Peroxisome proliferators are an important class of tumor promoters that bind to members of the steroid hormone receptor superfamily named PPARs (Peroxisome Proliferator-Activated Receptors). Several subtypes of PPAR have been discovered (alpha, beta, gamma), although the predominant liver subtype (PPARalpha) is our primary interest due to the critical role it plays in carcinogenesis. Little is known about the biochemical properties of this important receptor. The central hypothesis of this proposal is that the ability of PPARalpha to regulate gene expression is modulated by protein kinase activity as well as protein-protein interactions. A component of these studies is to compare the biochemical properties of PPARalpha to that of PPARbeta and gamma. First, we will determine if phosphorylation through protein kinases pathways is an important regulator of PPARalpha function. Chemical and enzymatic digestion followed by phosphoamino and phosphopeptide analysis will be used to examine which residues are phosphorylated on PPARalpha and which are differentially affected by peroxisome proliferator treatment. The importance of each phosphorylated residue will be examined by introducing specific mutations in PPARalpha. The kinases that regulate PPARalpha activity in a subtype-specific manner will be sought. Second, the subcellular localization of PPARalpha will be examined. Whether PPARalpha is primarily cytosolic in the unactivated state is an important consideration in receptor function. Indirect immunofluorescence microscopy will be used to examine PPARalpha distribution in the unactivated and peroxisome proliferator-activated states. Third, the composition of the PPARalpha-protein complex will be determined. The specific interactions of PPARalpha with heat shock proteins and other factors often associated with steroid hormone receptors will be examined using several complimentary approaches. Comparison of the receptor complex before and after peroxisome proliferator treatment will be important in the overall understanding of the signal transduction pathway. Taken together the proposed studies will greatly increase our knowledge of PPARalpha's mechanism of action and hence our understanding of an important class of chemical carcinogens.
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