CHARACTERIZATION OF P150, AN ADAPTER PROTEIN FOR THE HUMAN PHOSPHATIDYLINOSITOL (PTDINS) 5-KINASE - SUBSTRATE PRESENTATION BY PHOSPHATEDYLINOSITOL TRANSFER PROTEIN TO THE P150-PTDINS 3-KINASE COMPLEX

Genetic and biochemical studies have shown that the phosphatidylinosit ol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is required for t he efficient sorting and delivery of proteins to the vacuole. A human homologue of the yeast VPS34 gene product has recently been characteri zed as part of a complex with a cellular protein of 150 kDa (Volinia, S., Dhand, R., Vanhaesebroeck, B., MacDougall, L. H., Stein, R,, Zvele bil, M. J., Domin, J., Panaretou, C,, and Waterfield, M. D. (1995) EMB O J. 14, 3339-3348). Here, cDNA cloning is used to show that the amino acid sequence of this protein, termed p150, is 29.6% identical and 53 % similar to the yeast Vps15p protein, an established regulator of Vps 34p, Northern blot analysis showed a ubiquitous tissue distribution fo r p150 similar to that previously observed with PtdIns 3-kinase. Recom binant p150 associated with PtdIns 3-kinase in vitro in a stable manne r, resulting in a 2-fold increase in lipid kinase activity. Addition o f phosphatidylinositol transfer protein (PI-TP) further stimulated the lipid kinase activity of the p150 . PtdIns 3-kinase complex 3-fold. A PtdIns 3-kinase activity could also be coimmunoprecipitated from huma n cell lysates using anti-PI-TP antisera. This observation demonstrate s that an interaction between a PtdIns 3-kinase and PI-TP occurs in vi vo, which further implicates lipid transfer proteins in the regulation of PtdIns 3-kinase activity. These results suggest that the Vps15p . Vps34p complex has been conserved from yeast to man and in both specie s is involved in protein trafficking.