Characterization of p150, an Adaptor Protein for the Human Phosphatidylinositol (PtdIns) 3-Kinase

Abstract

Genetic and biochemical studies have shown that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast gene is required for the efficient sorting and delivery of proteins to the vacuole. A human homologue of the yeast gene product has recently been characterized as part of a complex with a cellular protein of 150 kDa (Volinia, S., Dhand, R., Vanhaesebroeck, B., MacDougall, L. K., Stein, R., Zvelebil, M. J., Domin, J., Panaretou, C., and Waterfield, M. D. (1995) 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 Vps34p. Northern blot analysis showed a ubiquitous tissue distribution for p150 similar to that previously observed with PtdIns 3-kinase. Recombinant p150 associated with PtdIns 3-kinase in a stable manner, resulting in a 2-fold increase in lipid kinase activity. Addition of 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 co-immunoprecipitated from human cell lysates using anti-PI-TP antisera. This observation demonstrates that an interaction between a PtdIns 3-kinase and PI-TP occurs , 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 species is involved in protein trafficking.