Molecular dissection of PI3Kß synergistic activation by receptor tyrosine kinases, GßGγ, and Rho-family GTPases.

ABSTRACT

The class 1A phosphoinositide 3-kinase (PI3K) beta (PI3Kß) 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 PI3Kß 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 PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our understanding of PI3Kß regulation, we established an assay to directly visualize and decipher how three distinct protein interactions regulate PI3Kß 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 PI3Kß, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing the strength of membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1(GTP) through a mechanism consistent with allosteric regulation.