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

26 pagesThe class 1A phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), heterotrimeric guanine nucleotidebinding protein (G-protein)-coupled receptors (GPCRs), and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane tethered signaling inputs, however, remains unclear. Previous experiments have not been able to elucidate whether interactions with membranetethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our understanding of PI3Kβ regulation, we established an assay to directly visualize and decipher how three binding interactions regulate PI3Kβ 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 PI3Kβ, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ must first cooperatively engage a single RTK-derived tyrosine phosphorylated (pY) peptide before it can engage either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, they only modestly stimulate lipid kinase activity. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by the increase in membrane avidity for these complexes. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1(GTP) through a mechanism of allosteric regulation