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


CONCLUSIONS
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.
Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan.
Takeshi
T
Kanno
Ayako
A
Tsuchiya
Tadashi
T
Shimizu
Miyuki
M
Mabuchi
Akito
A
Tanaka
Tomoyuki
T
Nishizaki
D000818:Animals; D001665:Binding Sites / Q000187:drug effects*; D002210:Caprylates / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002474:Cell-Free System / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects* / Q000378:metabolism; D009154:Mutation / Q000187:drug effects; D016716:PC12 Cells; D010718:Phosphatidylserines / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects
C506022:8-(2-(2-pentyl-cyclopropylmethyl)cyclopropyl)octanoic acid; D002210:Caprylates; D010718:Phosphatidylserines; D011493:Protein Kinase C