Cargando…

Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Matsumoto, Naoki, Nemoto-Sasaki, Yoko, Oka, Saori, Arai, Seisuke, Wada, Ikuo, Yamashita, Atsushi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234217/
https://www.ncbi.nlm.nih.gov/pubmed/34089703
http://dx.doi.org/10.1016/j.jbc.2021.100851
_version_ 1783714032553820160
author Matsumoto, Naoki
Nemoto-Sasaki, Yoko
Oka, Saori
Arai, Seisuke
Wada, Ikuo
Yamashita, Atsushi
author_facet Matsumoto, Naoki
Nemoto-Sasaki, Yoko
Oka, Saori
Arai, Seisuke
Wada, Ikuo
Yamashita, Atsushi
author_sort Matsumoto, Naoki
collection PubMed
description 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.
format Online
Article
Text
id pubmed-8234217
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher American Society for Biochemistry and Molecular Biology
record_format MEDLINE/PubMed
spelling pubmed-82342172021-06-29 Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization Matsumoto, Naoki Nemoto-Sasaki, Yoko Oka, Saori Arai, Seisuke Wada, Ikuo Yamashita, Atsushi J Biol Chem Research Article 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. American Society for Biochemistry and Molecular Biology 2021-06-03 /pmc/articles/PMC8234217/ /pubmed/34089703 http://dx.doi.org/10.1016/j.jbc.2021.100851 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Matsumoto, Naoki
Nemoto-Sasaki, Yoko
Oka, Saori
Arai, Seisuke
Wada, Ikuo
Yamashita, Atsushi
Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization
title Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization
title_full Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization
title_fullStr Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization
title_full_unstemmed Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization
title_short Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization
title_sort phosphorylation of human phospholipase a1 ddhd1 at newly identified phosphosites affects its subcellular localization
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234217/
https://www.ncbi.nlm.nih.gov/pubmed/34089703
http://dx.doi.org/10.1016/j.jbc.2021.100851
work_keys_str_mv AT matsumotonaoki phosphorylationofhumanphospholipasea1ddhd1atnewlyidentifiedphosphositesaffectsitssubcellularlocalization
AT nemotosasakiyoko phosphorylationofhumanphospholipasea1ddhd1atnewlyidentifiedphosphositesaffectsitssubcellularlocalization
AT okasaori phosphorylationofhumanphospholipasea1ddhd1atnewlyidentifiedphosphositesaffectsitssubcellularlocalization
AT araiseisuke phosphorylationofhumanphospholipasea1ddhd1atnewlyidentifiedphosphositesaffectsitssubcellularlocalization
AT wadaikuo phosphorylationofhumanphospholipasea1ddhd1atnewlyidentifiedphosphositesaffectsitssubcellularlocalization
AT yamashitaatsushi phosphorylationofhumanphospholipasea1ddhd1atnewlyidentifiedphosphositesaffectsitssubcellularlocalization