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

The membrane phospholipid PI(4,5)P 2 has structural functions and is a substrate for PLC and PI3K enzymes to generate Ins(1,4,5)P 3 , DAG and PI(3,4,5)P 3 signals. PIP5Ks, that 5-phosphorylate PI4P, are the major source of PI(4,5)P 2 in mammalian cells. Current thinking suggests distinct pools of PI(4,5)P 2 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 PIP 3 production and whether they differentially support synthesis of a pool of PI(4,5)P 2 , 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 PIP 2 and EGF-stimulated PIP 3 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 PIP 3 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.