Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization.
Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization.
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DOI：
10.1016/j.jbc.2021.100851
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发表时间：
2021-07
期刊：
影响因子：
--
通讯作者：
Yamashita A
中科院分区：
文献类型：
--
作者：
Matsumoto N;Nemoto-Sasaki Y;Oka S;Arai S;Wada I;Yamashita A
磷脂酶A1 （PLA1）水解甘油磷脂的脂肪酸，甘油磷脂是细胞膜的结构成分。细胞内PLA1 - dhd1基因突变可导致人类遗传性痉挛性截瘫（HSP）。然而，对dddhd1活性的调控尚未详细阐明。在本研究中，我们检测了DDHD1的磷酸化并确定了负责的蛋白激酶。我们对HEK293细胞中的丙氨酸替代突变体进行了MALDI-TOF MS/MS分析和Phos-tag SDS-PAGE分析，发现人dddhd1中有多个磷酸化位点，主要是Ser8、Ser11、Ser723和Ser727。用蛋白磷酸酶抑制剂处理细胞诱导了DDHD1的过度磷酸化，这表明多位点磷酸化不仅发生在这些主要位点，也发生在次要位点。位点特异性激酶底物预测算法和体外激酶分析表明，周期蛋白依赖性激酶CDK1/cyclin A2磷酸化DDHD1中的Ser8、Ser11和Ser727，并优先磷酸化Ser11， CDK5/p35也磷酸化Ser11和Ser727，并优先磷酸化Ser11。此外，酪蛋白激酶CK2α1被发现磷酸化Ser104，尽管在培养的HEK293细胞中这不是一个主要的磷酸化位点。磷酸化效应的评估显示，磷酸化模拟突变体S11/727E仅显示PLA1活性降低20%。然而，磷酸化模拟物主要定位于局灶黏附，而磷酸化抗性突变体S11/727A则不是。这表明磷酸化改变了dddhd1的亚细胞定位，而不显著影响其PLA1活性。
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.
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DOI：
10.3390/antiox6020025
发表时间：
2017-04-05
期刊：
Antioxidants (Basel, Switzerland)
影响因子：
--
作者：
Gao J;Wang L;Liu J;Xie F;Su B;Wang X
通讯作者：
Wang X
影响因子：
7
作者：
Kinoshita, E;Kinoshita-Kikuta, E;Koike, T
通讯作者：
Koike, T
影响因子：
3.9
作者：
Aoki, Junken;Inoue, Asuka;Arai, Hiroyuki
通讯作者：
Arai, Hiroyuki
影响因子：
4.8
作者：
Higgs, HN;Glomset, JA
通讯作者：
Glomset, JA
影响因子：
16.6
作者：
Inoue N;Hagihara Y;Wright D;Suzuki T;Wada I
通讯作者：
Wada I