PIP5K isoforms and phosphoinositide molecular species in EGF-stimulated PIP3 synthesis

Description

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 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. We found EGF-stimulated Akt, EGF Receptor (EGFR) and MAPK phosphorylation were also reduced by PIP5K inhibitors. 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 PI3K and MAPKs in MCF10A cells.

Institutions

• University of Cambridge

  England, Cambridge
• Babraham Institute

  England, Cambridge

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