c-Fos Activates and Physically Interacts with Specific Enzymes of the Pathway of Synthesis of Polyphosphoinositides

ANDRES M. CARDOZO GIZZI, ADOLFO R. ALFONSO PECCHIO, BEATRIZ L. CAPUTTO

Irvine

Workshop; 6th LFD Workshop in Advanced Fluorescence Imaging and Dynamics; 2011

Laboratory for Fluorescence Dynamics

The onco-protein c-Fos is a well recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum (ER) and activates the synthesis of phospholipids. However, the mechanism whereby c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are yet unknown. We showed that induction of quiescent cells to re-enter growth promotes an increase in the labeling of polyphosphoinositides that is dependent on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives (PtdInsP’s) is dependent on the activation of enzymes of the PtdInsP biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol Synthase (CDS), and PtdIns 4 Kinase II alpha (PI4KIIα) in vitro whereas no activation of Phosphatidylinositol Synthase (PIS) or of PtdIns 4 kinase II beta (PI4KIIβ) was observed. In order to understand the molecular mechanism of enzyme activation, analysis of protein-protein interactions between c-Fos and these phospholipid synthesis enzymes were initialed performed by co-immunoprecipitation, a biochemical method. To go further in the examination of a possible participation of c-Fos in a physical interaction with particular phospholipid synthesizing enzymes, we examined this by a different approach: microscopic Fluorescence Resonance Energy Transfer (FRET), using an intensity-based technique called “sensitized emission”. FRET has been used extensively in confocal fluorescence microscopy to quantify protein-protein interactions. Since FRET is dependent on proximity, fluorophores must lie within 1-10 nm of each other for energy transfer to occur, a distance range that is typical of protein-protein interaction. This requirement for FRET to take place practically excludes the possibility of a third party protein being involved. Both co-immunoprecipitation and FRET experiments consistently evidenced a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.