Type II diabetics are at a greater risk of exibiting abnormal lipid profiles. As the major cholesterol disposal pathway, bile acid (BA) synthesis exerts a powerful influence on the levels of serum lipids. BA regulate gene expression both by binding the nuclear hormone receptor, FXR, and by activating cellular kinases. BA-activation of FXR/RXR results in the activation of transcription of several genes, including alpha-fetoprotein transcription factor (FTP), which binds the bile acid response element of CYP7A1. This gene encodes the rate limiting step in BA synthesis, cholesterol 7 alpha-hydroxylase. c-Jun N-terminal kinase (JNK) is transiently activated by bile acids, and, here, we give evidence that FTF can be phosphorylated by JNK and protein kinase A (PKA) in vitro, and that gpCYP7 is phosphorylated by JNK, PKA, PKC's and AMP-activated protein kinase (AMPK). This research examines the mechanisms by which covalent modification of proteins by phosphorylation effects activity by a novel combination of biochemical and biophysical techniques. HYPOTHESIS: Kinases rapidly and transiently modulate gene expression via modification of transcription factor FTF structure in a manner analogous to ligand binding.
SPECIFIC AIMS : 1) Characterize the effect of phosphorylation on recombinant FTF structure and function and 2) Characterize the phosphorylation state of FTF/CPF in vivo. METHODS The effect of the phosphorylation on wild type and mutant FTF will be assayed by the electromobility shift assay, protease sensitivity assays, infrared spectroscopy and calorimetry. The physical state of FTF in the cell will be determined by mass spectroscopy and immune precipitation and immunofluorescence using antibodies specific for the phosphorylated and non-phosphorylated versions of the protein. The temporal relationships and magnitude of changes in transcription and protein expression will be measured by quantitative polymerase chain reaction, immuno-detection and enzyme activity assays. The data will serve as a basis for further studies using the proteomics techniques of 2D HPLC and SELDI.
