Phosphorylation of a distinct structural form of phosphatidylinositol transfer protein α at ser166 by protein kinase C disrupts receptor-mediated phospholipase c signaling by inhibiting delivery of phosphatidylinositol to membranes

Clive P. Morgan, Alison Skippen, Bruno Segui, Andrew Ball, Victoria Allen-Baume, Banafshe Larijani, Judith Murray-Rust, Neil McDonald, Gopal Sapkota, Nick Morrice, Shamshad Cockcroft

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Abstract

Phosphatidylinositol transfer protein α (PITPα) participates in the supply of phosphatidylinositol (PI) required for many cellular events including phospholipase C (PLC) β and γ signaling by G-protein-coupled receptors and receptor-tyrosine kinases, respectively. Protein kinase C has been known to modulate PLC signaling by G-protein-coupled receptors and receptor-tyrosine kinases, although the molecular target has not been identified in most instances. In each case phorbol myristate acetate pretreatment of HL60, HeLa, and COS-7 cells abrogated PLC stimulation by the agonists formyl-Met-Leu-Phe, ATP, and epidermal growth factor, respectively. Here we show that phosphorylation of PITPα at Ser166 resulted in inhibition of receptor-stimulated PLC activity. Ser166 is localized in a small pocket between the 165-172 loop and the rest of the protein and was not solvent-accessible in either the PI- or phosphatidylcholine-loaded structures of PITPα. To allow phosphorylation at Ser166, a distinct structural form is postulated, and mutation of Thr59 to alanine shifted the equilibrium to this form, which could be resolved on native PAGE. The elution profile observed by size exclusion chromatography of phosphorylated PITPα from rat brain or in vitro phosphorylated PITPα demonstrated that phosphorylated PITPα is structurally distinct from the non-phosphorylated form. Phosphorylated PITPα was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPα structure has to relax to allow access to the Ser166 site, and this may occur at the membrane surface where PI delivery is required for receptor-mediated PLC signaling.

Original languageEnglish
Pages (from-to)47159-47171
Number of pages13
JournalJournal of Biological Chemistry
Volume279
Issue number45
DOIs
Publication statusPublished - 5 Nov 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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