Casein Kinase I gamma 2 Down-Regulates Trafficking of Ceramide in the Synthesis of Sphingomyelin

Intracellullar trafficking of lipids is fundamental to membrane biogenesis. For the synthesis of sphingomyelin, ceramide is transported from the endoplasmic reticulum to the Golgi apparatus by the ceramide transfer protein CERT. CERT is phosphorylated by protein kinase D at S132 and subsequently multiple times in a serine-repeat motif, resulting in its inactivation. However, the kinase involved in the multiple phosphorylation remains unclear. Here, we identify the gamma 2 isoform of casein kinase I (CKI gamma 2) as a kinase whose overexpression confers sphingomyelin-directed toxin-resistance to Chinese hamster ovary cells. In a transformant stably expressing CKI gamma 2, CERT was hyperphosphorylated, and the intracellular trafficking of ceramide was retarded, thereby reducing de novo sphingomyelin synthesis. The reduction in the synthesis of sphingomyelin caused by CKI gamma 2 was reversed by the expression of CERT mutants that are not hyperphosphorylated. Furthermore, CKI gamma 2 directly phosphorylated CERT in vitro. Among three gamma isoforms, only knockdown of gamma 2 isoform caused drastic changes in the ratio of hypo-to hyperphosphorylated form of CERT in HeLa cells. These results indicate that CKI gamma 2 hyperphosphorylates the serine-repeat motif of CERT, thereby inactivating CERT and down-regulating the synthesis of sphingomyelin.