Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases

The class 1A phosphoinositide 3-kinase (PI3K) beta (PI3K{beta}) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), heterotrimeric guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCRs), and Rho-family GTPases. The mechanism by which PI3K{beta} prioritizes interactions with various membrane tethered signaling inputs, however, remains unclear. Previous experiments have not been able to elucidate whether interactions with membrane-tethered proteins primarily control PI3K{beta} localization versus directly modulate lipid kinase activity. To address this gap in our understanding of PI3K{beta} regulation, we established an assay to directly visualize and decipher how three distinct protein interactions regulate PI3K{beta} when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling membrane localization of PI3K{beta}, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3K{beta} prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either G{beta}G{gamma} or Rac1(GTP). Although pY peptides strongly localize PI3K{beta} to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/G{beta}G{gamma} or pY/Rac1(GTP), PI3K{beta} activity is dramatically enhanced beyond what can be explained by simply increasing the strength of membrane localization. Instead, PI3K{beta} is synergistically activated by pY/G{beta}G{gamma} and pY/Rac1(GTP) through a mechanism consistent with allosteric regulation.