The PIK3CA gene codes for the 110 kDa catalytic subunit (p110α) of phosphoinositide 3-kinase (PI3Ka). Together with its regulatory component, p85α (encoded by PIK3R), they comprise the powerful PI3Ka heterodimer. This heterodimer conducts growth impulses from multiple cell surface receptor tyrosine kinases (RTKs) to the interior of the cell.
It is known that individual missense mutations in p110α can lead to dysregulated PI3K gain of function in various cancers. Additional, distinct, cis mutations in PIK3CA enhance this overactivation. Characterizing the mechanisms of these mutations provides an opportunity for discovery of PIK3CA mutant selective inhibitors.
Some Backstory on p110α & p85α
From start to finish, p110α contains an adapter binding domain (ABD) that interacts with p85α’s nSH2 domain, a RAS binding domain (RBD), C2 domain (C2), helical domain (HD), a kinase domain (KD), consisting of an N lobe and a C lobe, and a C-terminal activation loop. The catalytic core includes everything C-terminal of the ABD domain. p85α consists of a N-terminal Src homology 3 domain (SH3), a breakpoint-cluster region homology domain (BH), and 3 sequential Src homology 2 domains (nSH2, iSH2, and cSH2). The tight association between p110α and p85α in the cytosol keeps this complex inactive. However, recruitment to membranes and full activation require binding to GTP loaded RAS and the binding of p85α’s nSH2 and cSH2 domains to phosphorylated moieties on RTKs.
While more than 80% of individual missense mutations in P110α occur in the helical domain or C-terminal region of the kinase domain, the exact mechanics of these and other upregulating mutations require further investigation.
The Burke Lab Employs Transcreener Assay to Characterize Oncogenic PIK3CA Mutations
The Burke lab, at the University of Victoria in Canada, employed three novel analytical tests to determine the structural and functional changes in several important oncogenic missense mutations. Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) accurately determines which protein surfaces are more exposed and available to interact. Protein-Lipid FRET (FRET) assays utilize reflected light to assess the ability of proteins to bind to membranes. Finally, to assess the impact of specific mutations on ATPase activity, they employed BellBrook Labs’ signature Transcreener ADP2 Fluorescence Intensity (FI) assay that measures catalytic release of ADP. In this way, each oncogenic mutation is characterized by changes in surface or motif exposure, membrane affinity, and ATPase function due to its unique conformational alterations.
The research by Jenkins et al (2023) at UVic revealed the unique mechanics behind the conformationally induced regulatory changes caused by p110α mutations and how they mimic typical PI3Kα activation. HD mutants (E542K or E545K) disrupt the inhibitory nSH2 contacts with p110α in the same way that p110α/p85α activates by binding phosphorylated RTKs. Mutations affecting the ABD, ABD-RBD linker, or the C2-iSH2 interface surfaces on p110α lead to greater ABD-p85α disengagement and membrane affinity akin to typical activation. In wild type PI3Kα, full membrane engagement requires binding to membrane-localized RAS. Oncogenic mutants H1047R and G1049R in the KD not only evidenced increased ATPase activity, but also enhanced membrane binding via WIF motif and activation loop reorientation. Other KD mutants, E726K and 1068 fs, possess residues that can interact more effectively with negatively charged membranes. However, these mutants don’t have enhanced ATPase activity.
Applications of this Research
All in all, the techniques and analyses used in this research provide a model that describes the mechanisms of activation for over 98% of known PIK3CA mutants. It also explains the increased oncogenicity, arising from multiple cis mutations. This model can accelerate the search for precise PI3Kα inhibitors or activators that can selectively modulate receptor interaction, membrane affinity, and/or ATPase function.
The Transcreener ADP2 Assay is a technology of BellBrook Labs. The assay measures ADP produced during ATP hydrolysis using direct detection, lowering the chance of interference compared to coupling enzyme assays. Because it is an ADP detection method, the assay is universal in nature and can be used to measure activity of virtually any kinase. It is a single-addition, mix-and-ready assay available with an FP, FI, or TR-FRET readout.
- Jenkins, M.L. et al (2023) Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus. Nature Communications, 14:181. https://doi.org/10.1038/s41467-023-35789-6.