Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes (original) (raw)
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Genes & Development, 1991
Recent experiments have shown that the cellular E2F transcription factor is found in complexes with cellular proteins and that one such complex contains the cyclin-A protein. Isolation of a cellular activity, which we term E2F-BF, can reconstitute the E2F-cyclin-A complex and has permitted a more detailed analysis of the mechanism of E1A dissociation. Through the analysis of a series of EIA mutants, we find that sequences in conserved region 1 (CR1) and conserved region 2 (CR2) are important for dissociation of the E2F complex, whereas amino-terminal sequences are not required. In contrast to the requirements for dissociation, only the CR1 sequences are required to block formation of the complex if E1A is added when the components are combined. We have also identified an activity, termed E2F-I, that inhibits E2F binding to DNA, again apparently through the formation of a complex with E2F. This inhibitory activity is also blocked by E1A, dependent on the same elements of the E1A protein that disrupt the interaction with E2F-BF. Because the E1A sequences that are important for releasing E2F from these interactions are also sequences necessary for oncogenesis, we suggest that this activity may be a critical component of the transforming activity of E1A.
Identification of distinct roles for separate E1A domains in disruption of E2F complexes
Molecular and Cellular Biology
The adenovirus E1A protein can disrupt protein complexes containing the E2F transcription factor in association with cellular regulatory proteins such as the retinoblastoma gene product (Rb) and the Rb-related p107 protein. Previous experiments have shown that the CR1 and CR2 domains of E1A are required for this activity. We now demonstrate that the CR2 domain is essential for allowing E1A to interact with the E2F-Rb or the E2F-p107-cyclin A-cdk2 complex. Multimeric complexes containing E1A can be detected when the CR1 domain has been rendered inactive by mutation. In addition, the E1A CR1 domain, but not the CR2 domain, is sufficient to prevent the interaction of E2F with Rb or p107. On the basis of these results, we suggest a model whereby the CR2 domain brings E1A to the E2F complexes and then, upon a normal equilibrium dissociation of Rb or p107 from E2F, the E1A CR1 domain is able to block the site of interaction on Rb or p107, thereby preventing the re-formation of the complexes.
The EMBO Journal, 1995
Transcription of the c-jun gene is up-regulated by either retinoic acid (RA) or adenovirus ElA during the differentiation of F9 cells. We show here that RA and ElA induce phosphorylation of the ElA-associated 300 kDa protein (p300) during the differentiation of F9 cells. The region of ElA that is required for interaction with cellular protein p300 overlaps with the region of ElA required for ElA to induce expression of the c-jun gene. Treatment of F9 cells with RA or infection of the cells by adenovirus led to a decrease in the electrophoretic mobility of p300. Phosphatase treatment of p300 from RA-treated or adenovirusinfected F9 cells reversed the changes in migration of p300, indicating that RA-and ElA-mediated changes in the mobility of p300 were due to phosphorylation. We also found factors, designated DRF1 and DRF2, that bound specifically to a sequence element that is necessary and sufficient for RAand ElA-mediated up-regulation of the c-jun gene. The mobility of DRF complexes was changed by ElA or RA and the complexes were supershifted by addition of a polyclonal p300 antiserum. Moreover, overexpression of p300 resulted in an increase in the level of DRF1 complex. p300 fused to the DNA binding domain of the E2 protein of papilloma virus stimulated E2-dependent reporter activity in response to RA or ElA in F9 cells. Our results suggest that p300 is part of the DRF complexes, that it is differentially phosphorylated in undifferentiated versus differentiated cells and that it is likely involved in regulating transcription of the c-jun gene during F9 cell differentiation.
Oncogene, 2000
Ectopic expression of the CDK inhibitors (CKIs) p16 INK4a and p27 Kip1 in Rat1 ®broblasts induces dephosphorylation and activation of Retinoblastoma-family proteins (pRb, p107 and p130), their association with E2F proteins, and cell cycle arrest in G1. The growth-inhibitory action of p16, in particular, is believed to be mediated essentially via pRb activation. The 12S E1A protein of human Adenovirus 5 associates with pRb-family proteins via residues in its Conserved Regions (CR) 1 and 2, in particular through the motif LXCXE in CR2. These interactions are required for E1A to prevent G1 arrest upon co-expression of CKIs. We show here that mutating either of two conserved motifs adjacent to LXCXE in CR2, GFP and SDDEDEE, also impairs the ability of E1A to overcome G1 arrest by p16 or p27. Strikingly, however, these mutations aect neither the association of E1A with pRb, p07 and p130, nor its ability to derepress E2F-1 transcriptional activity in transient transfection assays. One of the E1A mutants, however, is defective in derepressing several endogenous E2F target genes in the presence of p16 or p27. Thus, CR2 possesses an essential function besides pRb-binding. We speculate that this function might be required for the full derepression of E2F-regulated genes in their natural chromatin context.
Adenovirus E1A Directly Targets the E2F/DP-1 Complex
Journal of Virology, 2011
Deregulation of the cell cycle is of paramount importance during adenovirus infection. Adenovirus normally infects quiescent cells and must initiate the cell cycle in order to propagate itself. The pRb family of proteins controls entry into the cell cycle by interacting with and repressing transcriptional activation by the E2F transcription factors. The viral E1A proteins indirectly activate E2F-dependent transcription and cell cycle entry, in part, by interacting with pRb and family members to free the E2Fs. We report here that an E1A 13S isoform can unexpectedly activate E2F-responsive gene expression independently of binding to the pRb family of proteins. We demonstrate that E1A binds to E2F/DP-1 complexes through a direct interaction with DP-1. E1A appears to utilize this binding to recruit itself to E2F-regulated promoters, and this allows the E1A 13S protein, but not the E1A 12S protein, to activate transcription independently of interaction with pRb. Importantly, expression of E1A 13S, but not E1A 12S, led to significant enhancement of E2F4 occupancy of E2F sites of two E2F-regulated promoters. These observations identify a novel mechanism by which adenovirus deregulates the cell cycle and suggest that E1A 13S may selectively activate a subset of E2F-regulated cellular genes during infection.
Genes & Development, 1996
E2F transcription factors are key regulators of transcription during the cell cycle. E2F activity is regulated at the level of transcription and DNA binding and by complex formation with the retinoblastoma pocket protein family. We show here that free E2F-1 and E2F-4 transcription factors are unstable and that their degradation is mediated by the ubiquitin-proteasome pathway. Both E2F-I and E2F-4 are rendered unstable by an epitope in the carboxyl terminus of the proteins, in close proximity to their pocket protein interaction surface. We show that binding of E2F-1 to pRb or E2F-4 to p107 or p130 protects E2Fs from degradation, causing the complexes to be stable. The increased stability of E2F-4 pocket protein complexes may contribute to the maintenance of active transcriptional repression in quiescent cells. Surprisingly, adenovirus transforming proteins, which release pocket protein-E2F complexes, also inhibit breakdown of free E2F. These data reveal an additional level of regulation of E2F transcription factors by targeted proteolysis, which is inhibited by pocket protein binding and adenovirus early region 1 transforming proteins.
The EMBO Journal
Adenovirus E1A dependent trans-activation of transcription involves the utilization of cellular promoter specific transcription factors. One such factor termed E2F is important for the transcription of the viral E2 gene and appears to be a rate limiting component targeted during the trans-activation event. Since E2F is of cellular origin and likely to be involved in cellular gene control, we have identified E2F binding sites in cellular genes. Examples include the c-myc, c-myb and N-myc protoncogenes, the DHFR gene and the EGF receptor gene. The transcription of these genes is regulated by cell proliferation signals and each falls into the so-called immediate early class: genes that are activated independent of new protein synthesis. Because of these common properties of regulation, we have addressed the possible role of E2F in growth factor dependent activation of transcription. Expression of a c-myc promoter driven CAT gene, transfected into quiescent 3T3 cells, is stimulated by s...
Journal of virology, 1997
The transformation-defective Vero cell host range mutant CS-1 of the highly oncogenic adenovirus type 12 (Ad12) (Ad12-CS-1) has a 69-bp deletion in the early region 1A (E1A) gene that removes the carboxy-terminal half of conserved region 2 and the amino-terminal half of the Ad12-specific so-called spacer that seems to play a pivotal role in the oncogenicity of the virus. Despite its deficiency in immortalizing and transforming primary rodent cells, we found that the E1A 13S protein of Ad12-CS-1 retains the ability to bind p105-RB, p107, and p130 in nuclear extract binding assays with glutathione S-transferase-E1A fusion proteins and Western blot analysis. Like wild-type E1A, the mutant protein was able to dissociate E2F from retinoblastoma-related protein-containing complexes, as judged from gel shift experiments with purified 12S and 13S proteins from transfection experiments with an E1A expression vector or from infection with the respective virus. Moreover, in transient expressio...
The E2F transcription factor is a cellular target for the RB protein
Cell, 1991
it is generally believed that the product of the retinoblastoma susceptibility gene (RB7) is an important regulator of cell proliferation, the biochemical mechanism for its action is unclear. We now show that the RB protein is found in a complex with the E2F transcription factor and that only the underphosphorylated form of RB is in the E2F complex. Moreover, the adenovirus EIA protein can dissociate the EPF-RB complex, dependent on ElA sequence also critical for ElA to bind to RB. These sequences are also critical for El A to immortalize primary cell cultures and to transform in conjunction with other oncogenes. Taken together, these results suggest that the interaction of RB with E2F is an important event in the control of cellular proliferation and that the dissociation of the complex is part of the mechanism by which ElA inactivates RB function.