Identification of specific adenovirus E1A N-terminal residues critical to the binding of cellular proteins and to the control of cell growth

Abstract

Adenovirus early region 1A (E1A) oncogene-encoded sequences essential for transformation- and cell growth-regulating activities are localized at the N terminus and in regions of highly conserved amino acid sequence designated conserved regions 1 and 2. These regions interact to form the binding sites for two classes of cellular proteins: those, such as the retinoblastoma gene product, whose association with the E1A products is specifically dependent on region 2, and another class which so far is known to include only a large cellular DNA-binding protein, p300, whose association with the E1A products is specifically dependent on the N-terminal region. Association between the E1A products and either class of cellular proteins can be disrupted by mutations in conserved region 1. While region 2 has been studied intensively, very little is known so far concerning the nature of the essential residues in the N-terminal region, or about the manner in which conserved region 1 participates in the binding of two distinct sets of cellular proteins. A combination of site-directed point mutagenesis and monoclonal antibody competition experiments reported here suggests that p300 binding is dependent on specific, conserved residues in the N terminus, including positively charged residues at positions 2 and 3 of the E1A proteins, and that p300 and pRB bind to distinct, nonoverlapping subregions within conserved region 1. The availability of precise point mutations disrupting p300 binding supports previous data linking p300 with cell cycle control and enhancer function.

P21 and Retinoblastoma Protein Control the Absence of DNA Replication in Terminally Differentiated Muscle Cells

“…3 A, lane 2). However, after separately adding wild-type E1A or an E1A mutant (E1A.928) failing to bind Rb (Wang et al 1993) to analogous extracts, we found that immune complexes of cdk2 could now phosphorylate histone H1 (Fig. 3 A, lanes 3 and 4) to levels almost similar to that of cdk2 immune complexes from proliferating myoblasts.…”

Mitotic cycle reactivation in terminally differentiated cells by adenovirus infection

“…Furthermore, the availability of a wealth of adenovirus mutants should allow a fine dissection of the molecular mechanisms involved in the reactivation of the cell cycle in postmitotic cells. E1A exerts its cell cycle effects by binding a number of cellular proteins, such as p300, pRb, p107, p130, and cyclin A (Harlow et al, 1986;Wang et al, 1993;Whyte et al, 1989). We are in the process of determining the ability of mutant forms of E1A capable oS binding different subsets of these cellular proteins (Wang et al, 1993) to reactivate the cell cycle in terminally differentiated cells.…”

BS69, a novel adenovirus E1A-associated protein that inhibits E1A transactivation.

“…Activation of gene expression was also detected by the 243R ElA protein (Simon et al, 1987) and by domains encoded by exon 2 of Ad5 EIA (Mymryk and Bayley, 1993). CR1 and 2 are involved in transformation (Lillie et al, 1987;Whyte et al, 1989), suppression of enhancer activity (Borelli et al, 1984;Velcich and Ziff, 1985;Wang et al, 1993) and the induction of DNA synthesis (Lillie et al, 1987;Wang et al, 1991;Howe and Bayley, 1992). CR1 and CR2 are thought to exert their effect on cellular physiology by interacting with a number of cellular proteins (Yee and Branton, 1985;Harlow et al, 1986;Egan et al, 1987;Barbeau et al, 1994).…”