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.

神经酰胺激酶（CerK）将神经酰胺磷酸化为神经酰胺-1-磷酸酯（C1P），并且已经证明了CerK / C1P途径在调节细胞/生物学功能中的各种作用。CerK在几个丝氨酸（Ser，S）残基上组成性磷酸化，但是，尚未详细阐明Ser残基在CerK活性中的作用，包括其磷酸化作用。因此，我们进行了本研究以调查此问题。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以其磷酸化形式存在，并通过PMA处理以PKC-βI/ II介导的方式进一步磷酸化。我们检查了丙氨酸（丙氨酸，丙氨酸）对CerK中丝氨酸残基（72/300/340/403/408/427）的置换对其活性和磷酸化的影响。CerK中的三重突变（S340 / 408 / 427A），而不是单突变或双突变（S340 / 408A），显着降低了C1P的形成。在S340 / 408A-和S340 / 408 / 427A-CerK中，PMA诱导的磷酸化水平分别显着和最大程度降低，但在具有单个突变的CerK和野生型CerK中相似。测试的Ser残基突变（包括6个突变）对PMA诱导的C1P形成减少的影响没有超过预期。用蛋白磷酸酶抑制剂，冈田酸和环孢霉素A处理可减少C1P的形成。这些结果证明，CerK的活性在细胞中以磷酸化依赖性方式被调节。