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|Title:||Characterization of Protein COmplexes Interacting with Adenovirus Type 5 E1A Proteins and Identification of Novel E1A Phosphorylated Forms within these Complexes|
|Keywords:||protein;adenovirus;adenovirus type 5;phosphorylation|
|Abstract:||The transforming potential of adenovirus E1A oncogene products derives largely from the formation of complexes with cellular proteins, including the p105ᴿᵇ tumour suppressor and a related p107 species, p130 and p300 proteins, and cyclin A (p60ᶜʸᶜᴬ). Extensive quantitative analyses using E1A deletion mutants identified unique binding patterns for each of these polypeptides within the amino terminus and conserved regions 1 and 2 (CR1 and CR2) of E1A proteins. A novel protein, termed p400, was found by peptide mapping to be related to p300, and, like p300, to require the E1A amino terminus and a portion of CR1 for binding. p130 was shown to be related to p107, and like p107, to associate with p60ᶜʸᶜᴬ. p107, p130 and p105ᴿᵇ all interacted primarily with CR2, however, sequences within CRI and the amino terminus were capable of weak interactions and appeared to function cooperatively with CR2 to bind these proteins. Protein kinase activity exists in E1A complexes and probably derives in part from p33ᶜᵈᵏ²-p60ᶜʸᶜᴬ heterodimers associated with p107 and p130. In vitro phosphorylation of complexes purified by immunoprecipitation resulted in labeling of several proteins. p60ᶜʸᶜᴬ was phosphorylated to about the same extent in cyclin A complexes prepared from either AdS-or mock-infected KB cells, however, that of p 130 and p 107 was dramatically higher in p60ᶜʸᶜᴬ complexes from infected cells. p300 was also phosphorylated in complexes containing EIA proteins. Thus one role of E1A proteins in signal transduction and regulation of the cell cycle may be to control the biological activity ofpl07, p130 and p300 by enhancing their phosphorylation through complex formation. Both major proteins encoded by the E1A region of human adenoviruses are phosphorylated at serine residues located at amino acids 89, 96, 132 and 219 of the 289-residue E1A product and additional serine sites exist between residues 227 and 237. Mutants containing alanine residues in place of these serines formed complexes with a series of cellular polypeptides in an fashion analagous to 𝘸𝘵 Ad5. Phosphorylation at Ser-89 was known to cause a significant "shift" in mobility of E1A proteins in SDS polyacrylamide gels. A significant proportion of E1A proteins were phosphorylated at these additional sites near the carboxy terminus which induced a further "supershift" in gel mobility. However, whereas E1A proteins associated with the p105ᴿᵇ tumour suppressor in 𝘸𝘵-infected KB cells were found in both the normal and "supershifted" forms, the latter species were present preferentially in complexes containing p60ᶜʸᶜᴬ. These data suggested that phosphorylation near the carboxy terminus may play a role in complex formation or may be either enhanced or stablized through associations with protein complexes containing p60ᶜʸᶜᴬ.|
|Appears in Collections:||Digitized Open Access Dissertations and Theses|
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