Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization
| Autor: | Atsushi Yamashita, Yoko Nemoto-Sasaki, Seisuke Arai, Saori Oka, Ikuo Wada, Naoki Matsumoto |
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| Rok vydání: | 2021 |
| Předmět: |
IB
immunoblot 0301 basic medicine environment and public health Biochemistry IMAC immobilized metal affinity chromatography PA phosphatidic acid Phosphorylation LPI lysophosphatidylinositol CDK cyclin-dependent kinase biology Kinase Chemistry IP immunoprecipitation cyclin-dependent kinase (CDK) GSK-3 glycogen synthase kinase-3 Cell biology Phos-tag Casein kinase 1 Casein kinase 2 λPP lambda protein phosphatase LPA lysophosphatidic acid Research Article inorganic chemicals Hyperphosphorylation Glycerophospholipids 03 medical and health sciences Cyclin-dependent kinase CDC2 Protein Kinase Humans Molecular Biology MALDI-TOF MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry Cyclin-dependent kinase 1 HSP hereditary spastic paraplegia 030102 biochemistry & molecular biology Spastic Paraplegia Hereditary Cyclin-dependent kinase 5 Cell Membrane DDHD domain containing 1 (DDHD1) Cell Biology Phospholipases A1 CK2 casein kinase 2 enzymes and coenzymes (carbohydrates) HEK293 Cells 030104 developmental biology Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization phospholipase A1 (PLA1) biology.protein bacteria Cyclin A2 hereditary spastic paraplegia (HSP) |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| Popis: | Phospholipase A1 (PLA1) hydrolyzes the fatty acids of glycerophospholipids, which are structural components of the cellular membrane. Genetic mutations in DDHD1, an intracellular PLA1, result in hereditary spastic paraplegia (HSP) in humans. However, the regulation of DDHD1 activity has not yet been elucidated in detail. In the present study, we examined the phosphorylation of DDHD1 and identified the responsible protein kinases. We performed MALDI-TOF MS/MS analysis and Phos-tag SDS-PAGE in alanine-substitution mutants in HEK293 cells and revealed multiple phosphorylation sites in human DDHD1, primarily Ser8, Ser11, Ser723, and Ser727. The treatment of cells with a protein phosphatase inhibitor induced the hyperphosphorylation of DDHD1, suggesting that multisite phosphorylation occurred not only at these major, but also at minor sites. Site-specific kinase-substrate prediction algorithms and in vitro kinase analyses indicated that cyclin-dependent kinase CDK1/cyclin A2 phosphorylated Ser8, Ser11, and Ser727 in DDHD1 with a preference for Ser11 and that CDK5/p35 also phosphorylated Ser11 and Ser727 with a preference for Ser11. In addition, casein kinase CK2α1 was found to phosphorylate Ser104, although this was not a major phosphorylation site in cultivated HEK293 cells. The evaluation of the effects of phosphorylation revealed that the phosphorylation mimic mutants S11/727E exhibit only 20% reduction in PLA1 activity. However, the phosphorylation mimics were mainly localized to focal adhesions, whereas the phosphorylation-resistant mutants S11/727A were not. This suggested that phosphorylation alters the subcellular localization of DDHD1 without greatly affecting its PLA1 activity. |
| Databáze: | OpenAIRE |
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