PI3Ks are a family of lipid kinases that phosphorylate PIP2 to PIP3, to which the downstream signaling target Akt binds to regulate an array of cellular activities, including cell growth, proliferation, differentiation, migration, mobility and apoptosis. Unlike protein kinases where the substrate abuts the ATP, crystal structures indicate that in PI3Ka, the distance between the g phosphate of the ATP and the PIP2 lipid substrate is over 6 A, much too far for the phosphoryl transfer, raising the question of how catalysis is executed. PI3Ka has two subunits, the catalytic p110a and the regulatory p85a. Our results show that release of the autoinhibition exerted by the nSH2 domain triggers significant conformational change in p110a. Structural rearrangement in the kinase domain reduces the distance between the ATP g-phosphate and the substrate, offering an explanation to how phosphoryl transfer is executed. This mechanism not only explains how oncogenic mutations promote PI3Ka activation by facilitating nSH2 release, or nSH2-release-induced, allosteric motions; it also offers an innovative, PI3K isoform-specific drug discovery principle. Rather than competing with nanomolar range ATP in the ATP-binding pocket and contending with ATP pocket conservation and massive binding targets, this mechanism suggests blocking PI3Ka sequence-specific cavity between the ATP-binding pocket and the substrate binding site. Targeting isoformspecific residues in the cavity may prevent PIP2 phosphorylation. This work has been funded in whole or in part with Federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.