Phosphorylation of a distinct structural form of phosphatidylinositol transfer protein alpha at Ser166 by protein kinase C disrupts receptor-mediated phospholipase C signaling by inhibiting delivery of phosphatidylinositol to membranes.
Phosphatidylinositol transfer protein alpha (PITPalpha) participates in the supply of phosphatidylinositol (PI) required for many cellular events including phospholipase C (PLC) beta and gamma 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 PITPalpha 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 PITPalpha. 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 PITPalpha from rat brain or in vitro phosphorylated PITPalpha demonstrated that phosphorylated PITPalpha is structurally distinct from the non-phosphorylated form. Phosphorylated PITPalpha was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPalpha 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.
Morgan
Clive P
CP
Department of Physiology, University College London, London WC1E 6JJ.
Skippen
Alison
A
Segui
Bruno
B
Ball
Andrew
A
Allen-Baume
Victoria
V
Larijani
Banafshe
B
Murray-Rust
Judith
J
McDonald
Neil
N
Sapkota
Gopal
G
Morrice
Nick
N
Cockcroft
Shamshad
S
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000196:embryology / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing; D050356:Lipid Metabolism; D018613:Microscopy, Confocal; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D049294:Phospholipid Transfer Proteins / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism
D010455:Peptides; D049294:Phospholipid Transfer Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D013912:Threonine; D012694:Serine; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate