Phosphoinositide 3-kinase (PI3K) is a key regulator in cancer and inflammation. Surface receptor-coupled PI3Ks promote growth, cell cycle entry, cell migration and prevent apoptosis. Presently, major pharmacological efforts to inhibit PI3Ks are in progress. Current approaches all target the ATP-binding site of PI3K, to the generation of its “oncogenic” product, phosphatidylinositol(3,4,5)-trisphosphate [PtdIns(3,4,5)P3]. Genetic and pharmacological approaches have associated specific PI3K isoforms with cancer, chronic inflammation, allergy, metabolic disease and cardiovascular events. While consequences of PI3K activation are well understood, many aspects of PI3K activation have gained complexity, as simple “receptor activates PI3K” schemes are replaced by non-dogmatic inputs, and localized signaling yielding non-redundant PtdIns(3,4,5)P3 pools. Recently observed drug-induced feedback loops and drug-induced resistant mechanisms could be better understood, if PI3K activation would be fully explored. Here we propose to investigate two systems approaching novel regulatory mechanisms of the class I PI3K catalytic subunits of PI3Kalfa and PI3Kgamma: i) The catalytic subunit of PI3Kalfa is mutated frequently in cancer, and is key for tumor cell survival. Here we investigate PI3Kalfa interacting proteins to corroborate a missing mechanistic link to synergistic action of inhibitors targeting PI3K and poly(ADP-ribose) polymerase (PARP). ii) Signaling of G protein-coupled receptors (GPCRs) is mediated by the so-called PI3Kgamma downstream of trimeric G proteins. PI3Kgamma is a heterodimer composed of a catalytic p110gamma subunit, and one adapter subunit, which is either p84 or p101. We have recently demonstrated that p110gamma is a substrate for protein kinase Cbeta (PKCbeta). Phosphorylation of the helical domain of p110gamma is triggered by clustering of immunoglobulin E (IgE) receptors on mast cells. Ongoing studies suggest that multiple phosphorylation sites in the PI3Kgamma complex, and localized PtdIns(3,4,5)P3 signaling, provide switches between cell adhesion, migration, and degranulation. Recently established chemical-inducers for protein dimerization will be used to explore localized PI3Kgamma signaling. Moreover, novel approaches to target PI3Kgamma complex formation will be explored.