Identification of distinct roles for separate E1A domains in disruption of E2F complexes (original) (raw)
Related papers
Molecular and Cellular Biology, 1993
The transcription factor E2F is present in independent complexes with the product of the retinoblastoma susceptibility gene, pRB, and a related gene product, p107, in association with the cyclin A-cdk2 or the cyclin E-cdk2 kinase complex. pRB and p107 can negatively regulate E2F activity, since overexpression of pRB or p107 in cells lacking a functional pRB leads to the repression of E2F activity. The products of the adenovirus E1A gene can disrupt E2F complexes and result in free and presumably active E2F transcription factor. The regions of E1A required for this function are also essential for binding to a number of cellular proteins, including pRB and p107. Through the use of a number of glutathione S-transferase fusion proteins representing different regions of E1A, as well as in vivo expression of E1A proteins containing deletions of either conserved region 1 (CR1) or CR2, we find that CR2 of E1A can form stable complexes with E2F. E1A proteins containing both CR1 and CR2 also ...
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.
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.
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.
The CtBP binding domain in the adenovirus E1A protein controls CR1- dependent transactivation
Nucleic Acids Research, 1996
The adenovirus E1A-243R protein has the ability to force a resting cell into uncontrolled proliferation by modulating the activity of key targets in cell cycle control. Most of these regulatory mechanisms are dependent on activities mapping to conserved region 1 (CR1) and the non-conserved N-terminal region of E1A. We have previously shown that CR1 functions as a very potent transactivator when it is tethered to a promoter through a heterologous DNA binding domain. However, artificial DNA binding was not sufficient to convert full-length E1A-243R to a transactivator. Thus, an additional function(s) of the E1A-243R protein modulates the effect of CR1 in transcription regulation. Here we demonstrate that a 44 amino acid region at the extreme C-terminus of E1A inhibited transactivation by a Gal4-CR1 fusion protein. Inhibition correlated with binding of the nuclear 48 kDa C-terminal binding protein (CtBP), which has been implicated in E1Amediated suppression of the metastazing potential of tumour cells. This might suggest that CtBP binding can regulate E1A-mediated transformation by modulating CR1-dependent control of transcription.
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.
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.
Journal of Cancer Research and Clinical Oncology, 1995
REPRESSION OF THE c-JUN trans-ACTIVATION FUNCTION BY THE ADENOVIRUS TYPE 12 E1A 52R PROTEIN CORRELATES WITH THE INHIBITION OF PHOSPHORYLATION OF THE c-JUN trans-ACTIVATION DOMAIN. D. Brocknu an, G. KrOner, C. Bury and H. Esche. The cellular transcription factor complex AP-1 mediates growth factor signals on the level ofgene expression and is considered to be decisive in cell differentiation, proliferation and transformation. AP-1 consists mainly of proteins encoded by the jun gene family (c-jun, junB, junD) and the fos gene family (c-los, fosB) including thefos-related antigensfral andfra2. The early region 1A (E1A) 52R polypeptide, a protein expressed exclusively by the in vivo oncogenic Adenovims subtype 12 (Adl2), represses the trans-activating activity of AP-1 consisting of c-Jun:c-Jun homodimers. Repression is accompanied by a direct physical interaction of the adenovirus protein with the bZIP domain of c-Jun essential for dimerization and DNA-binding. Interestingly this interaction does not lead to the prevention of the promoter bindung of c-Jun/AP-1. Moreover, the association between c-JUN and the TATA-box binding protein TBP is not disturbed by the 52R polypeptide. Down-regulation of c-Jun activity is rather due to the inhibition of the phosphorylatinn of its acidic trans-activation domain located at the amino terminal end. In vivo phosphorylation of the c-Jun trans-activation domain by JNK kinases-enzymes belonging to the mitogen-activated protein (MAP) kinase group-is necessary for the interaction of c-Jun with specific co-factors like CBP and therefore a prerequisite for the activation of specific target genes. Due to these results we propose a model in which the 52R protein represses the trans-activating activity of c-Jun by prevemijag its phosphorylation through a JNK kinase(s). (Supported by the Deutsche Forschungsgemeinschaft through SFB 354/TP3 and the Fonds der Chemischen Industrie.
Dual Level Inhibition of E2F-1 Activity by Adeno-associated Virus Rep78
Journal of Biological Chemistry, 2001
E2F-1, a major cellular transcription factor, plays a pivotal role in regulating the cell cycle. The activity of E2F-1 is negatively regulated by its interaction with retinoblastoma protein (pRB), and disruption of the pRB-E2F-1 complex, a hallmark of cellular transformation by DNA tumor viruses, leads to cell proliferation. Adeno-associated virus-2 (AAV) is known to have oncosuppressive properties against DNA tumor viruses. Here we provide, for the first time, the molecular basis for antioncogenic activity of AAV. Rep78, a major regulatory protein of AAV, interacts at the protein level with E2F-1 and stabilizes the pRB-E2F-1 complex. At the DNA level, Rep78 binds to a putative site on the E2F-1 promoter and down-regulates the adenovirus-induced E2F-1 transcription. This dual level of Rep78 activity leads to decreased cellular levels of free E2F-1, leading to its onco-suppressive properties.