DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain
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-...
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| Format: | Article |
| Language: | English |
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Cell Physiol Biochem Press GmbH & Co KG
2015-08-01
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| Series: | Cellular Physiology and Biochemistry |
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| Online Access: | http://www.karger.com/Article/FullText/430344 |
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| author | Takeshi Kanno Ayako Tsuchiya Tadashi Shimizu Miyuki Mabuchi Akito Tanaka Tomoyuki Nishizaki |
| author_facet | Takeshi Kanno Ayako Tsuchiya Tadashi Shimizu Miyuki Mabuchi Akito Tanaka Tomoyuki Nishizaki |
| author_sort | Takeshi Kanno |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-10T11:46:16Z |
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| id | doaj.art-7f9bfe1901da4eb3ac7e0d9a9f0381fb |
| institution | Directory Open Access Journal |
| issn | 1015-8987 1421-9778 |
| language | English |
| last_indexed | 2024-12-10T11:46:16Z |
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| publisher | Cell Physiol Biochem Press GmbH & Co KG |
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| series | Cellular Physiology and Biochemistry |
| spelling | doaj.art-7f9bfe1901da4eb3ac7e0d9a9f0381fb2022-12-22T01:50:04ZengCell Physiol Biochem Press GmbH & Co KGCellular Physiology and Biochemistry1015-89871421-97782015-08-0137119320010.1159/000430344430344DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like DomainTakeshi KannoAyako TsuchiyaTadashi ShimizuMiyuki MabuchiAkito TanakaTomoyuki NishizakiBackground/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.http://www.karger.com/Article/FullText/430344PKCεC2-like domainPhosphatidylserine binding/associating sitesDirect interactionDCP-LA |
| spellingShingle | Takeshi Kanno Ayako Tsuchiya Tadashi Shimizu Miyuki Mabuchi Akito Tanaka Tomoyuki Nishizaki DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain Cellular Physiology and Biochemistry PKCε C2-like domain Phosphatidylserine binding/associating sites Direct interaction DCP-LA |
| title | DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain |
| title_full | DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain |
| title_fullStr | DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain |
| title_full_unstemmed | DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain |
| title_short | DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain |
| title_sort | dcp la activates cytosolic pkcε by interacting with the phosphatidylserine binding associating sites arg50 and ile89 in the c2 like domain |
| topic | PKCε C2-like domain Phosphatidylserine binding/associating sites Direct interaction DCP-LA |
| url | http://www.karger.com/Article/FullText/430344 |
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