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|Title:||Characterization of phosphorylation sites on the E1A polypeptides of human adenovirus type 5|
|Authors:||Dumont, Joseph Daniel|
|Advisor:||Branton, Philip E.|
|Keywords:||Medical Sciences;Medical Sciences|
|Abstract:||<p>Human adenoviruses can induce tumors in rodents through the action of the products of two viral genes, termed E1A and E1B. E1A produces multiple transcripts as a result of differential splicing, however, only the products of the 13S and 12S mRNAs appear to play a role in oncogenicity and cell transformation. These mRNAs encode proteins of 289 and 243 residues that are identical except for the presence of a unique 46-amino internal sequence. Each of these species migrates in polyacrylamide gels as at least two electrophoretically separable forms which differ in apparent molecular mass by about 4 kDa. Previous studies had indicated that differential phosphorylation is largely responsible for this shift in gel migration. The objective of this thesis was to identify the phosphorylation events responsible for this mobility shift, and to determine the functional significance. These studies focussed on phosphorylation sites towards the amino terminus of E1A proteins. Mutants were generated by site-directed mutagenesis in which known phosphorylation sites at Ser89 and Ser96 were altered to alanine residues. Characterization of these and other mutants suggested that phosphorylation at Ser89 was largely responsible for the shift in gel migration of the E1A proteins, and may regulate phosphorylation at Ser96. Removal of the phosphorylation site at Ser89 was found to have no significant effect on the ability of E1A proteins either to transactivate E3 expression or to repress SV-40 enhancer activity, but it did reproducibly reduce E1A-mediated transforming activity by about three fold. These results suggested that phosphorylation at Ser89 may be of some regulatory significance. Ser89 as well as Ser219 were shown to be phosphorylated in vitro by the cell cycle protein p34^(cdc2Hs) which had been purified by immunoprecipitation using specific antiserum. In addition, E1A proteins were found to be most highly phosphorylated during mitosis, the period in the cell cycle of maximal p34^(cdc2Hs) activity. These results suggested that p34^(cdc2Hs) or a related protein kinase phosphorylates E1A proteins at these two sites in vivo.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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