p53 protein is involved in regulation of the cell cycle, apoptosis and cell differentiation. As a sensor of cellular homoestasis and DNA damage, p53 levels can be elevated by exposure to various environmental agents or in response to disease. Changes in phosphorylation state of p53 can regulate transcriptional activity after induction of p53 or sometimes without detectable change in p53 levels. The purpose of this project is to study how changes in p53 phosphorylation relate to function by several approaches. 1) 2D PAGE is being used to separate p53 phosphoisoforms, measure proportional changes and appearance of new species in response to DNA damaging agents. Individual isoforms are being related to p53-mediated cellular responses to identify bioactive isoforms. Production of recombinant p53 from the baculovirus expression system is being used to identify normal phosphorylated and hyperphosphorylated p53 digestion fragments by mass spectral (MS) analysis in collaboration with the MS group in LSB. MS analysis is being similarly applied to purified p53 from normal cells in response to DNA damaging agents. 2) Several collaborators are examining protein- protein interactions with these two types of r-p53 including PARP and in another project with NO producing tumors. 3) Normal and tumor cell lines expressing wild type p53 are being compared for different responses of p21-waf1s inhibition on cdk-cyclin kinase activity to growth arrest and DNA damaging agents. We are determining possible defects in p21-waf1 or downstream responses in tumor cells expressing wt p53. Ongoing and collaborative work will link specific biological activities of growth arrest and apoptosis to individual p53 isoforms and elucidate the role of p21-waf1 phosphorylation in abrogating wt p53 expressing tumors. Future work will incorporate cDNA microarray technology in studying these research problems. - p53 protein, tumor suppressor, p21 or waf1, okadaic acid, hydrogen peroxide, reactive oxygen species, irradiation, cancer
