# A new functional assay reveals that membrane binding is critical for overactivation of the phosphoinositide 3-kinase H1047R mutant

## TL;DR

A new experiment shows that the H1047R mutation in PI3Kα causes cancer by making the enzyme bind more tightly to cell membranes.

## Contribution

A novel liposome-based assay was developed to study PI3Kα activity and reveal membrane-dependent overactivation of the H1047R mutant.

## Key findings

- Membrane-bound substrate is essential for H1047R overactivation, unlike E545K.
- Molecular dynamics simulations show H1047R stabilizes membrane interactions.
- Surface plasmon resonance confirms increased membrane binding rate for H1047R.

## Abstract

PIK3CA encodes the catalytic subunit of class I PI3Kα (p110α), a key enzyme in receptor-mediated signaling that phosphorylates phosphatidylinositol-4,5-bisphosphate to the 3,4,5-triphosphate lipid. This gene is frequently mutated in cancers, with H1047R and E545K being the most prevalent mutations. However, the mechanisms of mutants’ overactivation remain incompletely understood. Here, we report the development of a PI3Kα activity assay using reconstituted liposomes, which we employed to address the role of lipid membranes on mutant overactivation. The assay was validated by assessing typical enzymatic features and by confirming the IC50 of well-known inhibitors and the activation by a phosphopeptide mimicking receptor-mediated stimulation. Additionally, we examined the impact of lipid membranes on mutant overactivation by comparing WT and mutant activities with soluble or liposomal PIP2. Interestingly, we discover that the membrane form of the substrate is crucial for the catalytic overactivation of H1047R, whereas E545K overactivation occurs independently of membranes. Consistently, molecular dynamics simulations of ΔABD p110α on a model membrane, performed for WT and H1047R, revealed structural and dynamic changes induced by H1047R, including stabilization of the C-terminal tail on the membrane and altered dynamics of membrane-binding loop 2 residues, suggesting enhanced membrane binding. Intriguingly, in agreement with the above findings, surface plasmon resonance assays reveal higher rate of association of H1047R-PI3Kα with the membrane. Altogether, our data suggest that the overactivation of the H1047R mutant is due to increased rate of membrane binding, providing novel mechanistic insights into how this hot spot mutation leads to catalytic overactivation and contributes to oncogenesis.