P53 protein is involved in regulation of the cell cycle, apoptosis and cell differentiation. In greater than 50% of human tumors, the negative regulatory activities of p53 can be inactivated by either gene mutation or gene deletion. The p53 tumor suppressor gene products are either inactive or absent contributing to malignant transformation. However, in remaining human tumors that contain normal p53 genes and express normal gene products, their negative growth activity is rendered inactive by unknown mechanisms within cancer cells. We are considering the hypothesis that normal functioning of tumor suppressor gene products in some tumors may be circumvented or functionally inactivated by differential phosphorylation or aberrant modification. Changes in phosphorylation state of p53 can regulate its transcriptional activity, sometimes without detectable changes in p53 levels. We are studying how changes in p53 phosphorylation might affect its biological activity. We are developing methods to assess p53 phosphorylation status. Recombinant-p53 from baculovirus was used as a model to develop a mass spectrometry (MS) means for site-specific determination of phosphorylation. Okadaic acid treatment created a hyperphosphorylated species. Six phosphorylation sites and N-terminal acetylation were identified by MS. 2D PAGE was also used separate p53 phosphoisoforms and we found that each isoform was a mixture of phosphorylated species. These MS studies set the stage for further analytical work linking changes in p53 phosphorylation with biological events such as proliferation, apoptosis and growth arrest. Altered phosphorylation of p53-dependent genes might also be an effective method of downstream p53 inactivation to escape growth control in tumor development. An MS approach for assessing the posttranslationally modified status of p53 in human tumors has been used by our collaborators at the University of Alabama. Their hypothesis was that many clinical gliomas contain wild type p53 protein that inactivated postranslationally. After discovering a high level of nitric oxide synthetase activity in these tumors, they showed immunoreactive nitrosylated tyrosine residues on p53. Using the purified p53 that our laboratory supplied them as a surrogate target, they were able to show tyrosine nitrosylated residues on r-p53 by MS using extracts from clinial gliomas. Further, they demonstrated that nitrosylated p53 bound poorly to DNA containing sequence specific binding sites suggesting posttranslational inactivation of p53. Our collective studies suggest that posttranslation inactivation by phosphorylation by key kinase modifying p53 or by other enzyme systems that alter the p53 molecule provide alternative means of circumventing the normal function of p53 without mutation or deletion of the p53 gene. These studies suggest alternate target sites for clinical therapy for those patients that possess malignancies containing normal p53 genes.
