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

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

Takeshi Kanno, Ayako Tsuchiya, Tadashi Shimizu, Miyuki Mabuchi, Akito Tanaka, Tomoyuki Nishizaki

Cell Physiol Biochem. 2015;37(1):193-200.

PMID: 26289043

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP28399317	DCP-LA		28399-31-7	Price

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