I plan to study the gene-regulating activities specifically associated with the transforming functions of the 12S product of the adenovirus E1A oncogene. This analysis is distinctly different from previous studies in many laboratories of the transcriptional transactivation function associated with the 13S E1A product. In spite of recent progress in identifying E1A functional domains and E1A associated proteins, including the retinoblastoma (Rb) product, there is as yet, very little insight into the specific steps by which the E1A products control the expression of cellular products in such a way as to activate resting cells. It is known that the E1A 12S protein carries two separate functions which regulate cell growth. Both are required to direct cells through even a single round of cell division, but expression of the N-terminal function (not involved in Rb binding) is sufficient to drive cells into S-phase and to stall at this point in the cell cycle. In this project, I plan to focus on a specific cellular product known to be regulated in response to E1A cell-cycle functions, and known to play a central role in cell cycle control, I will study the mammalian cdc2 product, determine the level of its E1A-mediated regulation, isolate and make a detailed analysis of its promoter, and correlate the activity of specific promoter elements with the activity of specific functional domains in the E1A products. The relationship between cdc2 activity and E1A-stimulated cell growth will be studied further by determining the cdc2 phosphorylation state, kinase activity and subunit structure in relation to expression of wild-type and mutant E1A products. The availability of mutants specifically inactivating individual E1A transforming domains makes this a particularly attractive system. In the second part of the project, I will focus on the effects of cdc2 activity. The cdc2 kinase appears to be the phosphorylating agent for the Rb gene product. Current models of Rb function propose that phosphorylation inactivates the cell growth-suppressing effect of Rb and that titration of active Rb by the E1A proteins mimics Rb inactivation and allows cell to leave the resting state and enter the cell cycle. In contrast to this model, it is clear that Rb binding by E1A is not at all essential for E1A-mediated induction of S-phase, although these cells fail to proceed to mitosis. I propose that induction of cdc2 protein expression and activation of some level of cdc2 kinase activity may be an alternate mechanism by which E1A can induce S-phase without physically binding Rb. Preliminary results suggest that expression of the N-terminal E1A function may be sufficient for E1A-mediated activation of cdc2. To explore this possibility and to elucidate the mechanisms underlying the biological events directed by E1A, I will characterize the post-translational modifications that occur on the product of the Rb gene in primary cells stimulated by wild-type E1A and selected E1A mutants which induce abortive cell cycle progression. E1A expression can also block the cell growth suppressing effects of TGFbeta. Preliminary results suggest that E1A induction of cdc2 activity may be part of this mechanism. I will characterize the properties of cdc2 in TGFbeta treated cells, in the presence and absence of E1A wild-type and mutant expression, to detail the mechanism by which E1A counteracts the effects of TGFbeta. I feel my plans to focus on limited systems with good preliminary indications of significance will enable me to proceed productively. This project also has the advantage of being able to utilize two important tools that are not always feasible in mammalian cell studies: normal primary cells, and a good genetic system.
Showing the most recent 10 out of 13 publications
