The long term goal of this laboratory is to understand the role of phosphoinositides in cellular regulation. The objective of this grant is to understand the mechanism of regulation of the enzymes that produce the various phosphoinositides. During the previous granting period it was discovered that the phosphatidylinositol kinase that co-purifies with protein-tyrosine kinases is in a new pathway. This enzyme phosphorylates the D-3 position of phosphoinositides to produce three lipids not previously known to exist, phosphatidylinositol (3)phosphate [PtdIns(3)P], phosphatidylinositol(3,4)bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol(3,4,5)trisphosphate [PtdIns(3,4,5)P3]. Mutational studies of growth factor receptors and oncogenes indicate that production of these lipids is essential for stimulation of cell growth and transformation. PtdIns(3,4)P2 can be produced by phosphorylation of PtdIns(4)P at the D-3 position by the PtdIns 3-kinase or in some cells by phosphorylation of PtdIns(3)P at the D-4 position by a recently discovered PtdIns(3)P 4-kinase. In order to understand the importance of these lipids for cell signalling and the regulation of their synthesis, the phosphoinositide kinases will be purified to homogeneity. The purified enzymes will be characterized with respect to subunit composition, substrate utilization, and association with and phosphorylation by purified protein-tyrosine kinases. The proteins will be sequenced and oligonucleotide probes based on the sequence will be used to obtain cDNA clones. Antibodies will be raised against the purified proteins. Once clones are available the proteins will be expressed and the role of phosphorylation in regulation of activity can be evaluated by site specific mutations. The PtdIns 3-kinase has been highly implicated in platelet derived growth factor responses and in cell transformation and might be a useful target for drug intervention of vascular smooth muscle growth and tumor growth.
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