Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes.

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 associate with E2F, although the presence of these proteins results in the release of free E2F from its complexes. In vitro reconstitution experiments indicate that E1A-E2F interactions are not direct and that pRB can serve to facilitate these interactions. Complexes containing E1A, p107, cyclin A, and E2F were identified in vivo, which indicates that E1A may associate with E2F through either p107 or pRB. Peptide competition experiments demonstrate that the pRB-binding domain of the human E2F-1 protein can compete with the CR1 but not CR2 domain of E1A for binding to pRB. These results indicate that E1A CR1 and E2F-1 may bind to the same or overlapping sites on pRB and that E1A CR2 binds to an independent region. On the basis of our results, we propose a two-step model for the release of E2F from pRB and p107 cellular proteins.

Characterization of the retinoblastoma binding proteins RBP1 and RBP2.

The retinoblastoma gene product, pRB, regulates cell proliferation by binding to and inhibiting the activity of key growth promoting proteins. Several cellular proteins have been shown to bind directly to pRB and the genes encoding a number of them have been isolated. The protein product of one of these genes is the transcription factor E2F. We have now isolated cDNA clones that contain the full-length coding sequence of two other proteins, RBP1 and RBP2, cloned originally by their interaction with pRB. The products of the RBP1 and RBP2 genes are ubiquitously expressed, large (200 kDa for RBP1 and 195 kDa for RBP2) nuclear phosphoproteins with structural motifs that suggest a role in transcriptional regulation. In addition we have been able to identify complexes of pRB and RBP1 in vivo that are dissociated in the presence of purified human papillomavirus E7 protein.

Synthesis, posttranslational modifications, and nuclear transport of polyomavirus major capsid protein VP1.

Polyomavirus major capsid protein VP1 synthesis was studied in infected primary baby mouse kidney cells. A standard curve of VP1 protein was used to quantitate VP1 in the cytoplasm and nucleus of infected cells during the time course of infection. Polyomavirus VP1 continued to be accumulated in the cytoplasm of the cells until 27 h postinfection, at which time the synthesis of VP1 leveled off. VP1 continued to accumulate in the nucleus of the infected cells throughout the course of infection. The presence of the six isospecies, A to F, of polyomavirus VP1 was also studied to determine the relative quantity of each species during the time course of infection. All six species were found in the cytoplasm and nucleus of infected cells at various times postinfection. However, the relative quantity of each species was different at early as compared with later times of infection. In addition, phosphorylated VP1 was found in isolated polyribosomes of infected cells, suggesting that phosphorylation of VP1 is a cotranslational modification. Examination of the effect of macromolecular synthesis on the transport of VP1 into the nucleus of infected baby mouse kidney cells as well as the rate of its nuclear accumulation during and after protein synthesis inhibition revealed that the continual transport and accumulation of VP1 in the nucleus required protein synthesis.

Heterodimerization of the transcription factors E2F-1 and DP-1 leads to cooperative trans-activation.

The E2F transcription factor has been implicated in the regulation of genes whose products are involved in cell proliferation. Two proteins have recently been identified with E2F-like properties. One of these proteins, E2F-1, has been shown to mediate E2F-dependent trans-activation and to bind the hypophosphorylated form of the retinoblastoma protein (pRB). The other protein, murine DP-1, was purified from an E2F DNA-affinity column, and it was subsequently shown to bind the consensus E2F DNA-binding site. To study a possible interaction between E2F-1 and DP-1, we have now isolated a cDNA for the human homolog of DP-1. Human DP-1 and E2F-1 associate both in vivo and in vitro, and this interaction leads to enhanced binding to E2F DNA-binding sites. The association of E2F-1 and DP-1 leads to cooperative activation of an E2F-responsive promoter. Finally, we demonstrate that E2F-1 and DP-1 association is required for stable interaction with pRB in vivo and that trans-activation by E2F-1/DP-1 heterodimers is inhibited by pRB. We suggest that "E2F" is the activity that is formed when an E2F-1-related protein and a DP-1-related protein dimerize.