Phosphoinositide 3-kinase (PI 3-kinase) was discovered because of its co- purification with activated protein-tyrosine kinases. Activation of this enzyme in vivo by growth factors and other cellular activators results in the production of PtdIns-3,4,5-P3, a lipid that is nominally absent in quiescent cells. The stimulation of PI 3-kinase has been implicated in a variety of cellular responses including cell growth, cell transformation, chemotaxis, membrane ruffling, activation of ribosomal S6 kinase, induction of fos, stimulation of glucose uptake, secretion of histamine, activation of ras, lysosomal degradation of receptors and irreversible aggregation of platelets. In several of these studies, the evidence for the importance of PI 3-kinase is quite strong. The yeast PI 3-kinase homolog (VPS34) is required for movement of proteins from the golgi to the vacuole. However, the biochemical mechanism by which this enzyme causes all or even any of these responses is still not understood. The goal of this research is to identify the biochemical pathway(s) by which PI 3-kinase mediates cellular responses. The hypothesis is that the lipid products of this enzyme directly bind to signaling proteins in the cytosol to nucleate specific event at the membrane where the lipid is generated. Two general approaches will be taken. 1) We will look for proteins that show selectivity in binding PtdIns-3,4,5-P3 and/or PtdIns- 3,4-P2 over the more abundant steroisomer PtdIns-4,5-P2. We have identified a subfamily of protein kinase C isoforms (PKCepsilon, PKCdelta and PKCcia) that bind PtdIns-3,4,5-P3 and PtdIns-3,4-P2 with high affinity and stereospecificity and will investigate the possibility that these enzymes are activated by PtdIns-3,4,5-P3 and/or Ptdins-3,4-P2 in vivo. Other proteins that have been reported to bind PtdIns-4,5-P2 will be investigated for their ability to bind PtdIns-3,4,5-P3 and/or PtdIns-3,4-P2 in vitro (in particular actin severing, capping and bunding protein). Affinity columns will be used to isolate novel phosphoinositide-binding proteins. 2) We will also develop in vivo assays for the importance of PI 3-kinase in cellular responses. In particular, we will inject reagents that inhibit or mimic PI 3-kinase activation into single cells and monitor rapid responses that have been attributed to this enzyme (membrane ruffling, chemotaxis). The targets identified in part 1 can be tested in this system.
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