Characterizing the Kinetics of Localization and Activation of PI3K Paralogs α and β in Immune Cell Signali
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Date
2023-05
Authors
Peabody, Sarah
Journal Title
Journal ISSN
Volume Title
Publisher
University of Oregon
Abstract
Cell polarization and migration are critical processes performed by immune cells to execute their
specialized functions. At the forefront of understanding these processes is the study of enzyme
that catalyze biochemical reaction that drive immune cell activation. In particular, the rapid
synthesis of the phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P 3 ) is an early step in many
immune cell signaling pathways. This reaction is catalyzed by phosphatidylinositol-3-kinase
(PI3K) and a collection of membrane associated factors that controlling the localization and
activity of PI3K. The proposed models in the field suggest that the activity of PI3K paralogs α
and β are controlled through synergistic binding of a variety of membrane proteins including
GβGγ, pY peptides, and the Rho-family small GTPases Rac1 or HRas. However, these previous
studies have not addressed which of these factors control PI3K localization to membranes and
which play a role in directly activating the enzyme. For my thesis, I used supported lipid bilayer
technology and Total Internal Reflection Fluorescence (TIRF) microscopy to study how different
PI3K regulatory factors drive the localization or activation of PI3Kα and PI3Kβ. Using these
techniques, I was able to determine that both PI3Kα and β are primarily localized to membranes
by RTK derived phosphorylated tyrosines (pY) peptides. Once bound to pY peptide, PI3Kα and
β can form secondary interactions with either HRas for PI3Kα or Rac1/GβGγ for PI3Kβ. These
secondary interactions can enhance membrane binding dynamics of PI3K, but they are unable to
drive localization on their own, and instead must work in tandem with pY peptides. Finally, I
found that activation of PI3Kα and β by the combination of inputs is not proportional to the
change in localization, suggesting that synergistic activation of PI3K is driven by an allosteric
mechanism.
Description
45 pages
Keywords
Cell Signaling, Polarization, PI3K, Synergy, Protein Interactions, Kinetics