VPS34P REQUIRED FOR YEAST VACUOLAR PROTEIN SORTING IS A MULTIPLE SPECIFICITY KINASE THAT EXHIBITS BOTH PROTEIN-KINASE AND PHOSPHATIDYLINOSITOL-SPECIFIC PI-3-KINASE ACTIVITIES

The Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase h ave been shown to function as a membrane-associated complex which faci litates the delivery of proteins to the vacuole in yeast, Biochemical characterization of the autophosphorylation reaction catalyzed by Vps1 5p demonstrates that it is a functional serine/threonine protein kinas e, In addition, we show that the Vps34 phosphatidylinositol 3-kinase u ndergoes an autophosphorylation event both in vivo and in vitro, indic ating that it represents a novel multiple specificity kinase capable o f phosphorylating both protein and lipid substrates, Vps34p is phospho rylated predominately on serine in vivo and is able to phosphorylate s erine, threonine, and tyrosine residues in vitro, Mutant Vps34 protein s containing alterations in conserved amino acids in the lipid kinase domain are severely defective for both PI 3-kinase activity and autoph osphorylation, Characterization of the PI 3-kinase activity of Vps34p demonstrates that it, unlike the mammalian p110 PI 3-kinase, is highly resistant to the PI 3-kinase inhibitors wortmannin and LY294002, We a lso find that Vps34p is a phosphatidylinositol specific 3-kinase, as i t is able to utilize phosphatidylinositol (PtdIns) but not PtdIns(4)P or PtdIns(4,5)P-2 as substrates in an in vitro PI kinase reaction, The substrate specificity, wortmannin resistance, and other biochemical c haracteristics of its PtdIns 3-kinase activity suggest that Vps34p is quite similar to a PtdIns-specific 3-kinase activity recently characte rized from mammalian cells, These data indicate the existence of a fam ily of PI 3-kinases composed of p110-like PI 3-kinases and Vps34p-like PtdIns-specific 3-kinases, On the basis of the role for Vps34p in vac uolar protein sorting, we propose that the production of a specific ph osphoinositide, PtdIns(3)P, is involved in regulating intracellular pr otein sorting reactions in eukaryotic cells.