Solar irradiation is a significant cause of human malignancy. Natural cell responses to UV induced damage involves activation of the tumor suppressor protein p53, which normally is preventive of carcinogenesis. p53 functions both to halt cell division to allow time for damage repair, and also to remove irreparably damaged cells through apoptosis. The means by which p53 becomes stimulated during the UV response is not known. Recent work in our lab has turned toward the study of the signaling response to cell stressors, including UV irradiation. We have characterized a new signaling cascade that results in activation of the Stress Activated Protein Kinase (SAPK) after phosphorylation effected by the MEKK signaling kinase. We hypothesize that the MEKK-SAPK cascade regulates the function of p53 via phosphorylation. Phosphorylated p53 is predicted to gain DNA binding activity in vitro and translation stimulating activity in vivo. Mutants of p53 that lack sites of phosphorylation should be unable to respond to UV irradiation, but may retain response to ionizing radiation. To test this hypothesis, we propose these Aims: 1. To identify sites of phosphorylation on p53 by SAPK. Preliminary results have shown p53 to be substrate of SAPK in vitro and in vivo, and have narrowed the possible sites of phosphorylation. Relying heavily on site directed mutagenesis, we will identify these phosphorylation sites and ablate them. 2. To characterize effects of phosphorylation on p53 function. Using the non-phosphorylatable mutant as a critical control, we will examine the ability of phosphorylated verses on non phosphorylated p53 in inducing p53 dependent gene transcription and the cell responses of G1 growth arrest and apoptosis. 3. To assess residual properties of p53 retained in mutants of p53 phosphorylation sites. Since p53 is activated through diverse mechanisms, we predict that UV- nonresponsive mutants will retain the ability to be activated through alternate pathways. This could clarify the different means by which p53 can become activated. p53 deficient cell lines will be reconstituted with wild type or phosphorylation deficient p53 mutants and p53 response measured. In long range goals, p53 deficient mice could be similarly reconstituted.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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