C-MYC PHOSPHORYLATION AND GROWTH RELATED KINASES

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The nuclear proto-oncogene c-myc is thought to be involved in regulation of cell proliferation. Abnormal regulation of c-myc has been implicated in transformation of cells. Despite extensive work on the regulation of c-myc mRNA levels, little is known about the c-myc protein. This study will expand our knowledge about the protein. It will emphasize studies on post-translation modifications, particularly phosphorylation. Biochemical and genetic approaches will be used to examine the role of c-myc protein in the regulation of cellular proliferation. Gel-based enzymatic and chemical cleavage techniques will be used to analyze c-myc phosphorylation patterns under a number of growth condition. Specific phosphorylation sites will be determined by peptide analysis, high voltage electrophoresis of hydrolyzed amino acids and sequence analysis in a spinning cup sequenator. Polyclonal antibodies will be generated against a c- myc/bets-galactosidase fusion protein and used to immuno- precipitate native and denatured protein. Site specific mutants of c-myc will be constructed using oligonucleotide directed mutagenesis. These mutants will be tested for functional and biological properties. This study will also examine the effects of other oncogenes such as the polyoma T antigens on c-myc phosphorylation. Polyoma mutants defective for the stimulation of host cell DNA synthesis will be generated and tested for their effects on cellular growth related kinases and c-myc phosphorylation.