Phosphoinositide 3-kinases (PI3K)s regulate many cellular responses. The host laboratory has identified a novel PI3K in myeloid cells which, unlike known PI3Ks, is not activated by receptor protein-tyrosine kinases, but by the BY subunits of heterotrimeric G proteins.
The enzyme is a heterodimer of a 120 kDa and a 101 kDa protein; both subunits are now cloned. This proposal is aimed at characterizing the molecular structure of the novel PI3K, its regulation by ISY subunits, and its involvement in cellular signalling. The project is based on the generation of deletion mutants of both PI3K subunits for definition of the molecular basis of their interaction, the catalytic activity, and their interaction with BY subunits.
We shall introduce the enzyme into mammalian cells by transient transfection to determine its effect on cellular signalling and we shall attempt to generate dominant negative forms of the PI3K for selective inhibition of G protein BY subunit-mediated pathways.