Casein Kinase Iγ2 Down-Regulates Trafficking of Ceramide in the Synthesis of Sphingomyelin

Abstract

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 ␥2 isoform of casein kinase I (CKI␥2) as a kinase whose overexpression confers sphingomyelin-directed toxin-resistance to Chinese hamster ovary cells. In a transformant stably expressing CKI␥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␥2 was reversed by the expression of CERT mutants that are not hyperphosphorylated. Furthermore, CKI␥2 directly phosphorylated CERT in vitro. Among three ␥ isoforms, only knockdown of ␥2 isoform caused drastic changes in the ratio of hypo-to hyperphosphorylated form of CERT in HeLa cells. These results indicate that CKI␥2 hyperphosphorylates the serine-repeat motif of CERT, thereby inactivating CERT and down-regulating the synthesis of sphingomyelin.

Phosphoregulation of the Ceramide Transport Protein CERT at Serine 315 in the Interaction with VAMP-associated Protein (VAP) for Inter-organelle Trafficking of Ceramide in Mammalian Cells

“…4C). We expected this band to be VAP-A because the S315E mutation enhanced the interaction between CERT and VAP-A (14,31). Experiments were performed at least three times, and similar results were obtained.…”

“…The treatment of cells with ISP-1 or sphingomyelinase induced the de-phosphorylation of the SRM (14) and also the phosphorylation of Ser-315 (this study), which rendered CERT in a more active state. These results suggested that CERT was post-translationally regulated by at least two different kinase systems as follows: a system involving protein kinase D and casein kinase I, which is relevant to the phosphorylation of the SRM (30,31), and a system relevant to the phosphorylation of Ser-315, whose responsible kinase(s) has yet to be identified.…”

Transmembrane BAX Inhibitor Motif Containing (TMBIM) Family Proteins Perturbs a trans-Golgi Network Enzyme, Gb3 Synthase, and Reduces Gb3 Biosynthesis

“…Isolation of Stx-resistant Genes-HeLa-mCAT#8 cells (1 ϫ 10 6 cells in a 10-cm plate) were infected with the pLIB HeLa cell cDNA-library packaged retroviral particles (Clontech, Mountain View, CA), as described previously (18,19). After 1 week of infection, the infected cells (4.5 ϫ 10 6 cells in three 15-cm plates) were cultured with 200 ng/ml of Stx1 for 1 week.…”

Osh proteins regulate COPII-mediated vesicular transport of ceramide from the endoplasmic reticulum in budding yeast

“…For protein extraction from CHO cells, the cells cultured in dishes, were washed with phosphate-buffered saline and harvested in buffer A (40 mM Tris-HCl, pH 7.4, 180 mM NaCl and 1 mM EDTA) by scraping (Tomishige et al, 2009). After precipitation by centrifugation, cells were suspended in buffer B [10 mM HEPES-NaOH, pH 7.4, 250 mM sucrose, 1 mM EDTA and protease inhibitor cocktail (Complete; Roche Diagnostics)], and lysed by sonication.…”