Poster Sessions

MOLBIO/BIOCHEM/BIOPHYS-7

Naveen Bojjireddy

N. Bojjireddy, A. Balla, T. Balla

Development of a Fluorescence-based Phosphatidylinositol 4-kinase Assay for High-throughput Screening to Find Phosphatidylinositol 4-kinase Inhibitors

Phosphatidylinositol 4-kinases (PI4Ks) are emerging as critical components of various cellular processes ranging from vesicular trafficking, cytoskeletal rearrangements exo-and endocytosis to cellular signaling. PI4Ks catalyze the phosphorylation of phosphatidylinositol (PI) at the hydroxyl group of the 4th position of the myo-inositol ring. There are four PI4Ks classified into two classes, namely type II and type III forms each having alpha and beta isoforms. Sofar PI 3-kinase inhibitors have been used to inhibit type III PI4Ks. Knockdown studies were unable to answer the physiological significance of the individual PI4Ks, as complete knockdown cannot be achieved and a small fraction of the PI4Ks is sufficient to carry out some of the cellular processes. Therefore, it is becoming increasingly important to develop isoform specific PI4Ks inhibitors to address the physiological significance of each PI4K isoform. We are developing a PI4K activity assay adapting the alpha screen for high throughput screening. The assay is based on the principle that the FAPP1-PH domain specifically binds to PI4P. We have created a His6- tagged FAPP1PH and used biotinylated-PI as a substrate to set up the assay. We will present data on the optimization of the various parameters in the alpha screen-based PI4K assay.