The long term objective of this proposal is to understand the biochemical steps which transform a normal resting cell to one with malignant characteristics. This proposal focuses on the mechanism by which the Adenovirus E1A oncogene induces proliferation of quiescent primary cells. Recent evidence indicates that this process is related to E1A binding of an unidentified 300kDa cellular product.
The specific aims of this project are to characterize the 300kDa product by (1) purifying and raising antibodies to it; (2) cloning and overexpressing it; (3) characterizing its posttranslational modifications, and (4) determining its intrinsic biological activities. The identification and characterization of p300 will focus largely on obtaining specific antibodies that recognize this product. When antibodies or some sequence are obtained, cDNA libraries will be screened to identify a p300 clone. The isolation and expression of a p300 clone will permit more extensive characterization including in vitro mutagenesis to study its biological activity. The biochemical and biological properties of the 300kDa protein will be characterized with particular emphasis on properties related to cell growth control and regulation of gene expression. The 300kDa product is a phosphoprotein which appears to undergo specific hyperphosphorylation during mitosis (preliminary results). The phosphorylation patterns of interphase and mitotic p300 will be mapped using techniques such as phosphopeptide analysis. Ultimately, phosphorylation events will be correlated with biological activity. The overall goal of the present proposal is to determine what biological activity of p300 makes it an apparent target for E1A cell growth regulating activity. Obtaining purified p300 will allow us to test its intrinsic biological activity. We will determine, for example, whether it is a DNA binding protein. We will determine the subcellular localization of p300, and the level of protein expression in various normal and aberrantly regulated cells. Possible interactions between p300 and other cellular products will be probed to identify cellular factors that may influence p300 function. Recent discoveries (reviewed in Green, 1989) have emphasized the relationship between E1A transforming functions and human cancer. The transforming mechanisms of adenoviruses are closely related to those of the human papillomaviruses, implicated in human malignancies. E1A association with the retinoblastoma protein provides further evidence that study of the E1A-associated 300-kilodalton product will promote our understanding of human tumorigenesis.
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