We report the identification and characterization of a novel lipid kinase that phosphorylates multiple substrates. This enzyme, which we term MuLK for multisubstrate lipid kinase, does not belong to a previously described lipid kinase family. MuLK has orthologs in many organisms and is broadly expressed in human tissues. Although predicted to be a soluble protein, MuLK co-fractionates with membranes and localizes to an internal membrane compartment. Recombinant MuLK phosphorylates diacylglycerol, ceramide, and 1-acylglycerol but not sphingosine. Although its affinity for diacylglycerol and ceramide are similar, MuLK exhibits a higher V max toward diacylglycerol in vitro, consistent with it acting primarily as a diacylglycerol kinase. MuLK activity was inhibited by sphingosine and enhanced by cardiolipin. It was stimulated by calcium when magnesium concentrations were low and inhibited by calcium when magnesium concentrations were high. The effects of charged lipids and cations on MuLK activity in vitro suggest that its activity in vivo is tightly regulated by cellular conditions.The membranes of eukaryotic organelles contain a shifting constellation of lipids that, in addition to their structural role, also serve as modulators of protein function, scaffolding molecules, and ligands for G protein-coupled receptors. The generation, phosphorylation, and dephosphorylation of monoacylglycerol, diacylglycerol (DG), 1 sphingosine, and ceramide produce signaling lipids that modulate a vast array of cellular processes (1-5). In animal cells, the kinases that act on DG (6), ceramide (7), and sphingosine (8) constitute three different families of enzymes, each of which demonstrates substrate selectivity. In contrast, Escherichia coli expresses a lipid kinase that phosphorylates all three substrates (9). Although animal cell lipid kinases are specific and clearly belong to distinct families based on their amino acid sequences, they share a similar catalytic domain that was first identified in DG kinase ␣ (10) and is therefore known as a DG kinase domain.Sequencing of the human and mouse genomes has revealed multiple putative lipid kinases, several of which contain a DG kinase domain. In an attempt to identify cDNAs encoding a calcium-activated ceramide kinase that co-purifies with neurotransmitter-containing (synaptic) vesicles (11), we searched sequence databases for orphan lipid kinases that might encode a ceramide kinase. We report here the characterization of a lipid kinase that phosphorylates ceramide but also demonstrates significant activity toward DG and monoacylglycerol. This enzyme, which we term MuLK for multi-substrate lipid kinase, is a novel lipid kinase. EXPERIMENTAL PROCEDURESMaterials-Restriction endonucleases were purchased from Fermentas (Hanover, MD). The cloning vector pFLAG-CTC, the anti-FLAG agarose affinity gel, the 3X-FLAG peptide, and the anti-FLAG M2 monoclonal antibody were obtained from Sigma. Primers were purchased from Sigma Genosys (The Woodlands, TX). Expressed sequence tag clones of human (...

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