DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain

Cell Physiol Biochem. 2015;37(1):193-200. doi: 10.1159/000430344. Epub 2015 Aug 20.

Abstract

Background/aims: The linoleic acid derivative DCP-LA selectively and directly activates PKCε. The present study aimed at understanding the mechanism of DCP-LA-induced PKCε activation.

Methods: Point mutation in the C2-like domain on PKCε was carried out, and each kinase activity was monitored in PC-12 cells using a föerster resonance energy transfer (FRET) probe with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at the N- and C-terminal ends of PKCε, respectively, or in the cell-free systems using a reversed phase high-performance liquid chromatography (HPLC). Intracellular PKCε mobilization was monitored in PC-12 cells using mRuby-conjugated PKCε. DCP-LA binding to PKCε was assayed using a fluorescein conjugated to DCP-LA at the carboxyl-terminal end (Fluo-DCP). Uptake of DCP-LA into cells was measured in PC-12 ells.

Results: In the FRET analysis, DCP-LA decreased the ratio of YFP signal intensity/CFP signal intensity in PC-12 cells and in the cell-free kinase assay, DCP-LA increased area of phosphorylated PKC substrate peptide, indicating DCP-LA-induced PKCε activation. These effects were significantly suppressed by replacing Arg50 and Ile89 by Ala or Asn in the C2-like domain of PKCε. In the fluorescent cytochemistry, DCP-LA did not affect intracellular PKCε distribution. In the cell-free binding assay, Fluo-DCP, that had no effect on the potential for PKCε activation, bound to PKCε, and the binding was inhibited only by mutating Ile89. Extracellularly applied DCP-LA was taken up into cells in a concentration-dependent manner. Although no activation was obtained in the cell-free kinase assay, the broad PKC activator PMA activated PKCε in PC-12 cells in association with translocation towards the cell surface, which was inhibited by mutating I89A.

Conclusion: Unlike PMA DCP-LA activates cytosolic PKCε by binding to the phosphatidylserine binding/associating sites Arg50 and Ile89, possibly at the carboxyl-terminal end and the cyclopropane rings, respectively.

MeSH terms

  • Animals
  • Binding Sites / drug effects*
  • Caprylates / pharmacology*
  • Cell Line, Tumor
  • Cell-Free System / drug effects
  • Cell-Free System / metabolism
  • Cytosol / drug effects*
  • Cytosol / metabolism
  • Mutation / drug effects
  • PC12 Cells
  • Phosphatidylserines / metabolism*
  • Phosphorylation / drug effects
  • Protein Kinase C / metabolism*
  • Protein Structure, Tertiary
  • Rats
  • Signal Transduction / drug effects

Substances

  • 8-(2-(2-pentyl-cyclopropylmethyl)cyclopropyl)octanoic acid
  • Caprylates
  • Phosphatidylserines
  • Protein Kinase C