Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases
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Date
2023-05-01
Authors
Duewell, Benjamin R.
Wilson, Naomi E.
Bailey, Gabriela M.
Peabody, Sarah E.
Hansen, Scott D.
Journal Title
Journal ISSN
Volume Title
Publisher
bioRxiv
Abstract
The 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.
Description
26 pages
Keywords
1A phosphoinositide 3-kinase, 1A phosphoinositide 3-kinase beta, G-protein, GPCR
Citation
Duewell, B. R., Wislon, N. E., Bailey, G. M., Peabody, S. E., & Hansen, S. D. (2023). Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases. bioRxiv, 1—26. https://doi.org/10.1101/2023.05.01.538969