Phosphatidylinositol 4-kinases (PI4Ks) catalyze the phosphorylation of the D-4 hydroxyl of PI to yield PI(4)P. PI(4)P itself is an important regulator of Golgi budding and sphingolipid metabolism, in addition to its function as a precursor in the synthesis of PI(4,5)P2 and PI(3,4,5)P3l two lipids which control a vast array of cellular processes, including cytoskeletal assembly, membrane trafficking, ion channel opening, and many aspects of cell signaling. There are two classes of PI4K, Types II and III, which are essentially unrelated in sequence and have distinct catalytic properties. The Type II kinases (PI4KMs) were recently cloned and expressed in our laboratory. In mammalian cells they exist as ? and ? isoforms. PI4Kll? is found almost exclusively as an integral membrane protein in intracellular organelles. In contrast, PI4Kll? is distributed evenly between cytosol and membranes, and partially redistributes to the plasma membrane in response to growth factor receptor activation. Our long-term goal is to define the functions and regulation of the Type II PI 4-kinases. In this proposal we seek to understand the functions and mechanism of regulation of PI4Kll?, which we believe to be primarily involved in phosphoinositide signaling at the plasma membrane and in regulation of the cortical cytoskeleton. To this end we will examine: 1. How perturbation of PI4Kll? activity affects cell signaling, adhesion and endocytic trafficking. To explore the function of the single PI4KII enzyme in Drosophila in these contexts we will use loss-of-function models generated by RNAi and genomic mutations. 2. How PI4Kll? translocates to the plasma membrane in a stimulus-dependent manner, and whether it is recruited to membrane subdomains and signaling complexes;and 3. How PI4Kll? is regulated by palmitoylation as a dynamic post-translational modification, and how binding to the chaperone, Hsp90, influences palmitoylation. Results of this study should significantly advance our understanding of this novel family of lipid kinases.
