Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-32P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD- C 6 -ceramide) as substrate. Its ？？ ？？ value (4？μM) was comparable to that of N-hexanoyl-sphingenine ( C 6 -ceramide). The produced fluorescent NBD- C 6 -ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126; I C 5 0 4？μM) and ceramide analogues (e.g., fenretinide, AMG-9810; I C 5 0 1.1？μM) affect CERK in vitro. 1. Introduction Ceramide, the initial product of the sphingomyelin cycle, functions as a key component in the regulation of various cellular functions like differentiation, proliferation, apoptosis, and inflammation [1, 2]. In the past years, ceramide-1-phosphate (Cer-1-P), a metabolite of ceramide and sphingomyelin, also gained more attention and turned out to be a powerful bioactive sphingolipid. Cer-1-P has been implicated as a regulator of different cellular processes, like mitosis, apoptosis, phagocytosis, and inflammation [3, 4]. In mice lacking CERK, neutrophil homeostasis is defective leading to more severe disease upon pulmonary infection [5]. Until now, CERK is the only mammalian enzyme known to phosphorylate ceramide, but the residual Cer-1-P levels in tissues of the CERK-deficient mice [5], indicate the existence of alternative pathways to generate Cer-1-P. CERK was first described in 1989 as a Ca2+-dependent lipid kinase [6] and cloned in 2002 by Sugiura et al. [7], based on similarity to sphingosine kinase. A similar cloning strategy was followed by others [8]. CERK appears to associate with (endo)membranes via a pleckstrin domain [9, 10] and is highly selective for the D-erythro configuration of ceramides [8, 11]. CERK activity is commonly determined using a radioactivity-based assay [7–11] based on [γ-32P]ATP. After extraction of lipids, the amount of radiolabeled ceramide-1-phosphate in the organic
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