity were detected at the G1/S, moderate levels at the G1 and S phases, and undetectable activity at the M phase of synchronized CV-1 cells, suggesting that SPase activity is regulated through the cell cycle. Degradation of RB correlated with SPase activity throughout the cell cycle, suggesting that SPase regulates RB, which has a functional role in regulating cell cycle. These results demonstrated that SPase plays an integral role in regulating the nuclear regulator, RB, in controlling cell cycle progression.

• Knudsen ES, Wang JY
• Hyperphosphorylated p107 and p130 bind to T-antigen: identification of a critical regulatory sequence present in RB but not in p107/p130.
• Oncogene. 1998; 16: 1655-63
• Display abstract

• The retinoblastoma tumor suppressor RB and its related proteins, p107 and p130, are targets of several viral oncoproteins, including SV40 large T-antigen (T-Ag) and adenovirus E1A. T-Ag and E1A each contains an LXCXE-peptide motif through which binding to the conserved 'A/B pocket' present in RB, p107 and p130 is achieved. The LXCXE-binding activity of RB, we have previously shown, is inhibited by phosphorylation at Thr 821 and 826 in the C-terminal region of RB. Thr 821 and its surrounding sequence is unique to RB and not found in p107 or p130. Interestingly, hyperphosphorylation of p107 does not disrupt its ability to bind T-Ag or to inhibit the transactivating function of E1A. Insertion of a fourteen amino acid sequence of RB containing Thr 821 into p107 prevents binding of T-Ag and E1A to phosphorylated p107. These results show that the RB sequence surrounding Thr 821 plays a critical role in the regulation of the 'A/B pocket' function. In addition, these data indicate that the protein binding functions of RB and p107 are not equivalently regulated by phosphorylation.

• Gutierrez C
• The retinoblastoma pathway in plant cell cycle and development.
• Curr Opin Plant Biol. 1998; 1: 492-7
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• The activity of cyclin-dependent kinases (CDKs) on specific targets mediates the temporal regulation of plant cell cycle transitions. The sequential activity of CDKs and the spatial regulation of cell proliferation during plant development, however, are still poorly understood. Understanding these aspects depends on the identification of the downstream targets and upstream modulators of CDKs and their regulation in response to mitogenic and/or differentiation signals. Current efforts to elucidate the answers to these questions are very promising; in particular, recent works reveal the essential role that the retinoblastoma pathway plays in controlling cell cycle progression and, presumably, some developmental events.

• Bravo J, Staunton D, Heath JK, Jones EY
• Crystal structure of a cytokine-binding region of gp130.
• EMBO J. 1998; 17: 1665-74
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• The structure of the cytokine-binding homology region of the cell surface receptor gp130 has been determined by X-ray crystallography at 2.0 A resolution. The beta sandwich structure of the two domains conforms to the topology of the cytokine receptor superfamily. This first structure of an uncomplexed receptor exhibits a similar L-shaped quaternary structure to that of ligand-bound family members and suggests a limited flexibility in relative domain orientation of some 3 degrees. The putative ligand-binding loops are relatively rigid, with a phenylalanine side chain similarly positioned to exposed aromatic residues implicated in ligand binding for other such receptors. The positioning and structure of the N-terminal portion of the polypeptide chain have implications for the structure and function of cytokine receptors, such as gp130, which contain an additional N-terminal immunoglobulin-like domain.

• Brehm A, Miska EA, McCance DJ, Reid JL, Bannister AJ, Kouzarides T
• Retinoblastoma protein recruits histone deacetylase to repress transcription.
• Nature. 1998; 391: 597-601
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• The retinoblastoma protein (Rb) silences specific genes that are active in the S phase of the cell cycle and which are regulated by E2F transcription factors. Rb binds to the activation domain of E2F and then actively represses the promoter by a mechanism that is poorly understood. Here we show that Rb associates with a histone deacetylase, HDAC1, through the Rb 'pocket' domain. Association with the deacetylase is reduced by naturally occurring mutations in the pocket and by binding of the human papilloma virus oncoprotein E7. We find that Rb can recruit histone deacetylase to E2F and that Rb cooperates with HDAC1 to repress the E2F-regulated promoter of the gene encoding the cell-cycle protein cyclin E. Inhibition of histone deacetylase activity by trichostatin A (TSA) inhibits Rb-mediated repression of a chromosomally integrated E2F-regulated promoter. Our results indicate that histone deacetylases are important for regulating the cell cycle and that active transcriptional repression by Rb may involve the modification of chromatin structure.

• Kelly BL, Wolfe KG, Roberts JM
• Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein.
• Proc Natl Acad Sci U S A. 1998; 95: 2535-40
• Display abstract

• Considerable advances have been made in characterizing the cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through the cell cycle, but there has been relatively lesser success in identifying the specific biochemical pathways and cell cycle events that are directly under CDK control. To identify physiologically significant CDK substrates we generated mutations in cyclin E that altered the ability of the cyclin to direct the cyclin-CDK holoenzyme to specific in vivo substrates. We show that one of these mutations defines a domain in cyclin E necessary for phosphorylation of the retinoblastoma protein (Rb). These observations confirm the idea that cyclins contribute to substrate recognition by cyclin-CDK complexes, demonstrate the utility of targeting mutants in the identification of essential cyclin-CDK substrates, and put cyclin E squarely into the family of proteins designed to regulate Rb.

• Norton JD, Deed RW, Craggs G, Sablitzky F
• Id helix-loop-helix proteins in cell growth and differentiation.
• Trends Cell Biol. 1998; 8: 58-65
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• Id helix-loop-helix proteins function at a general level as positive regulators of cell growth and as negative regulators of cell differentiation. They act as dominant-negative antagonists of other helix-loop-helix transcription factors, which drive cell lineage commitment and differentiation in diverse cell types of higher eukaryotes. In addition, the functions of Id proteins are integrated with cell-cycle-regulatory pathways orchestrated by cyclin-dependent kinases and the retinoblastoma protein. Here, some of the recent advances that highlight the importance of Id proteins as regulatory intermediates for coordinating differentiation-linked gene expression with cell-cycle control in response to extracellular signalling are reviewed.

• Trimarchi JM, Fairchild B, Verona R, Moberg K, Andon N, Lees JA
• E2F-6, a member of the E2F family that can behave as a transcriptional repressor.
• Proc Natl Acad Sci U S A. 1998; 95: 2850-5
• Display abstract

• The E2F family of proteins is required to establish the correct cell-cycle-dependent transcription of genes that direct the process of cell division. All previously identified E2F proteins can act in a similar manner; depending on whether or not they are associated with the cell cycle inhibitors the retinoblastoma protein (pRB), p107, or p130, they can either repress or activate the transcription of E2F-responsive genes. We now report the cloning and characterization of another E2F family member, E2F-6, whose structure is reminiscent of the dominant inhibitors of other transcription factor families. The dimerization and DNA binding properties of E2F-6 are similar to those of the other E2F family members. However, it is not regulated by pRB, p107, or p130, and it is unable to activate transcription. Instead, it can act to repress the transcription of E2F responsive genes by countering the activity of the other E2F complexes via a pRB-, p107-, or p130-independent mechanism.

• Trouche D
• [Control of cell proliferation by the retinoblastoma gene product]
• Pathol Biol (Paris). 1997; 45: 5-8
• Display abstract

• Transformed cells proliferate abnormally, due to the unregulated activation of oncogenes and the inactivation of anti-oncogenes. The molecular mechanisms by which the product of the RB anti-oncogene, the RB protein, regulates cell proliferation begin to be understood. Major targets of RB include proteins involved in cell cycle entry, like the E2F transcription factor, and effectors of terminal differentiation. The effect of RB is thus to block cells into the G1 phase of the cell cycle and to induce them to terminally differentiate. Recently, a new role has been shown for RB. RB is able to repress the activity of the RNA polymerases I and III, thereby modulating the protein biosynthesis capacities of the cell. RB appears thus to control directly the balance between DNA and protein synthesis during the cell cycle.

• Berezutskaya E, Yu B, Morozov A, Raychaudhuri P, Bagchi S
• Differential regulation of the pocket domains of the retinoblastoma family proteins by the HPV16 E7 oncoprotein.
• Cell Growth Differ. 1997; 8: 1277-86
• Display abstract

• The human papillomavirus E7 oncoprotein binds to the retinoblastoma (Rb) tumor suppressor protein, and the binding to Rb correlates with the oncogenic potential of E7. Recent studies from several laboratories indicated that the half-life of the Rb protein is reduced in cells that are stably transformed with E7, suggesting that E7 could induce the proteolytic degradation of Rb. To investigate whether the Rb degradation is a primary effect of E7 or a result of altered cell phenotype, we sought to develop assays that can distinguish between the two possibilities. Using recombinant adenovirus expressing the human papillomavirus type 16 E7 protein, we show that the expression of E7 leads to an increased rate of decay of the Rb protein. Moreover, Rb degradation immediately follows the expression of E7 suggesting that it is an early and primary effect. Consistent with a previous study, we observed that the E7-induced degradation of Rb can be blocked by the inhibitors of the 26S proteasome. We have also developed a transient transfection assay for the E7-induced degradation of Rb. Using this assay, we show that the pocket domain of Rb is necessary and sufficient for the E7-induced degradation. However, the proteolysis is relatively specific for Rb because the level of p107 or p130 was not significantly altered by the expression of E7. Thus, although E7 binds to all three members of the Rb family of proteins, the proteolysis is much more efficient in the case of Rb. In the transient transfection assays, adenovirus E1A and SV40 large T antigen failed to induce degradation of Rb, suggesting that the Rb degradation is a unique property of the E7 oncoprotein.

• Doonan J, Fobert P
• Conserved and novel regulators of the plant cell cycle.
• Curr Opin Cell Biol. 1997; 9: 824-30
• Display abstract

• Cell division is highly regulated, both spatially and temporally, during plant development. Recent evidence implicates cyclin-dependent kinases (cdks) and their associated proteins as the principal temporal regulators of cell division. It is now known that plants contain an extended family of cdks, some of which appear to be unique to this group. Positive rate-limiting regulators of cell proliferation and growth include mitotic or B-type cyclins whose transcription is restricted to the G2 and M phases. Current research suggests that MYB-related transcription factors may be responsible for this restriction. Cdk-interacting proteins, such as cdk inhibitors and suc1 homologues, have been isolated using yeast two-hybrid approaches.

• Nevins JR, Leone G, DeGregori J, Jakoi L
• Role of the Rb/E2F pathway in cell growth control.
• J Cell Physiol. 1997; 173: 233-6

• Trouche D, Le Chalony C, Muchardt C, Yaniv M, Kouzarides T
• RB and hbrm cooperate to repress the activation functions of E2F1.
• Proc Natl Acad Sci U S A. 1997; 94: 11268-73
• Display abstract

• Forced expression of the retinoblastoma (RB) gene product inhibits the proliferation of cells in culture. A major target of the RB protein is the S-phase-inducing transcription factor E2F1. RB binds directly to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To induce complete G1 arrest, RB requires the presence of the hbrm/BRG-1 proteins, which are components of the coactivator SWI/SNF complex. This cooperation is mediated through a physical interaction between RB and hbrm/BRG-1. We show here that in transfected cells RB can contact both E2F1 and hbrm at the same time, thereby targeting hbrm to E2F1. E2F1 and hbrm are indeed found within the same complex in vivo. Furthermore, RB and hbrm cooperate to repress E2F1 activity in transient transfection assays. The ability of hbrm to cooperate with RB to repress E2F1 is dependent upon several distinct domains of hbrm, including the RB binding domain and the NTP binding site. However, the bromodomain seems dispensable for this activity. Taken together, our results point out an unexpected role of corepressor for the hbrm protein. The ability of hbrm and RB to cooperate in repressing E2F1 activity could be an underlying mechanism for the observed cooperation between hbrm and RB to induce G1 arrest. Finally, we demonstrate that the domain of hbrm that binds RB has transcriptional activation potential which RB can repress. This suggest that RB not only targets hbrm but also regulates its activity.

• Mayol X, Grana X
• pRB, p107 and p130 as transcriptional regulators: role in cell growth and differentiation.
• Prog Cell Cycle Res. 1997; 3: 157-69
• Display abstract

• The mammalian cell cycle engine, which is composed of cyclin/CDK holoenzymes, controls the progression throughout the cell cycle by regulating, at least in part, the transcription of two types of genes: genes whose protein products are required for DNA metabolism and genes whose protein products are involved in cell cycle control. Among the targets of cyclin/CDKs, there is a family of negative growth regulators collectively known as pocket proteins. This family of pocket proteins includes the product of the retinoblastoma tumor suppressor gene, pRB and the functionally and structurally related proteins p107 and p130. In this review, the mechanisms by which pocket proteins are thought to regulate cell growth and differentiation are discussed.

• Peeper DS et al.
• Ras signalling linked to the cell-cycle machinery by the retinoblastoma protein.
• Nature. 1997; 386: 177-81
• Display abstract

• The Ras proto-oncogene is a central component of mitogenic signal-transduction pathways, and is essential for cells both to leave a quiescent state (G0) and to pass through the G1/S transition of the cell cycle. The mechanism by which Ras signalling regulates cell-cycle progression is unclear, however. Here we report that the retinoblastoma tumour-suppressor protein (Rb), a regulator of G1 exit, functionally links Ras to passage through the G1 phase. Inactivation of Ras in cycling cells caused a decline in cyclin D1 protein levels, accumulation of the hypophosphorylated, growth-suppressive form of Rb, and G1 arrest. When Rb was disrupted either genetically or biochemically, cells failed to arrest in G1 following Ras inactivation. In contrast, inactivation of Ras in quiescent cells prevented growth-factor induction of both immediate-early gene transcription and exit from G0 in an Rb-independent manner. These data suggest that Rb is an essential G1-specific mediator that links Ras-dependent mitogenic signalling to cell-cycle regulation.

• Mavromatis KO, Jones DL, Mukherjee R, Yee C, Grace M, Munger K
• The carboxyl-terminal zinc-binding domain of the human papillomavirus E7 protein can be functionally replaced by the homologous sequences of the E6 protein.
• Virus Res. 1997; 52: 109-18
• Display abstract

• The carboxyl-terminus is necessary for the functional and structural integrity of the human papillomavirus (HPV) E7 oncoprotein. Since many mutations in this domain of E7 result in the formation of unstable proteins, we have evaluated the importance of this region by replacing it with structurally related domains derived from HPV E6 proteins. Biological analysis of these mutant chimeric E7/E6 proteins showed that they retained E7-specific biological activities including cooperation with the ras oncogene to transform primary baby rat kidney cells and transcriptional activation of an E2F responsive reporter plasmid. One of the chimeric proteins was impaired in its ability to physically disrupt pRB/E2F complexes in vitro suggesting that there are defined molecular determinants in the carboxyl-terminus of E7 for this activity. In contrast, none of these proteins exhibited E6-like properties including binding to p53 and/or degradation of associated proteins.

• Lee WH, Lee EY
• [The retinoblastoma gene: from its basic understanding as a signal mediator for growth and differentiation to its use in the treatment of cancer]
• Gan To Kagaku Ryoho. 1997; 24: 1368-80
• Display abstract

• The retinoblastoma gene (RB) plays important roles in the genesis of human cancers. Several pieces of evidence have shown that the retinoblastoma protein (Rb) has dual roles in gating cell cycle progression and promoting cellular differentiation. The molecular mechanisms involved in these roles have been elucidated in some biological systems: Rb sequesters the transcription factors of E2F-1 to regulate entry of cell cycle, but enhances the activities of transcription factors such as C/ EBPs to initiate terminal differentiation. Thus, the Rb protein can serve as a mediator of extracellular signals for growth or differentiation. RB also has a tumor suppression function. Introduction of wild-type RB into human tumor cells deficient for RB suppresses their tumorigenicity in nude mice. Similarly, mice with RB+/- genotypes created by gene knockout methods spontaneously develop melanotrophic tumors with complete penetrance. Immune-competent RB+/- mice benefited significantly from adenovirus-mediated RB gene therapy. The fundamental knowledge of Rb function will allow us to take the next steps toward the use of tumor suppressor genes in the treatment of human cancer.

• Wang JY
• Retinoblastoma protein in growth suppression and death protection.
• Curr Opin Genet Dev. 1997; 7: 39-45
• Display abstract

• Puzzling new information indicates an inadequacy in our understanding of the retinoblastoma protein (RB). RB and the transcription factor E2F appear to be collaborators. RB-E2F interaction is necessary but not sufficient for growth suppression. Unbecoming of a tumor suppressor, RB has an active role in antagonizing the death response. How RB integrates its multiple functions into a tumor suppression program is still an open issue.

• Shi Y, Hata A, Lo RS, Massague J, Pavletich NP
• A structural basis for mutational inactivation of the tumour suppressor Smad4.
• Nature. 1997; 388: 87-93
• Display abstract

• The Smad4/DPC4 tumour suppressor is inactivated in nearly half of pancreatic carcinomas and to a lesser extent in a variety of other cancers. Smad4/DPC4, and the related tumour suppressor Smad2, belong to the SMAD family of proteins that mediate signalling by the TGF-beta/activin/BMP-2/4 cytokine superfamily from receptor Ser/Thr protein kinases at the cell surface to the nucleus. SMAD proteins, which are phosphorylated by the activated receptor, propagate the signal, in part, through homo- and hetero-oligomeric interactions. Smad4/DPC4 plays a central role as it is the shared hetero-oligomerization partner of the other SMADs. The conserved carboxy-terminal domains of SMADs are sufficient for inducing most of the ligand-specific effects, and are the primary targets of tumorigenic inactivation. We now describe the crystal structure of the C-terminal domain (CTD) of the Smad4/DPC4 tumour suppressor, determined at 2.5 A resolution. The structure reveals that the Smad4/DPC4 CTD forms a crystallographic trimer through a conserved protein-protein interface, to which the majority of the tumour-derived missense mutations map. These mutations disrupt homo-oligomerization in vitro and in vivo, indicating that the trimeric assembly of the Smad4/DPC4 CTD is critical for signalling and is disrupted by tumorigenic mutations.

• Jones DL, Munger K
• Interactions of the human papillomavirus E7 protein with cell cycle regulators.
• Semin Cancer Biol. 1996; 7: 327-37
• Display abstract

• Human papillomavirus (HPVs) critically depend on the cellular machinery for the replication of their genome. Viral replication is restricted to the differentiated strata of the skin that are normally growth arrested. Hence, the HPVs have developed strategies to subvert cellular growth regulatory pathways and are able to uncouple cellular proliferation and differentiation. The HPV E7 protein can overcome the activity of some cyclin-dependent kinase inhibitors, associate with cyclin/cyclin dependent kinase complexes and bind to and destabilize the retinoblastoma tumor suppressor protein. These biological activities contribute to the carcinogenic potential of the high risk HPV E7 proteins which are consistently expressed in HPV-positive cervical carcinomas.

• Ikeda MA, Jakoi L, Nevins JR
• A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation.
• Proc Natl Acad Sci U S A. 1996; 93: 3215-20
• Display abstract

• Examination of the interactions involving transcription factor E2F activity during cell growth and terminal differentiation suggests distinct roles for Rb family members in the regulation of E2F accumulation. The major species of E2F in quiescent cells is a complex containing the E2F4 product in association with the Rb-related p130 protein. As cells enter the cell cycle, this complex disappears, and there is a concomitant accumulation of free E2F activity of which E2F4 is a major component. E2F4 then associates with the Rb-related p107 protein as cells enter S phase. Rb can be found in interactions with each E2F species, including E2F4, during G1, but there appears to be a limited amount of Rb with respect to E2F, likely due to the maintenance of most Rb protein in an inactive state by phosphorylation. A contrasting circumstance can be found during the induction of HL60 cell differentiation. As these cells exit the cell cycle, active Rb protein appears to exceed E2F, as there is a marked accumulation of E2F-Rb interactions, involving all E2F species, including E2F4, which is paralleled by the conversion of Rb from a hyperphosphorylated state to a hypophosphorylated state. These results suggest that the specific ability of Rb protein to interact with each E2F species, dependent on concentration of active Rb relative to accumulation of E2F, may be critical in cell-growth decisions.

• Chow KN, Dean DC
• Domains A and B in the Rb pocket interact to form a transcriptional repressor motif.
• Mol Cell Biol. 1996; 16: 4862-8
• Display abstract

• The retinoblastoma protein (Rb) is a tumor suppressor that regulates progression from the G1 phase to the S phase of the cell cycle. Previously, we found that Rb is a transcriptional repressor that is selectively targeted to promoters through an interaction with the E2F family of cell cycle transcription factors--when Rb is tethered to a promoter through E2F, it not only blocks E2F activity, it also binds surrounding transcription factors, preventing their interaction with the basal transcription complex, thus resulting in a dominant inhibitory effect on transcription of cell cycle genes. Here we examine the repressor motif of Rb. The two domains in the Rb pocket, A and B, which are conserved across species and in the Rb-related proteins p107 and p130, are both required for repressor activity. The nonconserved spacer separating A and B is not required. Although neither A nor B alone had any repressor activity, surprisingly, repressor activity was observed when the domains were coexpressed on separate proteins. Transfection assays suggest that one domain can recruit the other to the promoter to form a repressor motif that can both interact with E2F and have a dominant inhibitory effect on transcription. Using coimmunoprecipitation and in vitro binding assays, we show that A and B interact directly and that mutations which disrupt this interaction inhibit repressor activity. The Rb pocket was originally defined as the binding site for oncoproteins from DNA tumor viruses such as adenovirus E1a. We present evidence that E1a interacts with a site formed by the interaction of A and B and that this interaction with A and B induces or stabilizes the A-B interaction.

• Zhu W, Zeng Q, Colangelo CM, Lewis M, Summers MF, Scott RA
• The N-terminal domain of TFIIB from Pyrococcus furiosus forms a zinc ribbon.
• Nat Struct Biol. 1996; 3: 122-4
• Display abstract

• The three-dimensional structure of the N-terminal domain of an archaeal TFIIB, which has high sequence homology with eucaryal analogues, is strikingly similar to that of the C-terminal zinc ribbon of the eucaryal transcription elongation factor TFIIB.

• Hirai H, Sherr CJ
• Interaction of D-type cyclins with a novel myb-like transcription factor, DMP1.
• Mol Cell Biol. 1996; 16: 6457-67
• Display abstract

• The cyclin D-dependent kinases CDK4 and CDK6 trigger phosphorylation of the retinoblastoma protein (RB) late in G1 phase, helping to cancel its growth-suppressive function and thereby facilitating S-phase entry. Although specific inhibition of cyclin D-dependent kinase activity in vivo can prevent cells from entering S phase, it does not affect S-phase entry in cells lacking functional RB, implying that RB may be the only substrate of CDK4 and CDK6 whose phosphorylation is necessary for G1 exit. Using a yeast two-hybrid interactive screen, we have now isolated a novel cyclin D-interacting myb-like protein (designated DMP1), which binds specifically to the nonamer DNA consensus sequences CCCG(G/T)ATGT to activate transcription. A subset of these DMP1 recognition sequences containing a GGA trinucleotide core can also function as Ets-responsive elements. DMP1 mRNA and protein are ubiquitously expressed throughout the cell cycle in mouse tissues and in representative cell lines. DMP1 binds to D-type cyclins directly in vitro and when coexpressed in insect Sf9 cells. In both settings, it can be phosphorylated by cyclin D-dependent kinases, suggesting that its transcriptional activity may normally be regulated through such mechanisms. These results raise the possibility that cyclin D-dependent kinases regulate gene expression in an RB independent manner, thereby serving to link other genetic programs to the cell cycle clock.

• Bazan JF
• Helical fold prediction for the cyclin box.
• Proteins. 1996; 24: 1-17
• Display abstract

• The smooth progression of the eukaryotic cell cycle relies on the periodic activation of members of a family of cell cycle kinases by regulatory proteins called cyclins. Outside of the cell cycle, cyclin homologs play important roles in regulating the assembly of transcription complexes; distant structural relatives of the conserved cyclin core or "box" can also function as general transcription factors (like TFIIB) or survive embedded in the chain of the tumor suppressor, retinoblastoma protein. The present work attempts the prediction of the canonical secondary, supersecondary, and tertiary fold of the minimal cyclin box domain using a combination of techniques that make use of the evolutionary information captured in a multiple alignment of homolog sequences. A tandem set of closely packed, helical modules are predicted to form the cyclin box domain.

• Cobrinik D
• Regulatory interactions among E2Fs and cell cycle control proteins.
• Curr Top Microbiol Immunol. 1996; 208: 31-61

• Farnham PJ
• Conclusions and future directions.
• Curr Top Microbiol Immunol. 1996; 208: 129-37

• Geng Y et al.
• Regulation of cyclin E transcription by E2Fs and retinoblastoma protein.
• Oncogene. 1996; 12: 1173-80
• Display abstract

• Cyclin E is critical for the advance of cells through the G1 phase of their growth cycle. Transcription of the cyclin E gene is known to be cell cycle-dependent. We have shown previously that mRNA levels of cyclin E are regulated positively by mitogens and negatively by TGF-beta. Much circumstantial evidence implicates both E2F transcription factors and the retinoblastoma protein (pRB) in the control of cyclin E expression. However, the molecular basis of this control has remained unclear. We report here the cloning of the cyclin E promoter and the identification of several putative E2F binding sites within the promoter sequence. We have found that cell cycle regulation of cyclin E transcription is mediated by E2F binding sites present in the promoter. The activity of this promoter can be regulated negatively by pRB. Our results suggest the operation of a positive-feedback loop in late G1 that functions to ensure continued cyclin E expression and pRB inactivation.

• Gerloff DL, Cohen FE
• Secondary structure prediction and unrefined tertiary structure prediction for cyclin A, B, and D.
• Proteins. 1996; 24: 18-34
• Display abstract

• We present heuristic-based predictions of the secondary and tertiary structures of cyclins A, B, and D, representatives of the cyclin superfamily. The list of suggested constraints for tertiary structure assembly was left unrefined in order to submit this report before an announced crystal structure for cyclin A becomes available. To predict these constraints, a master sequence alignment over 270 positions of cyclin types A, B, and D was adjusted based on individual secondary structure predictions for each type. We used new heuristics for predicting aromatic residues at protein-protein interfaces and to identify sequentially distinct regions in the protein chain that cluster in the folded structure. The boundaries of two conjectured domains in the cyclin fold were predicted based on experimental data in the literature. The domain that is important for interaction of the cyclins with cyclin-dependent kinases (CDKs) is predicted to contain six helices; the second domain in the consensus model contains both helices and a beta-sheet that is formed by sequentially distant regions in the protein chain. A plausible phosphorylation site is identified. This work represents a blinded test of the method for prediction of secondary and, to a lesser extent, tertiary structure from a set of homologous protein sequences. Evaluation of our predictions will become possible with the publication of the announced crystal structure.

• Grafi G et al.
• A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.
• Proc Natl Acad Sci U S A. 1996; 93: 8962-7
• Display abstract

• Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a 105-kDa nuclear phosphoprotein. To date, RB genes have been isolated only from metazoans. We have isolated a cDNA from maize endosperm whose predicted protein product (ZmRb) shows homology to the "pocket" A and B domains of the Rb protein family. We found ZmRb behaves as a pocket protein based on its ability to specifically interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in vivo with the replication-associated protein, RepA, encoded by the wheat dwarf virus. The maize Rb-related protein undergoes changes in level and phosphorylation state concomitant with endoreduplication, and it is phosphorylated in vitro by an S-phase kinase from endoreduplicating endosperm cells. Together, our results suggest that ZmRb is a representative of the pocket protein family and may play a role in cell cycle progression. Moreover, certain plant monopartite geminiviruses may operate similarly to mammalian DNA viruses, by targeting and inactivating the retinoblastoma protein, which otherwise induces G1 arrest.

• Knudsen ES, Wang JY
• Differential regulation of retinoblastoma protein function by specific Cdk phosphorylation sites.
• J Biol Chem. 1996; 271: 8313-20
• Display abstract

• The retinoblastoma tumor suppressor protein, RB, contains at least three distinct protein binding domains. The A/B pocket binds proteins with the LXCXE motif, the C pocket binds the nuclear c-Abl tyrosine kinase, and the large A/B pocket binds the transcription factor E2F. Dissociation of RB from its targets is observed as RB becomes phosphorylated during G1/S progression. There are 16 Cdk consensus phosphorylation sites in RB. It was previously unknown whether the many phosphorylation sites had redundant or distinct functions in the regulation of RB. Using RB mutant proteins lacking specific phosphorylation sites, we show that each of the binding domains is inhibited by different sites. Thr-821/826 phosphorylation is required to inhibit the binding to LXCXE containing proteins. Mutation of these two sites does not interfere with the hyperphosphorylation of RB. However, this phosphorylated mutant retains the ability to bind T-Ag, E7, and Elf-1, all of which contain the LXCXE motif. In contrast, Ser-807/811 phosphorylation is required to disrupt c-Abl binding. Mutation of Ser-807/811 and Thr-821/826 does not abolish the regulation of E2F binding. Taken together, these results show that the protein binding domains of RB are each regulated by distinct Cdk phosphorylation sites.

• Taya Y
• [Study of the cell cycle-dependent phosphorylation of the RB protein to the development of a method to cure cancer]
• Tanpakushitsu Kakusan Koso. 1996; 41: 1737-41

• Klotzbucher A, Stewart E, Harrison D, Hunt T
• The 'destruction box' of cyclin A allows B-type cyclins to be ubiquitinated, but not efficiently destroyed.
• EMBO J. 1996; 15: 3053-64
• Display abstract

• The destruction of mitotic cyclins by programmed proteolysis at the end of mitosis is an important element in cell cycle control. This proteolysis depends on a conserved motif of nine residues known as the 'destruction box', which is located 40-50 residues from the N-terminus. The sequences of the A- and B-type destruction boxes are slightly different, which might account for the differences in timing of their destruction. When the cyclin A-type destruction box was substituted for the normal one in cyclin B1 or B2, however, the resulting constructs were unexpectedly stable, although the converse substitution of B-type destruction boxes in cyclin A permitted normal degradation. We compared the ubiquitination of various cyclin constructs, and found that whereas mutation of the highly conserved residues in the destruction box strongly reduced the level of ubiquitinated intermediates, the stable destruction box 'swap' constructs did form such adducts. Thus, while ubiquitination is probably necessary for cyclin destruction, it is not sufficient. We also found that poly-ubiquitinated cyclin derivatives are still bound to p34cdc2, which is not detectably ubiquitinated itself, raising the questions of how cyclin and cdc2 dissociate from one another, and at what stage, in the process of degradation.

• Zwicker J, Muller R
• Cell cycle-regulated transcription in mammalian cells.
• Prog Cell Cycle Res. 1995; 1: 91-9
• Display abstract

• The periodic, phase-specific transcription of defined sets of genes is a hallmark of cell cycle progression in all organisms (1-3). In this article, we will summarise our current knowledge and views of the mechanisms governing the cross-coupling of cell cycle control and transcriptional regulation in mammalian cells, with particular emphasis on the transcription factor E2F and the retinoblastoma protein pRb (1-3). Excluded from this review will be the genomic response to mitogenic stimulation, which is part of the mitogen-triggered signal transduction cascades rather than a reflection of cell cycle regulation (4).

• Weinberg RA
• The retinoblastoma protein and cell cycle control.
• Cell. 1995; 81: 323-30

• Itoh A, Levinson SF, Morita T, Kourembanas S, Brody JS, Mitsialis SA
• Structural characterization and specificity of expression of E2F-5: a new member of the E2F family of transcription factors.
• Cell Mol Biol Res. 1995; 41: 147-54
• Display abstract

• Members of the E2F gene family are transcription factors that have been implicated in the control of genes essential for cell cycle progression. Regulation of E2F function is finely tuned by the retinoblastoma tumor suppressor gene product and a small family of related "pocket proteins," with the participation of a number of cyclins and cyclin-dependent kinases. Perturbations of this regulatory network can lead to oncogenic transformation and, in certain systems, to the loss of the ability to maintain terminal differentiation. We describe here the cloning, structural characterization, and tissue expression pattern of a new member of the E2F family, E2F-5. We show that this protein is highly conserved between human and rat but exhibits considerable divergence from E2F-1, E2F-2, or E2F3. Together with the recently reported E2F-4, E2F-5 defines a new branch of the E2F family. The distribution of E2F-5 mRNA among adult rat tissues and the temporal pattern of its expression during the cell cycle of vascular smooth muscle cells are distinctly different from that of E2F-1. The structural divergence between the two branches of the E2F family may thus reflect participation in different regulatory networks.

• Kouzarides T
• Transcriptional control by the retinoblastoma protein.
• Semin Cancer Biol. 1995; 6: 91-8
• Display abstract

• The retinoblastoma gene product is an abundant nuclear protein whose 'pocket domain' mediates numerous protein-protein interactions. A substantial proportion of the RB-interacting proteins are transcription factors suggesting that RB plays a fundamental role in the regulation of transcription. Via these interactions, RB can influence both the progression through the cell cycle and the expression of lineage specific products. In this review I discuss some of the likely mechanisms by which RB regulates cell proliferation and differentiation.

• Radulescu RT
• The 'LXCXE' hydropathic superfamily of ligands for retinoblastoma protein: a proposal.
• Med Hypotheses. 1995; 44: 28-31
• Display abstract

• The present study reports structural similarities between viral oncoproteins, growth factors belonging to the insulin family, members of the steroid/thyroid receptor superfamily, a D-type cyclin, the Elf-1 transcription factor and Bcl oncoproteins in regions that have been shown or proposed to mediate complex formation of these proteins with the tumor suppressor retinoblastoma protein (RB). This relationship predicts a common intracellular pathway for mitogenic signals and molecules promoting cell survival. Conversely, the structural evidence described here suggests that RB may play a central role both at the boundary between negative and positive cell growth regulation as well as in developmental decisions between cell death and cell survival.

• Soni R, Carmichael JP, Shah ZH, Murray JA
• A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif.
• Plant Cell. 1995; 7: 85-103
• Display abstract

• A new family of three related cyclins has been identified in Arabidopsis by complementation of a yeast strain deficient in G1 cyclins. Individual members show tissue-specific expression and are conserved in other plant species. They form a distinctive group of plant cyclins, which we named delta-type cyclins to indicate their similarities with mammalian D-type cyclins. The sequence relationships between delta and D cyclins include the N-terminal sequence LXCXE. This motif was originally identified in certain viral oncoproteins and is strongly implicated in binding to the retinoblastoma protein pRb. By analogy to mammalian cyclin D, these plant homologs may mediate growth and phytohormonal signals into the plant cell cycle. In support of this hypothesis, we show that, on restimulation of suspension-cultured cells, cyclin delta 3 is rapidly induced by the plant growth regulator cytokinin and cyclin delta 2 is induced by carbon source.

• Nikolov DB et al.
• Crystal structure of a TFIIB-TBP-TATA-element ternary complex.
• Nature. 1995; 377: 119-28
• Display abstract

• The crystal structure of the transcription factor IIB (TFIIB)/TATA box-binding protein (TBP)/TATA-element ternary complex is described at 2.7 A resolution. Core TFIIB resembles cyclin A, and recognizes the preformed TBP-DNA complex through protein-protein and protein-DNA interactions. The amino-terminal domain of core TFIIB forms the downstream surface of the ternary complex, where it could fix the transcription start site. The remaining surfaces of TBP and the TFIIB can interact with TBP-associated factors, other class II initiation factors, and transcriptional activators and coactivators.

• Chen PL, Riley DJ, Lee WH
• The retinoblastoma protein as a fundamental mediator of growth and differentiation signals.
• Crit Rev Eukaryot Gene Expr. 1995; 5: 79-95
• Display abstract

• The retinoblastoma gene (RB) is the prototype of the tumor suppressor genes, which play critical roles in the genesis of cancer in humans. Mouse models created through gene knock-out and transgenic methods were established for exploring and manipulating RB in vivo. These models and several other pieces of evidence have shown that the retinoblastoma protein (Rb) plays dual roles in gating cell cycle progression and promoting cellular differentiation. The molecular mechanisms involved in these roles are becoming more obvious in some biological systems: Rb sequesters the transcription factor of E2F to regulate entry of cell cycle but enhances the activities of another class of the transcription factors, exemplified by NF-IL6, to initiate terminal cellular differentiation. Thus, the Rb protein can serve as a mediator for extracellular signals of growth or differentiation. The fundamental question of why only limited cell types are susceptible to tumor formation when Rb expression is lost, however, remains unanswered at present.

• Radulescu RT, Bellitti MR, Ruvo M, Cassani G, Fassina G
• Binding of the LXCXE insulin motif to a hexapeptide derived from retinoblastoma protein.
• Biochem Biophys Res Commun. 1995; 206: 97-102
• Display abstract

• Peptides corresponding to retinoblastoma protein (RB) fragment 649-654 (LFYKKV) were tested for their ability to recognize the LXCXE sequence motif in human papilloma virus type 16E7 protein (HPV-16E7) encompassing E7 residues 21-26 (DLYCYE) and an identical motif in human insulin comprising insulin B-chain residues 16-21 (YLVCGE), respectively. Interaction between these complementary peptide sequences was observed by several approaches, including direct and competitive ELISA as well as affinity chromatography. Moreover, we demonstrated that immobilized RB649-654 displays specific recognition properties towards full-length insulin. Hence, this study provides a first experimental support for the previously anticipated complex formation between insulin and RB.

• Fagan R, Flint KJ, Jones N
• Phosphorylation of E2F-1 modulates its interaction with the retinoblastoma gene product and the adenoviral E4 19 kDa protein.
• Cell. 1994; 78: 799-811
• Display abstract

• The transcription factor E2F is regulated through its cyclical interaction with a spectrum of cellular proteins. One such protein is the product of the retinoblastoma gene (Rb); association of E2F with Rb inhibits its transactivation potential. However, in adenovirus-infected cells, E2F is complexed to the 19 kDa product of the adenovirus E4 gene. We have studied the interaction of E2F-1 with the Rb and adenovirus E4 proteins and show that phosphorylation of E2F-1 on serine residues 332 and 337 prevented its interaction with Rb but was a prerequisite for interaction with E4. These residues were phosphorylated in vivo and by p34cdc2 kinase in vitro. Upon stimulation of serum-starved cells, phosphorylation was induced in the late G1 phase of the cell cycle. These observations suggest that phosphorylation of E2F-1 is important in the regulation of its activity during the cell cycle and during infection of cells by adenovirus.

• La Thangue NB
• DP and E2F proteins: components of a heterodimeric transcription factor implicated in cell cycle control.
• Curr Opin Cell Biol. 1994; 6: 443-50
• Display abstract

• In mammalian cells, DRTF1/E2F is a transcription factor widely believed to integrate cell-cycle progression with the transcription apparatus through its cyclical interactions with important regulators of the cell cycle, such as the retinoblastoma tumour-suppressor gene product, cyclins and cyclin-dependent kinases. Recently, a number of exciting developments have uncovered the heterodimeric nature of DRTF1/E2F by defining two distinct families of proteins, DP and E2F, which comprise its activity; efficient DNA-binding activity arises when a DP protein interacts with an E2F protein. Combinatorial interactions generate a surprising array of sequence-specific heterodimers, a diversity that is probably necessary to enable different cell cycle regulating proteins to integrate their activities with transcription.

• Riley DJ, Lee EY, Lee WH
• The retinoblastoma protein: more than a tumor suppressor.
• Annu Rev Cell Biol. 1994; 10: 1-29

• La Thangue NB
• DRTF1/E2F: an expanding family of heterodimeric transcription factors implicated in cell-cycle control.
• Trends Biochem Sci. 1994; 19: 108-14
• Display abstract

• During the cell cycle, the transcription of certain genes is integrated with cell-cycle progression, thus providing an important level of control. In mammalian cells, DRTF1/E2F is a transcription activity comprising a group of related heterodimeric transcription factors that function in this integration process. The primary molecules involved in generating the afferent signals that converge on DRTF1/E2F belong to a class of proteins, exemplified by the retinoblastoma tumour suppressor gene product, whose activities are, in turn, regulated by cyclin-dependent kinases. The transcriptional activity of DRTF1/E2F is therefore regulated through a pathway that links the machinery of the cell cycle to the transcription apparatus. As such, it is likely to play a pivotal role in regulating cell-cycle progression.

• Gallant P, Nigg EA
• Identification of a novel vertebrate cyclin: cyclin B3 shares properties with both A- and B-type cyclins.
• EMBO J. 1994; 13: 595-605
• Display abstract

• Cyclins play a key role in controlling progression through the cell cycle. They act as regulatory subunits of p34cdc2/CDC28 and related cyclin-dependent protein kinases (cdks). In vertebrates, cyclins B1 and B2 function during M phase, whereas cyclin A is required for S phase as well as the G2 to M phase transition. Here, we describe the identification and characterization of a novel vertebrate cyclin, termed cyclin B3. The assignment of this cyclin to the B-type subfamily is based on its cDNA-derived sequence and its pattern of expression in synchronized cells, both suggesting a distant relationship to other B-type cyclins. Interestingly, however, cyclin B3 also displays properties that resemble those of A- rather than B-type cyclins. Specifically, cyclin B3 localizes to the cell nucleus throughout the cell cycle, and is able to associate in vivo with at least two kinase subunits, p34cdc2 and p33cdk2. Furthermore, deletion of 26 amino acids from the C-terminus of cyclin B3 impairs both its interaction with kinase catalytic subunits and its nuclear localization, reminiscent of recent results obtained with cyclin A. Based on these observations, we conclude that cyclin B3 may share functional properties with both A- and B-type cyclins.

• Pines J, Hunter T
• The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B.
• EMBO J. 1994; 13: 3772-81
• Display abstract

• We have shown previously that human cyclins A and B1 are localized differentially in the cell during interphase; cyclin A is nuclear and cyclin B1 is a cytoplasmic protein. To understand the basis of this difference we created deletion mutants and various chimeras between the two types of cyclin and expressed them in tissue culture cells by transient transfection. We find that the N-terminus of cyclin B1 contains a 42 amino acid region that is sufficient to retain the normally nuclear cyclin A in the cytoplasm. Conversely, deleting the cytoplasmic retention signal region from cyclin B1 causes the protein to become nuclear. Although the cytoplasmic retention signal region is outside the cyclin box, its sequence is well conserved in human cyclin B2, and is both necessary and sufficient to keep cyclin B2 in the cytoplasm. Thus we propose that the subcellular distribution of the B-type cyclins is determined primarily by a small region of the N-terminus which targets the cyclin--CDK complexes to particular structures in the cytoplasm.

• Boehmelt G, Ulrich E, Kurzbauer R, Mellitzer G, Bird A, Zenke M
• Structure and expression of the chicken retinoblastoma gene.
• Cell Growth Differ. 1994; 5: 221-30
• Display abstract

• The molecular cloning and initial characterization of the complementary DNA encoding the chicken homologue of the human retinoblastoma susceptibility gene product Rb are described. Chicken Rb (chRb) was found to be highly homologous to human, mouse, and Xenopus Rb in primary amino acid sequence, underlining its conserved function in cell cycle control in higher eukaryotes. The highest level of homology was found within the Rb "pocket domains" and at the Rb COOH terminus, whereas the extreme NH2 terminus of chRb is divergent. We also show that transcription of chRb initiates at multiple start sites within a DNA segment which represents a CpG island. ChRb is transcribed as a 4.7-kilobase (kb) mRNA species in a variety of normal chicken tissues and oncogene-transformed hematopoietic cells. However, in v-rel-transformed cell lines, a variant 3.1-kb Rb-specific mRNA was detected in addition to the 4.7-kb transcript. ChRb is expressed as a 104 kilodalton protein and is therefore slightly smaller than the mammalian Rb proteins.

• Ewen ME
• The cell cycle and the retinoblastoma protein family.
• Cancer Metastasis Rev. 1994; 13: 45-66
• Display abstract

• Tumor formation results from alterations in the control of normal cell proliferation. To further our understanding of the molecular mechanisms underlying the deregulation of cell proliferation much attention, over the past decade, has been focused on the function of proto-oncogenes. Cellular oncogenes are thought to be growth promoting. More recently, a class of genes known as tumor suppressors have come under intense study. Tumor suppressors are largely thought to restrain cell proliferation. The retinoblastoma protein (Rb) is one of a growing list of tumor suppressors. Concurrent with the study of tumor suppressor genes has been a rapid increase in our understanding of the cell cycle at the molecular level. Rb and a related protein p107 are involved in the processes of cell proliferation and differentiation. Each functionally interacts with and affects the activity of the transcription factor E2F as well as other transcription factors involved in cell proliferation and differentiation. Additionally, Rb and p107 are modified by, and/or form specific complexes with, several elements of the basic cell cycle machinery. Specifically, Rb and p107 interact with and are modified by various cyclins and cyclin dependent kinases (cdk), some of which have been shown to be essential for cell cycle progression and in some cases their deregulation has been implicated in the development of cancer. This review will attempt to convey our current functional and mechanistic understanding of the biological roles Rb and p107 play in proliferation, development and differentiation. A knowledge of the interplay between these positive and negative regulators of cell proliferation and differentiation, noted above, is central to our understanding of human cancer.

• Dunaief JL et al.
• The retinoblastoma protein and BRG1 form a complex and cooperate to induce cell cycle arrest.
• Cell. 1994; 79: 119-30
• Display abstract

• The retinoblastoma tumor suppressor protein (RB) binds several cellular proteins involved in cell cycle progression. Using the yeast two-hybrid system, we found that RB bound specifically to the protein BRG1. BRG1 shares extensive sequence similarity to Drosophila brahma, an activator of homeotic gene expression, and the yeast transcriptional activator SNF2/SW12. BRG1 contains an RB-binding motif found in viral oncoproteins and bound to the A/B pocket and the hypophosphorylated form of RB. BRG1 did not bind RB in viral oncoprotein-transformed cells. Coimmunoprecipitation experiments suggested BRG1 associates with the RB family in vivo. In the human carcinoma cell line SW13, BRG1 exhibited tumor suppressor activity by inducing formation of flat, growth-arrested cells. This activity depended on the ability of BRG1 to cooperate and complex with RB, as both an RB-nonbinding mutant of BRG1 and the sequestration of RB by adenovirus E1A protein abolished flat cell formation.

• Hagemeier C, Cook A, Kouzarides T
• The retinoblastoma protein binds E2F residues required for activation in vivo and TBP binding in vitro.
• Nucleic Acids Res. 1993; 21: 4998-5004
• Display abstract

• The retinoblastoma (RB) tumour suppressor protein is capable of repressing the activity of promoters containing DNA binding sites for the transcription factor E2F. Recently a protein which binds RB and possesses the DNA binding characteristics of E2F has been cloned. Here we show that the E2F activation domain is the target for RB-induced repression. RB can silence the 57 residue E2F activation domain but cannot effectively repress an E2F mutant which has reduced RB binding capacity. Extensive mutagenesis of E2F shows residues involved in RB binding are required for transcription activation. Mutations which affect both functions most dramatically lie within the minimal RB binding region. A further subset of sensitive residues lies within a new repeat motif E/DF XX L X P which flanks the minimum RB binding site. These data show that RB can mask E2F residues involved in the activation process, possibly by mimicking a component of the transcriptional machinery. Consistent with this model, we find that the TATA box binding protein TBP can bind to the E2F activation domain in vitro in a manner indistinguishable from that of RB.

• Tommasino M et al.
• HPV16 E7 protein associates with the protein kinase p33CDK2 and cyclin A.
• Oncogene. 1993; 8: 195-202
• Display abstract

• E7 is the major transforming protein of human papillomavirus type 16 (HPV16). It has been found to associate with the retinoblastoma protein Rb1. We investigated whether HPV16 E7 protein was associated with other cellular proteins, in particular with those involved in cell cycle control. Immunoprecipitates from CaSki cell extracts with an anti E7 monoclonal antibody contained a histone H1 kinase. Recombinant E7, synthesized in yeast, when mixed with protein extracts from epithelial cells bound histone H1 kinase activity in vitro. The in vivo and the in vitro-formed E7-kinase complex had the same periodicity of activity during the cell cycle, being most active in S and G2/M. Immunoblotting of E7 immunoprecipitates with an antibody raised against the p33CDK2, revealed a 33 kDa protein band not detected by an anti-p34cdc2 antibody, suggesting that the E7-associated kinase activity is due to the p33CDK2. The interaction appears to be via cyclin A, since probing of similar immunoblots showed a 50 kDa band corresponding to cyclin A. The association of E7 with cyclin A appeared to be direct, not involving Rb 1 or other proteins.

• Cress WD, Johnson DG, Nevins JR
• A genetic analysis of the E2F1 gene distinguishes regulation by Rb, p107, and adenovirus E4.
• Mol Cell Biol. 1993; 13: 6314-25
• Display abstract

• The cellular transcription factor E2F appears to be a target for the regulatory action of the retinoblastoma tumor suppressor gene product. The recent isolation of the E2F1 cDNA clone, which encodes a polypeptide with properties characteristic of E2F, has now allowed a more detailed analysis of the regulation of E2F function by Rb as well as the Rb-related p107 protein and the adenovirus 19-kDa E4 gene product. Previous experiments have shown that each of these regulatory proteins can modulate the activity of cellular E2F. We find that each of these regulatory events can be mediated through the E2F1 product. Moreover, an examination of various E2F1 mutations reveals distinct specificities for these regulatory proteins. For instance, the ability of E4 to alter E2F1 function is dependent upon sequences within a putative leucine repeat of E2F1 as well as within the C-terminal acidic domain. In contrast, the leucine repeat element was not important for Rb- or p107-mediated inhibition of E2F1 activity. Although the C-terminal acidic domain of E2F1, previously shown to be important for Rb binding, appears to be a site for regulation of E2F1 by Rb and p107, point mutations within this region distinguish recognition by Rb and p107. These results underscore the complexity of E2F regulatory interactions and also demonstrate a qualitative distinction in the interactions of Rb and p107 with E2F1, perhaps reflective of functional differences.

• Lees EM, Harlow E
• Sequences within the conserved cyclin box of human cyclin A are sufficient for binding to and activation of cdc2 kinase.
• Mol Cell Biol. 1993; 13: 1194-201
• Display abstract

• Cyclins are pivotal in the coordinate regulation of the cell cycle. By physical association, they are able to activate at least one of the cyclin-dependent kinases, cdc2. How this association between the catalytic moiety and cyclins leads to subsequent activation of the kinase remains unclear. In this report, we describe experiments to investigate this event at a physical level. Our approach was to map the regions required on the cyclin A molecule for interaction with cdc2. We have mapped the contact regions to two small noncontiguous stretches of amino acids, residues 189 to 241 and 275 to 320, both located within the conserved cyclin box domain of the protein. We have further shown that this region not only represents a contact site for cdc2 but apparently represents an intact functional domain with respect to cdc2 activation. This region alone is sufficient to stimulate maturation when injected into immature Xenopus laevis oocytes. This observation implies that events leading to the activation of cdc2 kinase can be mediated through small regions of the cyclin molecule that are located in the cyclin box. These regions contain some of the most highly conserved residues found between all the cyclin members so far identified. This suggests that the cyclin family members may have conserved a similar mechanism to bind and activate cyclin-dependent kinases.

• Ewen ME, Sluss HK, Sherr CJ, Matsushime H, Kato J, Livingston DM
• Functional interactions of the retinoblastoma protein with mammalian D-type cyclins.
• Cell. 1993; 73: 487-97
• Display abstract

• The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G1 cyclins (D2 and D3) in vitro. Binding depended upon the minimal regions of Rb necessary for its growth-suppressive activity, as well as upon the D-type cyclin sequence motif shared with Rb-binding DNA tumor virus oncoproteins. Coexpression of the three D-type cyclins with the cyclin-dependent kinase (cdk4) in insect cells generated Rb kinase activity. By contrast, cyclins D2 and D3, but not D1, activated another such kinase, cdk2. Introduction of cyclin D2 and Rb into the Rb-deficient cell line SAOS-2 led to overt Rb hyperphosphorylation, whereas Rb, expressed alone or together with cyclin D1, remained unphosphorylated. Cyclin D2-dependent phosphorylation inhibited its binding to the transcription factor E2F and reversed the Rb G1 exit block in the cell cycle. Thus, all D-type cyclins do not function equivalently, and one of them plays a major role in reversing the cycle-blocking function of a known tumor suppressor.

• Girling R, Bandara LR, Zamanian M, Sorensen TS, Xu FH, La Thangue NB
• DRTFI/E2F transcription factor: an integrator of cell-cycle events with the transcriptional apparatus.
• Biochem Soc Trans. 1993; 21: 939-42

• Hollingsworth RE Jr, Hensey CE, Lee WH
• Retinoblastoma protein and the cell cycle.
• Curr Opin Genet Dev. 1993; 3: 55-62
• Display abstract

• Deregulation of the cell cycle may contribute one of the primary mechanisms through which cancer arises. Eukaryotic cell division has been found to be a strictly controlled process, involving response to both positive and negative external signals and assessment of the cell's internal state. Several recent discoveries have strengthened and refined the theory that the retinoblastoma protein is involved in the decision between cell division and differentiation, and have begun to provide an outline of the nature of this involvement.

• Moran E
• DNA tumor virus transforming proteins and the cell cycle.
• Curr Opin Genet Dev. 1993; 3: 63-70
• Display abstract

• Studies using the transforming proteins of the small DNA tumor viruses point to the role of the E2F cellular transcription factor in regulating cell cycle specific gene expression. The evidence derived suggests that E2F is controlled by the retinoblastoma gene product and related proteins, including their associated cyclins. Studies using DNA tumor virus products further suggest the existence of additional pathways of cell-cycle activation.

• Figge J et al.
• The binding domain structure of retinoblastoma-binding proteins.
• Protein Sci. 1993; 2: 155-64
• Display abstract

• The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.

• Huang PS et al.
• Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein.
• Mol Cell Biol. 1993; 13: 953-60
• Display abstract

• Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

• Hollingsworth RE Jr, Chen PL, Lee WH
• Integration of cell cycle control with transcriptional regulation by the retinoblastoma protein.
• Curr Opin Cell Biol. 1993; 5: 194-200
• Display abstract

• Rapid progress in several areas of molecular biology has led to the realization that the retinoblastoma protein may play a pivotal role in the coordination between cell cycle control and regulation of gene expression. This role is a subtle one, and is important in only certain mammalian cell types. Exploring the details of these connections, and why only some cells rely on them, is already beginning to shed light on the regulation of cell multiplication.

• Mansur CP, Androphy EJ
• Cellular transformation by papillomavirus oncoproteins.
• Biochim Biophys Acta. 1993; 1155: 323-45

• Dowdy SF, Hinds PW, Louie K, Reed SI, Arnold A, Weinberg RA
• Physical interaction of the retinoblastoma protein with human D cyclins.
• Cell. 1993; 73: 499-511
• Display abstract

• The retinoblastoma protein (pRb) functions as a regulator of cell proliferation and in turn is regulated by cyclin-dependent kinases. Cyclins D1 and D3 can form complexes with pRb that resemble those formed by several viral oncoproteins and are disrupted by the adenovirus E1A oncoprotein and derived peptides. These cyclins contain a sequence motif similar to the pRb-binding conserved region II motif of the viral oncoproteins. Alteration of this motif in cyclin D1 prevents formation of cyclin D1-pRb complexes while enhancing the biological activity of cyclin D1 assayed in vivo. We conclude that cyclins D1 and D3 interact with pRb in a fashion distinct from cyclins A and E, which can induce pRb hyperphosphorylation, and that cyclin D1 activity may be regulated by its association with pRb.

• Bandara LR, Adamczewski JP, Zamanian M, Poon RY, Hunt T, Thangue NB
• Cyclin A recruits p33cdk2 to the cellular transcription factor DRTF1.
• J Cell Sci Suppl. 1992; 16: 77-85
• Display abstract

• Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.

• Radulescu RT, Wendtner CM
• Proposed interaction between insulin and retinoblastoma protein.
• J Mol Recognit. 1992; 5: 132-7
• Display abstract

• Retinoblastoma protein (RB) is a tumor suppressor gene product involved in embryogenesis and cell cycle progression. One of the major mechanisms leading to RB dysfunction is complex formation with viral oncoproteins using the common RB binding motif Leu X Cys X Glu (LXCXE) which has also been identified in cellular ligands, e.g., RBP-1 and RBP-2. p107, a cellular protein with RB sequence homology, has been shown to bind to the same viral oncoproteins associating with RB and is therefore thought to contribute to cell cycle regulation. It has recently been suggested that insulin stimulates gene transcription through direct association with an, as yet, unidentified intracellular transcription factor. Due to the central roles of RB and p107 in coupling external growth signals with the progression of the cell cycle clock, we have hypothesized that these two proteins might be candidates for mediating the effects of insulin on DNA. We report here the identification of a region in the B-chain of human insulin that has the sequence LXCXE. Based on this finding we predict that the insulin B-chain may interact with RB and/or p107. Since we have also identified sequences hydropathically related to LXCXE in insulin-like growth factor I (IGF-I) and II (IGF-II), but not in relaxin, nerve growth factor, epidermal growth factor, glucagon or beta-endorphin, we further propose that both IGF-I and -II may assemble with RB and/or p107, too. Moreover, binding sites on RB and p107 identical with those suggested for viral oncoproteins and cellular ligands are predicted for insulin/IGF-I/IGF-II by using the hydropathic complementarity approach.(ABSTRACT TRUNCATED AT 250 WORDS)

• Faha B, Ewen ME, Tsai LH, Livingston DM, Harlow E
• Interaction between human cyclin A and adenovirus E1A-associated p107 protein.
• Science. 1992; 255: 87-90
• Display abstract

• The products of the adenovirus early region 1A (E1A) gene are potent oncoproteins when tested in standard transformation and immortalization assays. Many of the changes induced by E1A may be due to its interaction with cellular proteins. Four of these cellular proteins are the retinoblastoma protein (pRB), p107, cyclin A, and p33cdk2. The pRB and p107 proteins are structurally related and have several characteristics in common, including that they both bind to the SV40 large T oncoprotein as well as to E1A. Cyclin A and p33cdk2 are thought to function in the control of the cell cycle. They bind to one another, forming a kinase that closely resembles the cell cycle-regulating complexes containing p34cdc2. Cyclin A is now shown to bind to p107 in the absence of E1A. The association of p107 with cyclin A suggests a direct link between cell cycle control and the function of p107.

• Hamel PA, Gallie BL, Phillips RA
• The retinoblastoma protein and cell cycle regulation.
• Trends Genet. 1992; 8: 180-5
• Display abstract

• Although the precise function of the retinoblastoma gene product, p110RB1, remains unknown, recent data suggest that it plays a role in the control of cellular proliferation by regulating transcription of genes required for a cell to enter or stay in a quiescent or G0 state, or for progression through the G1 phase of the cell cycle. However, it is difficult to rationalize the expression of p110RB1 in a wide range of tissues with the fact that mutations in the RB1 gene initiate cancers in a limited number of tissues.

• Ewen ME, Faha B, Harlow E, Livingston DM
• Interaction of p107 with cyclin A independent of complex formation with viral oncoproteins.
• Science. 1992; 255: 85-7
• Display abstract

• The p107 protein and the retinoblastoma protein (RB) both bind specifically to two viral oncoproteins, the SV40 T antigen (T) and adenoviral protein E1A (E1A). Like RB, p107 contains a segment (the pocket) that, alone, can bind specifically to T, E1A, and multiple cellular proteins. Cyclin A bound to the p107 pocket, but not the RB pocket. Although both pockets contain two, related collinear subsegments (A and B), the unique sequence in the p107 pocket that occupies the space between A and B is required for the interaction with cyclin A.

• Pagano M, Durst M, Joswig S, Draetta G, Jansen-Durr P
• Binding of the human E2F transcription factor to the retinoblastoma protein but not to cyclin A is abolished in HPV-16-immortalized cells.
• Oncogene. 1992; 7: 1681-6
• Display abstract

• The adenovirus E1A, SV40 large T and papillomavirus E7 proteins immortalize primary cells by virtue of their ability to bind the retinoblastoma gene product (pRB) and other cellular proteins, including cyclin A and the prRB-related protein, p107. It has been demonstrated that these viral oncogene products will prevent the inhibition of positive growth regulators by pRB, one of them being the E2F transcription factor. Here we show that the interactions of pRB and cyclin A with E2F are present also in normal keratinocytes and in primary human fibroblasts. In human keratinocytes immortalized by human papillomavirus 16 (HPV-16), expressing high levels of HPV-16 E7 protein, complexes between E2F and pRB are disrupted. In this cell line, as well as in HeLa cells which express HPV-18 E7, complexes containing E2F and cyclin A are maintained, indicating that this interaction is not sensitive to the viral oncoprotein and that cyclin A can associate with E2F independently of pRB. In vitro binding experiments suggest that the E7 gene product is able to preferentially abolish the interaction of pRB with E2F, leaving the cyclin A complexes intact. Our findings suggest that E7-dependent immortalization of human cells is associated with modifications of E2F multiprotein complexes.

• Pagano M, Pepperkok R, Verde F, Ansorge W, Draetta G
• Cyclin A is required at two points in the human cell cycle.
• EMBO J. 1992; 11: 961-71
• Display abstract

• Cyclins play a fundamental role in regulating cell cycle events in all eukaryotic cells. The human cyclin A gene was identified as the site of integration of hepatitis B virus in a hepatocarcinoma cell line; in addition, cyclin A is associated with the E2F transcription factor in a complex which is dissociated by the E1A oncogene product. Such findings suggest that cyclin A is a target for oncogenic signals. We have now found that DNA synthesis and entry into mitosis are inhibited in human cells microinjected with anti-cyclin A antibodies at distinct times. Cyclin A binds both cdk2 and cdc2, giving two distinct cyclin A kinase activities, one appearing in S phase, the other in G2. These results suggest that cyclin A defines novel control points of the human cell cycle.

• Bandara LR, Adamczewski JP, Hunt T, La Thangue NB
• Cyclin A and the retinoblastoma gene product complex with a common transcription factor.
• Nature. 1991; 352: 249-51
• Display abstract

• The retinoblastoma gene (Rb) product is a negative regulator of cellular proliferation, an effect that could be mediated in part at the transcriptional level through its ability to complex with the sequence-specific transcription factor DRTF1. This interaction is modulated by adenovirus E1a, which sequesters the Rb protein and several other cellular proteins, including cyclin A, a molecule that undergoes cyclical accumulation and destruction during each cell cycle and which is required for cell cycle progression. Cyclin A, which also complexes with DRTF1, facilitates the efficient assembly of the Rb protein into the complex. This suggests a role for cyclin A in regulating transcription and defines a transcription factor through which molecules that regulate the cell cycle in a negative fashion, such as Rb, and in a positive fashion, such as cyclin A, interact. Mutant loss-of-function Rb alleles, which occur in a variety of tumour cells, also fail to complex with E1a and large T antigen. Here we report on a naturally occurring loss-of-function Rb allele encoding a protein that fails to complex with DRTF1. This might explain how mutation in the Rb gene prevents negative growth control.

• Rustgi AK, Dyson N, Hill D, Bernards R
• The c-myc oncoprotein forms a specific complex with the product of the retinoblastoma gene.
• Cold Spring Harb Symp Quant Biol. 1991; 56: 163-7

• Pagano M, Draetta G
• Cyclin A, cell cycle control and oncogenesis.
• Prog Growth Factor Res. 1991; 3: 267-77
• Display abstract

• One of the most fundamental questions in biology is how a cell is able to regulate its division cycle. Initially it was thought that in mammalian cells control over entry into the cell cycle is exerted at a restriction point in G1; once past this point the cell would be free to undergo all the steps needed until the following division. Hence, for many years research on tumorigenesis focused on the mitogenic activation of quiescent cells by growth factors, peptide hormones and oncogene products (for reviews see [1, 2]). These studies investigated the initial steps required to induce a quiescent, nondividing cell to proliferate, and led to the identification of many growth factor receptors, of both the tyrosine kinase family and the G-protein coupled family. Receptors bearing protein tyrosine phosphatase or serine kinase catalytic domains were also identified via this route (for reviews see [3, 4, 5]). However more recent studies on the cooperation between different growth factors for mitogenesis have shown that multiple requirements exist for a cell to proceed through the entire division cycle. Indeed studies in several different organisms, pioneered by investigators working with Ascomycetes [6, 7, 8], have now clearly shown that the eukaryotic cell cycle proceeds through multiple check-points. Furthermore, it now appears that many of the regulatory elements and even pathways have been conserved throughout evolution. In this review we discuss the possible involvement of one of the transducing molecules, cyclin A, in abnormal cell proliferation.

• Imai Y, Matsushima Y, Sugimura T, Terada M
• Purification and characterization of human papillomavirus type 16 E7 protein with preferential binding capacity to the underphosphorylated form of retinoblastoma gene product.
• J Virol. 1991; 65: 4966-72
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• Human papillomavirus type 16 E7 is considered to be a major viral oncoprotein playing an important role(s) in cervical cancers. E7 protein was shown to bind to the protein product of the retinoblastoma gene (RB), while simian virus 40 large T and adenovirus E1A were also shown to possess binding activity to RB protein. The RB protein is a cell cycle regulator that is highly phosphorylated specifically in S, G2, and M, whereas it is underphosphorylated in G0 and G1. Recently, large T was demonstrated to bind preferentially to the underphosphorylated RB protein, which is considered to be an active form restricting cell proliferation. However, it is not known whether E7 can bind to phosphorylated RB protein. We successfully purified large quantities of unfused human papillomavirus type 16 E7 protein expressed in Escherichia coli by using a T7 promoter-T7 RNA polymerase system. The purified E7 protein was demonstrated to bind preferentially to the underphosphorylated RB protein.

• Templeton DJ, Park SH, Lanier L, Weinberg RA
• Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering.
• Proc Natl Acad Sci U S A. 1991; 88: 3033-7
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• We have examined the functional consequences of mutations present in defective alleles of the retinoblastoma susceptibility gene (RB1) isolated from two spontaneously arising tumors. Unlike cDNA clones expressing the wild-type protein p110Rb, those encoding the two mutant proteins failed to induce the appearance of senescent cells in transfected Saos-2 human osteosarcoma cells. The mutant proteins were also defective in binding to the E1A oncoprotein, were unable to become hyperphosphorylated, and failed to become tightly associated with nuclear structures. We conclude that mutations in two distinct regions of the protein concomitantly affect these four aspects of p110Rb function.

• Nevins JR et al.
• E2F transcription factor is a target for the RB protein and the cyclin A protein.
• Cold Spring Harb Symp Quant Biol. 1991; 56: 157-62

• Banks L, Edmonds C, Vousden KH
• Ability of the HPV16 E7 protein to bind RB and induce DNA synthesis is not sufficient for efficient transforming activity in NIH3T3 cells.
• Oncogene. 1990; 5: 1383-9
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• Previous studies have demonstrated that the HPV-16 E7 gene product encodes the major transforming activity of the virus in rodent cell systems. In this study we have generated a series of point mutations affecting the region of HPV-16 E7, which shows homology to adenovirus E1a conserved domain (CD)1. In conjunction with previously described mutants in the region of E7 with similarity to E1a CD2 and SV40 LT, we have investigated three known activities of the E7 protein; transformation, association with the cellular RB protein and induction of cellular DNA synthesis. The results show that RB binding correlates with the ability of E7 to induce cellular DNA synthesis and mediate cell transformation. In addition an unidentified function of E7, which is necessary for transformation of NIH3T3 cells, but does not affect RB binding or the ability to induce cellular DNA synthesis, has also been demonstrated. This study therefore identifies two separate regions of the E7 gene necessary for transformation of established cells. One of these, in the region of E7 which shows similarity to E1a CD2 and LT, is required for RB binding and DNA synthesis. The other region important for transformation, in the N-terminus of E7, is separable from the RB binding/DNA synthesis function.

• Jones RE et al.
• Identification of HPV-16 E7 peptides that are potent antagonists of E7 binding to the retinoblastoma suppressor protein.
• J Biol Chem. 1990; 265: 12782-5
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• Complex formation between the human papilloma virus type-16 E7 protein (HPV-16 E7) and the retinoblastoma suppressor protein (pRB) is believed to be important in the process of cellular transformation that leads to cervical carcinoma. Utilizing an in vitro solution assay as well as a plate binding assay that measures the association between HPV-16 E7 and pRB proteins, we have examined a series of synthetic HPV-16 E7 peptides. HPV-16 E7 peptides which lie between amino acid residues 14 and 32 were found to be potent inhibitors of E7/pRB binding. The minimal peptide structure that possessed full antagonist activity was N-acetyl-E7-(21-29)-peptide amide. This peptide inhibited 100% of E7/pRB binding and exhibited an IC50 of 40 nM in the plate binding assay. A purified beta-galactosidase-E7 fusion protein exhibited an IC50 of 2 nM in the same assay. These results suggest that other regions of the E7 molecule in addition to amino acids 21-29 may contributed to E7/pRB interaction. Analysis of E7-(20-29)-peptides containing single amino acid substitutions suggests that Cys24, Tyr23, Tyr25, Asp21, and Glu26 are important residues for maintaining maximal antagonist activity. This series of peptides should prove useful in analyzing the biological consequences of E7/pRB binding in HPV-infected cells.

• Dyson N, Howley PM, Munger K, Harlow E
• The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product.
• Science. 1989; 243: 934-7
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• Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.

• Munger K, Werness BA, Dyson N, Phelps WC, Harlow E, Howley PM
• Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.
• EMBO J. 1989; 8: 4099-105
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• The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line.

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