Phosphorylation and inhibition of ceramide kinase by protein kinase C-β: Their changes by serine residue mutations
Phosphorylation and inhibition of ceramide kinase by protein kinase C-β: Their changes by serine residue mutations
复制标题
蛋白激酶 C-β 对神经酰胺激酶的磷酸化和抑制：丝氨酸残基突变引起的变化
DOI：
10.1016/j.cellsig.2018.11.008
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发表时间：
2019
期刊：
影响因子：
4.8
通讯作者：
Murayama T
中科院分区：
文献类型：
--
作者：
Takahashi H;Ashikawa H;Nakamura H;Murayama T
神经酰胺激酶 (CerK) 将神经酰胺磷酸化为神经酰胺-1-磷酸 (C1P)，并且 CerK/C1P 途径在细胞/生物功能调节中的各种作用已被证明。 CerK 在几个丝氨酸 (Ser, S) 残基上被组成型磷酸化，然而，Ser 残基的作用，包括其磷酸化，在 CerK 活性中的作用尚未详细阐明。因此，我们进行了本研究来调查这个问题。在表达野生型 CerK 的 A549 细胞中，佛波醇 12-肉豆蔻酸酯 13-乙酸酯 (PMA) 处理以蛋白激酶 C (PKC)-βI/II 介导的方式减少了 C1P 的形成。在 Phos-tag SDS-PAGE 分析中，CerK 以磷酸化形式存在，并通过 PMA 处理以 PKC-βI/II 介导的方式进一步磷酸化。我们研究了丙氨酸 (Ala, A) 置换 CerK 中 Ser 残基 (72/300/340/403/408/427) 对其活性和磷酸化的影响。 CerK 中的三重突变 (S340/408/427A)，但不是单一或双重突变 (S340/408A)，显着减少了 C1P 的形成。 S340/408A-和S340/408/427A-CerK中PMA诱导的磷酸化水平分别显着且最大限度地降低，但在具有单一突变的CerK和野生型CerK中相似。测试的 Ser 残基突变（包括六种突变）对 PMA 诱导的 C1P 形成减少的影响并没有超过预期。用蛋白磷酸酶抑制剂、冈田酸和环孢菌素 A 治疗可减少 C1P 的形成。这些结果表明，CerK 的活性在细胞中以磷酸化依赖性方式受到调节。
Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), and various roles for the CerK/C1P pathway in the regulation of cellular/biological functions have been demonstrated. CerK is constitutively phosphorylated at several serine (Ser, S) residues, however, the roles of Ser residues, including their phosphorylation, in CerK activity, have not yet been elucidated in detail. Therefore, we conducted the present study to investigate this issue. In A549 cells expressing wild-type CerK, a treatment with phorbol 12-myristate 13-acetate (PMA) decreased the formation of C1P in a protein kinase C (PKC)-βI/II-mediated manner. In the Phos-tag SDS-PAGE analysis, CerK existed in its phosphorylated form and was further phosphorylated by the PMA treatment in a PKC-βI/II-mediated manner. We examined the effects of the displacement of Ser residues (72/300/340/403/408/427) in CerK by alanine (Ala, A) on its activity and phosphorylation. Triple mutations (S340/408/427A), but not a single or double mutations (S340/408A), in CerK significantly decreased the formation of C1P. PMA-induced phosphorylation levels in S340/408A- and S340/408/427A-CerK were significantly and maximally reduced, respectively, but were similar in CerK with a single mutation and wild-type CerK. Ser residue mutations tested, including six mutations, did not affect PMA-induced decreases in C1P formation more than expected. Treatments with the protein phosphatase inhibitors, okadaic acid and cyclosporine A, decreased the formation of C1P. These results demonstrated that the activity of CerK was regulated in a phosphorylation-dependent manner in cells.