Induction of susceptibility to tumor necrosis factor by E1A is dependent on binding to either p300 or p105-Rb and induction of DNA synthesis.

The introduction of the adenovirus early region 1A (E1A) gene products into normal cells sensitizes these cells to the cytotoxic effects of tumor necrosis factor (TNF). Previous studies have shown that the region of E1A responsible for susceptibility is CR1, a conserved region within E1A which binds the cellular proteins p300 and p105-Rb at nonoverlapping sites. Binding of these and other cellular proteins by E1A results in the induction of E1A-associated activities such as transformation, immortalization, DNA synthesis, and apoptosis. To investigate the mechanism by which E1A induces susceptibility to TNF, the NIH 3T3 mouse fibroblast cell line was infected with viruses containing mutations within E1A which abrogate binding of some or all of the cellular proteins to E1A. The results show that TNF susceptibility is induced by E1A binding to either p300 or p105-Rb. E1A mutants that bind neither p300 nor p105-Rb do not induce susceptibility to TNF. Experiments with stable cell lines created by transfection with either wild-type or mutant E1A lead to these same conclusions. In addition, a correlation between induction of DNA synthesis and induction of TNF sensitivity is seen. Only viruses which induce DNA synthesis can induce TNF sensitivity. Those viruses which do not induce DNA synthesis also do not induce TNF sensitivity. These data suggest that the mechanisms underlying induction of susceptibility to TNF by E1A are intimately connected to E1A's capacity to override cell cycle controls.     

Complementary functions of E1a conserved region 1 cooperate with conserved region 3 to activate adenovirus serotype 5 early promoters.

The amino-terminal region of the adenovirus type 5 E1a protein including conserved regions (CRs) 1 and 2 binds the 105-kDa retinoblastoma protein and a second, 300-kDa, cellular protein. We show that mutant viruses with deletions of CR1 which release the binding of either p105 or p300 still activate early promoters and infect cells productively. However, mutations which disrupt binding of both proteins disrupt early promoter activity and block the viral life cycle. Ela CR3, which has an established role in early promoter activation, can act in trans to the amino-terminal functions. This suggests that the amino terminus provides distinct, redundant functions related to p300 and Rb binding that synergize with CR3 to transactivate early genes.     

The E1A products of oncogenic adenovirus serotype 12 include amino-terminally modified forms able to bind the retinoblastoma protein but not p300.

The cell growth-regulating properties of the adenovirus type 5 (Ad5) E1A oncogene correlate closely with the binding of the E1A products to specific cellular proteins. These proteins include the products of the retinoblastoma tumor susceptibility gene and a 300-kDa product, p300. pRB binds to E1A sequences that are highly conserved among the E1A products of various serotypes, while p300 binding requires sequences in the E1A amino terminus, a region that is not highly conserved. To help evaluate the roles of the E1A-associated proteins in cell growth control, we have compared the p300-binding abilities of the E1A products of Ad5 and of the more oncogenic Ad12 serotype. We show here that despite encoding a sequence that varies somewhat from the p300-binding sequences of Ad5 E1A, the Ad12 E1A products associate with p300 with an affinity similar to that of the Ad5 E1A products. Both the 12S and 13S splice products of Ad12 E1A, like those of Ad5 E1A, encode proteins able to associate with p300. Interestingly, though, both also give rise to prominent forms that are amino terminally modified and unable to associate with p300. This modification, at least in the 13S product, does not appear to diminish the affinity of this product for the retinoblastoma protein.     

Inactivation of the retinoblastoma susceptibility protein is not sufficient for the transforming function of the conserved region 2-like domain of simian virus 40 large T antigen.

Simian virus 40 large T antigen interacts with three cellular proteins, pRb, p107, and p130, through a common binding site on the T antigen protein called the E1A conserved region 2-like (CR2-like) domain. Mutations in this domain inactivate the transforming activity of large T antigen. Since these mutations have been demonstrated to abolish binding to pRb and p107, and presumably therefore affect binding to p130, assessment of the relative roles of these three proteins in transformation of rodent fibroblasts by T antigen has been difficult. We have examined the role of T antigen-pRb interactions in transformation. We have introduced a mutant T antigen, which is unable to bind any of these three proteins, into primary mouse fibroblasts derived from the embryos of mice in which the Rb gene encoding the retinoblastoma protein had been disrupted. This mutant is unable to transform the Rb-negative fibroblasts, indicating that inactivation of pRb is not the sole function of the CR2-like domain in the induction of transformation of mouse fibroblasts by simian virus 40.     

DNA-binding properties of the E1A-associated 300-kilodalton protein.

One of the major E1A-associated cellular proteins is a 300-kDa product (p300) that binds to the N-terminal region of the E1A products. The p300 binding site is distinct from sequences involved in binding the retinoblastoma product and other E1A-associated cellular products such as p60-cyclin A and p107. p300 binding to E1A is linked genetically to the enhancer repression function of E1A and the other E1A-mediated gene-regulating functions as well as to the transforming functions of E1A. However, the biochemical properties of p300 have not yet been characterized. We report here that p300 has an intrinsic DNA-binding activity and shows a preferential affinity for specific DNA sequences. The sequences selectively bound by p300 are related to those of a series of enhancer elements that are recognized by NF-kappa B. The direct physical interaction of p300 with enhancer elements provides a biochemical basis for the genetic evidence linking the E1A-mediated enhancer repression function with the p300-binding activity of E1A.     

In vitro analysis of the E1A-homologous sequences of RIZ.

The RIZ (G3B/MTB-Zf) gene was first isolated based on its ability to bind to the retinoblastoma protein (Rb). An acidic, approximately 100-amino-acid region around the Rb-binding motif of RIZ has structural and antigenic similarity to the conserved sequences of the E1A viral oncogene. We show here that this region interacts specifically with the E1A-binding domain of Rb. This interaction could be disrupted by E1A or by a peptide of RIZ homologous to the CR2 motif of E1A which is involved in binding to Rb family proteins. Also like E1A, RIZ can form a ternary complex with Rb and E2F1. Despite this similarity to E1A, however, RIZ could not bind to the Rb family proteins p107 and p130 in vitro. The data show that the RIZ CR2 motif can mediate differential binding to Rb family proteins. We also mapped the shared antigenic determinant between RIZ and E1A to a conserved sequence, designated CE1, which is located in the C terminus of E1A. Unlike that of ETA, the CE1 motif of RIZ is located next to the CR2 motif. Despite this proximity, CE1 and CR2 appear to act independently. The data show similarities as well as differences between the homologous sequences of RIZ and E1A and contribute to an understanding of the biochemistry of these proteins.     

Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein

We have studied the domain structure of the A1 heterogeneous nuclear ribonucleoprotein using both partial proteolysis and photochemical cross-linking to oligodeoxynucleotides. Both the intact A1 protein and its proteolytic fragment, the UP1 protein, can be cleaved by Staphylococcus aureus V-8 protease to produce two polypeptides of 92 amino acids. These two polypeptides correspond to the internal repeat sequence previously noted by us to occur in UP1. The two polypeptides can be purified via single-stranded DNA cellulose chromatography and independently cross-linked to [32P]p(dT)8, indicating that each domain can bind to single-stranded nucleic acids. Purification and sequencing of A1 tryptic peptides that had been cross-linked to oligothymidylic acid revealed that 4 phenylalanine residues, phenylalanines 16, 58, 107, and 149 are the sites of covalent adduct formation, with phenylalanine 16 being the major site of cross-linking. These phenylalanine residues are internally homologous when the repeat sequences in A1 are aligned, that is, phenylalanines 16 and 107 occupy analogous positions in the 91-residue repeat, as do phenylalanines 58 and 149. An examination of the primary structures of a variety of eucaryotic RNA-binding proteins with sequence homology to A1 reveals that the cross-linked phenylalanines in A1 are highly conserved among all of these proteins. Our results provide the first experimental evidence that conserved residues in the 90-amino acid repeating domains shared by A1 and other single-stranded nucleic acid binding-proteins form part of an RNA-binding pocket.     

Mutagenesis of lysine 62, asparagine 64, and conserved region 1 reduces the activity of human ecto-ATPase (NTPDase 2)

The human ecto-ATPase (NTPDase 2) contains conserved motifs including five apyrase conserved regions (ACRs) and four conserved regions (CRs) as well as conserved lysine and arginine residues that are also present in other cell surface E-NTPDases. Some of the positively charged amino acids may be involved in ATP binding. The protein also contains six potential N-linked glycosylation sites. Results obtained with seven lysine and six arginine mutants indicate the importance of K62 that is located in CR1, K182, which is downstream of ACR3, and R155, which immediately follows CR3. Mutation of asparagine at the six potential N-linked glycosylation sites individually to glutamine established the importance of N64 in CR1 and N443 in ACR5 in protein function and expression. Mutation of N64, which is conserved in all cell surface NTPDases, results in the expression of an unstable protein, the activity of which is only manifested in the presence of concanavalin A. Both K62 and N64 reside in CR1 that is conserved in all cell surface NTPDases. In the sequence of the CR1 of human ecto-ATPase, 58WPADKENDTGIV69, 65DTG67 is similar to the phosphate-binding motif (DXG) in ACR1 and 4. The D65A and G67A mutants have reduced protein expression and activity. Mutations of other residues in CR1 to alanine led to partial to complete loss of protein expression and activity except for P59. The alanine mutants of the three acidic amino acid residues, D61, E63, and D65, all have decreased affinity for divalent ions. D61 can be substituted by glutamate, but E63 appears to be invariable. Taken together, these results indicate that CR1, which follows ACR1 in the cell surface NTPDases, is an essential structural element in these enzymes.