Al-Jarrah Seto, Sara; (2025) Regulation of PI3Kδ activity by mutation and phosphorylation at the nSH2–helical interface. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Phosphoinositide 3-kinase delta (PI3Kδ), comprising the p110δ catalytic and p85α regulatory subunits, plays a central role in immune cell signalling, activation, survival and function. Dysregulated PI3Kδ activity underlies immune disorders such as Activated PI3Kδ Syndrome (APDS), a primary immunodeficiency caused by gain-of-function mutations in PIK3CD or PIK3R1. The p110δ-Y524N variant was recently identified in a subset of APDS patients; however, its structural and functional impact remains poorly characterised. Additionally, while several phosphorylation sites have been reported in p110δ, the regulatory roles played by these modifications remain poorly understood. This thesis investigated the regulation of PI3Kδ by examining the biochemical and structural impact of the p110δ-Y524N variant and by exploring the regulatory role of the adjacent S520 phosphorylation site at the key regulatory p110δ-helical/p85α-nSH2 interface. Using a cell model and purified recombinant protein, p110δ-Y524N was shown to be a gain-of-function variant. p110δ-Y524N exhibits elevated basal kinase activity, enhanced membrane binding and increased AKT/mTOR signalling relative to p110δ-WT, consistent with effects of other known APDS variants. Structural modelling predicted p110δ-Y524N disrupts the autoinhibitory helical–nSH2 interface between the p110δ and p85α subunits. Phospho-proteomic and functional studies presented here identified S520 as a putative inhibitory autophosphorylation site in p110δ-WT. S520 may play a role in stabilising the autoinhibited conformation of PI3Kδ, as loss of phosphorylation at this site increased kinase activity, while phospho-mimetic variants suppressed its function. Furthermore, the S520 phosphorylation was absent in the APDS variants, Y524N and E1021K, suggesting its loss may contribute to pathological PI3Kδ activation. Together, this work provides mechanistic insight into PI3Kδ regulation via pathogenic mutation and post-translational modification. It supports the classification of p110δ-Y524N as an activating variant and highlights S520 phosphorylation as a potential regulatory switch in PI3Kδ signalling. These findings contribute to a deeper understanding of PI3Kδ regulation in immune cells under normal conditions and in disease states such as APDS.

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