PIP5K isoforms and phosphoinositide molecular species in EGF-stimulated PIP₃ synthesis: A multi-method approach using enzyme genetic suppression and chemical inhibition in MCF10A cells
- 1Babraham Institute, Cambridge, United Kingdom
- 2Astra Zeneca, Cambridge, United Kingdom
- 3Cancer Research Horizons Therapeutic Innovation, Babraham Research Campus, Cambridge, United Kingdom
Abstract
The membrane phospholipid PI(4,5)P2 has structural functions and is a substrate for PLC and PI3K enzymes to generate Ins(1,4,5)P3, DAG and PI(3,4,5)P3 signals. PIP5Ks, that 5-phosphorylate PI4P, are the major source of PI(4,5)P2 in mammalian cells. Current thinking suggests distinct pools of PI(4,5)P2 may be differentially generated by PIP5Kα, β and γ isoforms and involved in different physiological functions. Phosphoinositides are comprised of families of acyl-chain variants but are uniquely enriched in C38:4 species. There is evidence that PIP5Ks are selective for certain acyl chain species of PI(4)P. We aimed to understand how inhibition of PIP5Ks impacts PIP3 production and whether they differentially support synthesis of a pool of PI(4,5)P2, that may be marked by a distinct profile of molecular species variants, which supply class I PI3Ks in a non-transformed human cell line (MCF10A cells). Using a combination of siRNA, Cas9/CRISPR-mediated gene knockout (k/o), recently derived selective, small-molecule inhibitors of PIP5Ks and mass-spectrometry-based lipidomic techniques, we attempted to address these questions. We found that MCF10A cells only express PIP5Kα and γ and that neither siRNA-mediated reduction nor k/o of either protein changed the levels or acyl chain profiles of any of the phosphoinositides in Epidermal Growth Factor- (EGF) stimulated MCF10A cells. We found that selective PIP5K inhibition reduced levels of PIP2 and EGF-stimulated PIP3 and increased levels of PIP, however, the acyl-chain-profiles of none of these lipids were significantly altered in MCF10A cells. We found EGF-stimulated Akt, EGF Receptor (EGFR) and MAPK phosphorylation were also reduced by PIP5K inhibitors in this cell line. To deconvolute these results, we tested the impact of a selective PI3Kα inhibitor (A66) and found it inhibited PIP3 accumulation and Akt phosphorylation and reduced MAPK phosphorylation but was without effect on total EGFR or its phosphorylation, suggesting some cross-talk between EGF-stimulated activation of class I PI3K and MAPKs in MCF10A cells.
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BibTeX
@article{alexandrou2026pip5k,
title = {{PIP5K isoforms and phosphoinositide molecular species in EGF-stimulated PIP₃ synthesis: A multi-method approach using enzyme genetic suppression and chemical inhibition in MCF10A cells}},
author = {Iosif Alexandrou and Vikki Flemington and Martin E. Swarbrick and Sabina Cosulich and Phillip T. Hawkins and Len R. Stephens},
journal = {PLOS One},
year = {2026},
month = nov,
doi = {10.1371/journal.pone.0362260},
url = {https://doi.org/10.1371/journal.pone.0362260},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - PIP5K isoforms and phosphoinositide molecular species in EGF-stimulated PIP₃ synthesis: A multi-method approach using enzyme genetic suppression and chemical inhibition in MCF10A cells
AU - Iosif Alexandrou
AU - Vikki Flemington
AU - Martin E. Swarbrick
AU - Sabina Cosulich
AU - Phillip T. Hawkins
AU - Len R. Stephens
PY - 2026
DA - 2026/11/05
JO - PLOS One
DO - 10.1371/journal.pone.0362260
UR - https://doi.org/10.1371/journal.pone.0362260
AB - The membrane phospholipid PI(4,5)P2 has structural functions and is a substrate for PLC and PI3K enzymes to generate Ins(1,4,5)P3, DAG and PI(3,4,5)P3 signals. PIP5Ks, that 5-phosphorylate PI4P, are the major source of PI(4,5)P2 in mammalian cells. Current thinking suggests distinct pools of PI(4,5)P2 may be differentially generated by PIP5Kα, β and γ isoforms and involved in different physiological functions. Phosphoinositides are comprised of families of acyl-chain variants but are uniquely enriched in C38:4 species. There is evidence that PIP5Ks are selective for certain acyl chain species of PI(4)P. We aimed to understand how inhibition of PIP5Ks impacts PIP3 production and whether they differentially support synthesis of a pool of PI(4,5)P2, that may be marked by a distinct profile of molecular species variants, which supply class I PI3Ks in a non-transformed human cell line (MCF10A cells). Using a combination of siRNA, Cas9/CRISPR-mediated gene knockout (k/o), recently derived selective, small-molecule inhibitors of PIP5Ks and mass-spectrometry-based lipidomic techniques, we attempted to address these questions. We found that MCF10A cells only express PIP5Kα and γ and that neither siRNA-mediated reduction nor k/o of either protein changed the levels or acyl chain profiles of any of the phosphoinositides in Epidermal Growth Factor- (EGF) stimulated MCF10A cells. We found that selective PIP5K inhibition reduced levels of PIP2 and EGF-stimulated PIP3 and increased levels of PIP, however, the acyl-chain-profiles of none of these lipids were significantly altered in MCF10A cells. We found EGF-stimulated Akt, EGF Receptor (EGFR) and MAPK phosphorylation were also reduced by PIP5K inhibitors in this cell line. To deconvolute these results, we tested the impact of a selective PI3Kα inhibitor (A66) and found it inhibited PIP3 accumulation and Akt phosphorylation and reduced MAPK phosphorylation but was without effect on total EGFR or its phosphorylation, suggesting some cross-talk between EGF-stimulated activation of class I PI3K and MAPKs in MCF10A cells.
SN - 1932-6203
ER -