Enzyme Activities of the Ceramide Synthases CERS2–6 Are Regulated by Phosphorylation in the C-terminal Region

Abstract

Ceramide and complex sphingolipids regulate important cellular functions including cell growth, apoptosis, and signaling. Dysregulation of sphingolipid metabolism leads to pathological consequences such as sphingolipidoses and insulin resistance. Ceramides in mammals vary greatly in their acyl-chain composition: six different ceramide synthase isozymes (CERS1-6) that exhibit distinct substrate specificity and tissue distribution account for this diversity. In the present study, we demonstrated that CERS2-6 were phosphorylated at the cytoplasmic C-terminal regions. Most of the phosphorylated residues conformed to a consensus motif for phosphorylation by casein kinase 2 (CK2), and treatment of cells with the CK2-specific inhibitor CX-4945 lowered the phosphorylation levels of CERS2, -4, -5, and -6. Phosphorylation of CERS2 was especially important for its catalytic activity, acting mainly by increasing its V max value. Phosphorylation modestly increased the catalytic activities of CERS4 and -5 and mildly increased those of CERS3 and -6. Dephosphorylation of endogenous ceramide synthases in the mouse brain led to severely reduced activity toward the Cers2 substrates C22:0/C24:0-CoAs and modestly reduced activity toward the Cers5/6 substrate C16:0-CoA. These results suggest that the phosphorylation of ceramide synthases may be a key regulatory point in the control of the distribution and levels of sphingolipids of various acyl-chain lengths.Sphingolipids, one of the major lipid constituents of eukaryotic membranes, mediate a number of cellular and physiological functions such as cell growth, apoptosis, immune responses, and the epidermal permeability barrier, whereas their abnormal metabolism is involved in diseases such as sphingolipidoses, neural disorders, and diabetes (1-7). Ceramides are located in the hub of sphingolipid metabolism. Ceramides are precursors for complex sphingolipids, whereas their hydrolysis releases a sphingoid long-chain base and a fatty acid (FA).2 The released long-chain base can then be recycled to synthesize new ceramide, or metabolized further into other lipids such as sphingosine 1-phosphate (8 -10). Ceramide synthases catalyze the formation of an amide bond between the long-chain base and the FA in the endoplasmic reticulum. In most tissues, the chain lengths of the FA moieties of ceramides are C16 -

Probing de novo sphingolipid metabolism in mammalian cells utilizing mass spectrometry

“…In vitro Vmax values established in the literature consistently range in the pmole/min/mg for CerS activity [44,45].…”

Ceramides and mitochondrial fatty acid oxidation in obesity

“…Compound ST1072 can inhibit CerS4 and CerS6 (105), but there are no data yet on in vivo effects under an HFD challenge. The new data on regulation of CerS activity by phosphorylation or deacetylation (106, 107) open up novel therapeutic options to control C16:0 ceramide production and its negative effects on health.…”

Biosynthesis of the anti‐lipid‐microdomain sphingoid base 4,14‐sphingadiene by the ceramide desaturase FADS3

“…Six ceramide synthases (CERS1-6) exist in mammals, and each exhibits characteristic substrate specificity toward acyl-CoAs with different chain lengths (CERS1, C18; CERS2, C22-C24; CERS3, ≥C18; CERS4, C18-C22; CERS5 and CERS6, C16). 5,32,41,42 While the substrate specificities of CERS1-6 toward acyl-CoAs are known, those toward sphingoid bases remained unclear. To reveal this, we performed an in vitro ceramide synthase assay toward SPH, DHS, PHS, and SPD, using membrane fractions prepared from HEK 293T cells overexpressing each of the CERS enzymes.…”

“…Although CERS3 exhibits activity toward acyl-CoAs with ≥C18, its preferred substrates are ≥C26. [42][43][44] However, such ≥C26 substrates are unsuitable for in vitro assay, due to low solubility in the reaction buffer. We, therefore, used a C18:0 substrate to measure the CERS3 activity.…”