Non-canonical activation of PI3Kγ by Ca²⁺/PKCβ in mast cells

Mast cells are key effector cells in allergic disease triggering inflammation through mediator release.
Allergens activate mast cells through the high-affinity receptor for IgE (FcεRI), which initiates
signalling pathways that regulate the release of inflammatory mediators from secretory granules and
the production of lipid mediators and cytokines. Receptor aggregation is coupled to the activation
of protein tyrosine kinases (PTK) that coordinate Ca2+ mobilisation and protein kinase C (PKC)
activation. Also essential is the activation of phosphoinositide 3-kinase (PI3K), as mast cell
degranulation is blocked by pan PI3K inhibition in cells or genetic inactivation of class IB PI3Kγ in
mice. Analysis of bone marrow-derived mast cells (BMMCs) showed that PI3Kγ regulates mast cell
hyperactivaton by boosting PIP3 production via autocrine-paracrine adenosine/G protein-coupled
receptor (GPCR) signalling.
Here we demonstrate a direct pathway from the FcεRI to PI3Kγ. Degranulation triggered by
IgE/antigen or stimulators of Ca2+ mobilisation such as Thapsigargin is blocked in PI3Kγ–/–
BMMCs. FcεRI- and Thapsigargin-induced PI3Kγ activation depends on a high threshold
concentration of intracellular Ca2+ and subsequent PKCβ activation. Thapsigargin to PI3Kγ
signalling occurs completely in an adenosine- and GPCR-independent manner. Active PKCβ
interacts with and phosphorylates PI3Kγ in vitro and in vivo on Ser582. This residue sits at the
beginning of the helical domain, a region that has attracted attention in PI3Kα as mutation hot-spot
in cancer. Analyses of phosphorylation-mimicking mutants indicate that Ser582 functions as switch-
site for PI3Kγ activation. Furthermore, we show that PKCβ-regulated PI3Kγ operates free of the
adaptor protein p84. By biochemical and structural approaches we mapped the p84 binding
interphase on PI3Kγ to the region around S582 and demonstrate that PI3Kγ-bound p84 blocks
S582 phosphorylation.
This study is the first to unravel a pathway and mechanism by which a protein tyrosine kinase
(PTK)-coupled receptor engages PI3Kγ. Contemporaneously we defined the role of PKC in mast
cell degranulation. The results of this work change our view on PI3-kinase regulation, opening
fascinating new insights into mechanisms of isoform-specific PI3K activation.