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Originally published In Press as doi:10.1074/jbc.M405932200 on July 13, 2004

J. Biol. Chem., Vol. 279, Issue 37, 38228-38235, September 10, 2004
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MuLK, a Eukaryotic Multi-substrate Lipid Kinase*

David W. Waggoner, Laura Beth Johnson{ddagger}, Philip C. Mann, Valerie Morris, John Guastella, and Sandra M. Bajjalieh§

From the Department of Pharmacology, University of Washington, Seattle, Washington 98195

Received for publication, May 27, 2004 , and in revised form, July 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We report the identification and characterization of a novel lipid kinase that phosphorylates multiple substrates. This enzyme, which we term MuLK for multi-substrate 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 Vmax 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha} (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 PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—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 (BE889885 [GenBank] ) and mouse (AW321722 [GenBank] ) MuLK and mouse CERK (6400872) were obtained from ResGen (Invitrogen). The normalized human cDNA library used for expression analysis and the pIRES-EGFP vector were purchased from BD Biosciences (Clontech). Ceramide (bovine brain), 1,2-dioleoyl-sn-glycerol, sn-1-monooleoylglycerol, tetraoleoylcardiolipin, and sphingosine were obtained from Avanti Polar Lipids. 1-Arachidonylglycerol (1-AG) and 2-arachidonylglycerol (2-AG) were purchased from Cayman Chemical Company. Expressed sequence tag clones of human MuLK (BE889885 [GenBank] ), mouse MuLK (AW321722 [GenBank] ), and CERK (6400872) were obtained from ResGen (Invitrogen). LipofectAMINE was from Invitrogen. The monoclonal anti-GFP antibody (B-2) was obtained from Santa Cruz Biotechnologies, and horseradish peroxidase-conjugated goat anti-mouse secondary antibody was from Zymed Laboratories Inc.. The chemiluminescent detection reagent was DuraWest from Pierce. Fluoromount G and 16% paraformaldehyde were from Electron Microscopy Sciences. [{gamma}-32P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Triton X-100, {beta}-octylglucoside, aprotinin, pepstatin, leupeptin, and Complete-Mini protease inhibitors (without EGTA) were obtained from Roche Applied Science. Recombinant E. coli DG kinase, Hoechst stain, and other reagents were from Sigma.

Genome Search for Potential Ceramide Kinases—The human sphingosine kinase, sphingosine kinase 2, was used as a query sequence in a TBLASTN search of the human genome data base. Ten hits with expect values of 7 x 10–8 or less were obtained. As anticipated, the highest scoring hits represented the known mammalian sphingosine kinase genes as well as related DG kinase genes. However, the search also revealed a gene with a region that is 44% homologous to human sphingosine kinase 2 (hSPK2). The region of highest homology corresponded to a potential kinase catalytic domain that is present in both sphingosine and DG kinases. This highest scoring sequence was used in a BLASTP search of the non-redundant protein data base, which revealed three unpublished entries. One entry was identified as a putative lipid kinase but did not describe any functional data. The full-length protein sequence of the putative lipid kinase was then used to re-query the human genome data base, and three homologous sequences were identified.

Sequence Comparisons and Domain Predictions—The sequences of MuLK, CERK, sphingosine, and DG kinases were aligned using ClustalW with the BLOSUM protein weight matrix. A phylogenetic tree was constructed, and genetic distances were calculated (percent divergence) using the neighbor-joining method of Saitou and Nei (12). The sequences included were Homo sapiens MuLK (CAB93536 [GenBank] , Mus musculus MuLK (CAC06108 [GenBank] , H. sapiens sphingosine kinase 1 (Q9NYA1), M. musculus sphingosine kinase 1 (AAH37710 [GenBank] , H. sapiens sphingosine kinase 2 (Q9NRAO), M. musculus sphingosine kinase 2 (Q9JIA7), H. sapiens ceramide kinase (BAC01154 [GenBank] , M. musculus ceramide kinase (NP_663450 [GenBank] ), H. sapiens DG kinases {alpha} (AAH223523), {delta} (Q16760 [GenBank] ), (P52429 [GenBank] ), {theta} (NP_001338 [GenBank] ), and {xi} (Q13574 [GenBank] ), and E. coli DG Kinase (P00556 [GenBank] ). Gene products of putative MuLKs from Xenopus laevis (AAH43761 [GenBank] and Danio rerio (AAH45347 [GenBank] were identified using a BLASTP search and compared with human and mouse MuLK sequences using ClustalW. ClustalW identifies conservative changes by dividing amino acid residues into five groups as follows: (i) small and hydrophobic; (ii) acidic; (iii) basic; (iv) hydroxyl amine and basic; and (v) others (www.ebi.ac.uk.clustalw/). Phosphorylation consensus sites and protein motifs across species were predicted using SwissProt-Prosite.

MuLK-EGFP Expression and Subcellular Fractionation—A carboxyl-terminal fusion of enhanced green fluorescent protein (EGFP) to MuLK (MuLK-EGFP) was generated by ligating an EGFP PCR product into a plasmid already containing MuLK. To create the carboxyl-terminal fusion of EGFP to CERK, CERK was amplified using PCR from IMAGE clone 6400872 (Invitrogen) and inserted into the pMuLK-EGFP construct, replacing MuLK. To evaluate cellular localization, a 60-cm plate of 80% confluent human embryonic kidney 293T cells was transfected with 1.6 µg of DNA of each construct (pIRES-EGFP, pMuLK-EGFP, and pCERK-EGFP) using LipofectAMINE 2000. A transfection efficiency of 60–70% was determined by fluorescent microscopy. After 48 h, cells were washed with ice-cold phosphate-buffered saline and scraped into phosphate-buffered saline containing 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 0.2 mM phenylmethylsulfonyl fluoride and then lysed by sonication. Intact cells and nuclei were removed by centrifuging at 2000 x g for 5 min. Soluble and insoluble fractions of the cleared lysate were then generated by ultracentrifugation at 100,000 x g for 1 h at 4 °C. Equal volumes of these samples were separated on a 12.5% Tris-glycine gel under reducing and denaturing conditions and then transferred to polyvinylidene difluoride. Blots were probed with monoclonal anti-GFP (B-2) antibody followed by horseradish peroxidase-conjugated goat anti-mouse secondary antibody and detected using the Pierce DuraWest enhanced chemiluminescence reagent. To visualize MuLK-EGFP expression in situ, transfected cells were fixed 48 h after transfection (4% paraformaldehyde for 20 min), washed three times in phosphate-buffered saline and 0.1 M glycine, incubated with Hoechst dye to label nuclei, and attached to coverslips using Fluoromount G as a mounting medium. EGFP fluorescence was visualized using an Applied Precision Deltavision microscope, and images were captured using Softworx software.

mRNA Expression Analysis—To determine the expression levels of MuLK in various human tissues, a 528-base pair product encompassing the 3'-end of the coding sequence and 119 base pairs of the 3'-untranslated region of human MuLK was amplified from a BD Biosciences normalized cDNA panel. Reactions were conducted according to the manufacturer's directions. Two 29-base pair primers, 5'-GGCACAACCACAGGATGCCCTTTCCCAAG-3' and 5'-TGCCATGAAAATGCCCTGGGGACCCTCTG-3', were used. MuLK reactions were incubated at 94 °C for 30s followed by 28 cycles of 94 °C for 5 s and 68 °C for 2 min. As a control for template loading, a portion of glyceraldehyde 3-phosphate dehydrogenase was also amplified using manufacturer-supplied primers and cycle parameters. The relative amounts of the glyceraldehyde 3-phosphate dehydrogenase product generated from each tissue were similar to the levels reported by the manufacturer. PCR products were separated in a 2% agarose gel and stained with ethidium bromide. Product band net intensity was quantified on a Kodak Image Station 440CF.

Expression of Carboxyl-terminal FLAG-tagged MuLK—A carboxylterminal FLAG-epitope-tagged mouse MuLK (MuLK-FLAG) was created by inserting MuLK into pFLAG-CTC. This construct was expressed in BL21, a protease-deficient strain of E. coli. To generate protein, cultures of transformed bacteria were grown under selection overnight, diluted 10-fold, and then grown to an A600 of 2, at which point protein expression was induced by adding 100 µM isopropyl-1-thio-{beta}-D-galactopyranoside for 4 h. After induction, bacteria were harvested by centrifugation, resuspended in ice-cold 10 mM MOPS, pH 7.2, containing 25 mM NaCl, 1 mM EGTA, and protease inhibitors (1 pellet per 10 ml of buffer) and probe-sonicated. The lysate was centrifuged at 20,000 x g for 20 min, and the resulting supernatant was incubated for 1–14 h with anti-FLAG IgG-coated agarose resin at 4 °C with continual turning. MuLK was eluted with 3–5 column volumes of MOPS buffer (see above) containing 0.1 mg/ml 3X-FLAG. This produced a preparation enriched in MuLK. An unrelated pFLAG-CTC fusion protein, which served as a negative control, was generated and purified using an identical procedure. Glycerol or ethylene glycol and dithiothreitol were added to the eluate to a final concentration of 20% (v/v) and 1 mM, respectively. For kinetic assays, the recombinant protein was stored at 4 °C and used within a week. Expression and purification of a 47-kDa protein was confirmed by Western analysis using the anti-FLAG M2 antibody. Expression levels of MuLK in E. coli varied between preparations, and protein yields were generally low.

Lipid Kinase Activity Assay—The activity of purified, recombinant mouse MuLK was assayed in {beta}-octylglucoside as described previously (13) or as follows. In a final volume of 100 µl, purified recombinant MuLK was combined with lipid-detergent mixed micelles containing the indicated substrate, detergent, and cardiolipin at one-tenth the concentration of the lipid substrate (unless stated otherwise). This mixture was buffered with 10 mM MOPS (pH 7.2) containing 100 mM NaCl, 1 mM EGTA, and 3 mM total CaCl2 (2 mM free calcium, unless indicated). The reaction was initiated by the addition of ATP/MgCl2 (1 mM [{gamma}-32P]ATP (5 µCi) and 5 mM MgCl2 in the final assay). The assay was incubated with constant agitation at 37 °C for 20 min. The reaction was stopped by the addition of 1 ml of chloroform/methanol (1:1). After brief vortexing, 350 µl of 2 M KCl and 2 mM H3PO4 was added to generate two phases. The two phases were separated after vigorous mixing, the lower (organic) phase was removed to a second tube, the volume was reduced under a stream of nitrogen gas, and the entire sample was analyzed by thin layer chromatography. Thin layer chromatography plates were developed for 80 min in chloroform/methanol/acetic acid (65:15:5). Phosphorylated reaction products were identified by co-migration with standards generated using E. coli DG kinase and quantified by scraping and scintillation counting or by using STORM image analysis (Molecular Dynamics) and Imagequant software. An unrelated purified pFLAG-CTC fusion protein containing the first 163 amino acids of synaptic vesicle protein 2A (SV2AN) was assayed as a control for contamination by endogenous E. coli lipid kinases. Under these conditions, all reactions were linear with time, proportional to the added enzyme, and substrate conversion was <2%. Calcium concentrations were calculated using the MaxChelator Program (www.stanford.edu.~cpatton/webmaxcS.htm). Kinetic parameters were calculated using Prism Graph Pad software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Two Novel Lipid Kinases by Homology Search—Based on the hypothesis that ceramide kinases would share structural and functional domains with sphingosine kinase, we performed a data base search using one of the human sphingosine kinases, sphingosine kinase 2, as a query sequence as described under "Experimental Procedures." This search revealed a gene that contained both a region that is 44% homologous to SPK2 and a potential lipid kinase catalytic domain that is present in sphingosine and DG kinases (6). The predicted full-length protein encoded by this gene was obtained from a BLASTP search. As described below, we have named this protein MuLK. The sequence of MuLK was then used to re-query the human genome data base. Three additional gene sequences were identified. All three are distantly related to sphingosine and DG kinases, with sequence homologies of 10–22%. One of these was recently described by Sugiura et al. as a ceramide-specific kinase that they have termed CERK (7). The other two share 68–87% amino acid sequence identity with MuLK. These three proteins therefore appeared to constitute a gene family. A BLASTP search using human MuLK as a query sequence revealed a mouse homolog as well as putative homologs in multiple organisms.

The Sequence of MuLK—Fig. 1 shows the sequence of mouse MuLK. The most notable feature of the protein is a DG kinase domain near the amino terminus. MuLK also contains a putative nuclear localization sequence that overlaps the amino-terminal portion of the catalytic domain. The nuclear localization domain is found in all MuLK orthologs except Caenorhabditis elegans. It is also found in type IV and type V DG kinases ({xi}, {iota}, and {theta}) (15) and has been shown to mediate the nuclear localization of DG kinase {xi} (16). This nuclear localization sequence occurs with high frequency in the protein data base, however, so it remains to be determined whether it regulates the subcellular localization of MuLK.



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  		FIG. 1.
The sequence of MuLK. The predicted amino acid sequence of mouse MuLK is shown with the DG kinase catalytic domain indicated by a black line and a putative nuclear localization sequence indicated by a gray line. ClustalW was used to determine the similarity among representative MuLK orthologs, including mouse (M. musculus, CAC06108 [GenBank] , human (H. sapiens; CAB93536), frog (X. laevis; AAH43761 [GenBank] /I>), and zebrafish (D. rerio; AAH45347). Stars indicate identical amino acids across the four species, and double dots indicate conservative changes as determined by ClustalW and described under "Experimental Procedures." Additional orthologs were also found in D. melanogaster (AAF52895 [GenBank] , C. elegans (T16422 [GenBank] ), A. thaliana (AAM91597 [GenBank] , and O. sativa (BAC65388 [GenBank] .

 
A comparison of putative MuLKs across selected vertebrate species revealed that the amino acid sequence identity is well conserved among vertebrate MuLK proteins, especially within the amino-terminal region that includes the DG kinase catalytic domain (Fig. 1). Additional MuLK orthologs were identified in Drosophila melanogaster (AAF52895 [GenBank] and C. elegans (T16422 [GenBank] ), Arabidopsis thaliana (AAM91597 [GenBank] , and Oryza sativa (BAC65388 [GenBank] , indicating that MuLK-like proteins are expressed in most eukaryotes. There was no MuLK-related protein sequence in S. cerevisiae however, suggesting that, like DG and ceramide kinases, MuLK is expressed only in higher eukaryotes.

The human MuLK gene is located on chromosome 7q34 and contains 15 exons. The mouse gene is located on chromosome 6B1 and also contains 15 exons. The other two human MuLK genes found in our original query are on the X chromosome (Xq26) and the Y chromosome (4q11.2). These genes lack many of the introns present in the chromosome 7 MuLK gene and are riddled with nonsense, missense, and frameshift mutations, suggesting that they are non-functional retroposons. Similarly, the mouse genome has an intronless MuLK sequence on chromosome 13C3 that has two frameshift mutations.

Several protein kinase substrate consensus sites in MuLK were identified using SwissProt-Prosite. Mouse MuLK contains nine casein kinase II sites, seven protein kinase C sites, and two protein kinase A sites. These sites are conserved between human and mouse but are not present in MuLK orthologs from zebrafish, fruit fly, nematode, Arabidopsis, and rice. It is not known if any are bona fide phosphorylation sites, although the regulation of lipid kinases by protein kinase C isoforms reported for DG kinase types I (17), IV (16), and V (15) and for sphingosine kinase I (18) suggests that MuLK may be also regulated by phosphorylation.

To determine whether MuLK is a member of a previously identified lipid kinase family, we compared MuLK sequences to those of other lipid kinases. Fig. 2 shows a phylogram comparison of MuLK to DG, ceramide, and sphingosine kinases. Bootstrap analysis of the aligned sequences revealed that human and mouse MuLK segregated to a unique branch. This indicates that MuLK is a distinct lipid kinase that is not a member of a previously described family.



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  		FIG. 2.
Phylogram analysis reveals MuLK to be a distinct lipid kinase. The BLOSUM weight matrix of ClustalW (origin 2) was used to align H. sapiens MuLK (CAB93536 [GenBank] , M. musculus MuLK (CAC06108 [GenBank] , H. sapiens sphingosine kinase 1 (Q9NYA1), M. musculus sphingosine kinase 1 (AAH37710 [GenBank] , H. sapiens sphingosine kinase 2 (Q9NRAO), M. musculus sphingosine kinase 2 (Q9JIA7), H. sapiens ceramide kinase (BAC01154 [GenBank] , M. musculus ceramide kinase (NP_ 663450), H. sapiens DG kinases {alpha} (AAH223523), {delta} (Q16760 [GenBank] ), (P52429 [GenBank] ), {theta} (NP_001338 [GenBank] ), and {xi} (Q13574 [GenBank] ), and E. coli DG Kinase (P00556 [GenBank] ). The neighbor-joining algorithm was used to evaluate genetic distances (12) and construct the dendrogram shown. Bootstrap analysis by PAUP confirmed that MuLK segregated from other lipid kinases to its own branch in 96 of 100 iterations.

 
MuLK Is a Ubiquitously Expressed, Membrane-associated Protein—Analysis of the amino acid sequence of MuLK predicts no signal sequence, transmembrane domains, or lipid modifications. This suggests that MuLK is a cytosolic, soluble protein. To test this possibility, we expressed MuLK fused to EGFP in human embryonic kidney 293T cells and compared the proportion of MuLK in the cytosolic and membrane fractions as described under "Experimental Procedures." As a control for the effects of EGFP on protein localization, we also analyzed the localization of mouse CERK-EGFP, because untagged CERK was shown previously to be largely membrane-associated (7). Western analyses of cytosolic and membrane fractions revealed that the majority of MuLK was found in the particulate fraction (100,000 x g pellet), a distribution similar to that of CERK. EGFP, expressed alone, was largely cytosolic (Fig. 3A). When expressed in Chinese hamster ovary cells, a fibroblast cell line, MuLK-EGFP localized to an internal membrane compartment (Fig. 3B). This result indicates that in unstimulated fibroblasts the enzyme is associated with endomembranes rather than being cytoplasmic or nuclear as suggested by its sequence.



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  		FIG. 3.
MuLK is a membrane-associated protein. A, MuLK cofractionates with membranes. Shown is an immunoblot probed with an anti-GFP antibody demonstrating the presence of an EGFP-immunoreactive protein in nuclei-free cell homogenates (H), cytosol (C), and membrane (M) fractions from cells expressing either EGFP alone, MuLK-EGFP, or CERK-EGFP. Both MuLK-EGFP and CERK-EGFP partitioned primarily to the membrane fraction, suggesting that both are membrane-associated. B, MuLK localizes to an internal membrane compartment. Shown are fluorescent images of Chinese hamster ovary cells expressing either EGFP or EGFP fused to the carboxyl terminus of MuLK (MuLK-EGFP). MuLK-EGFP localized to an internal membrane compartment.

 
To determine the tissue distribution of MuLK, we surveyed RNA expression levels by the amplification of normalized cDNA pools from various tissues as described under "Experimental Procedures." Fig. 4 shows that MuLK was expressed in all tissues surveyed. Expression was highest in neuroendocrine organs such as pancreas and brain and relatively lowest in skeletal muscle, kidney, and lung.



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  		FIG. 4.
MuLK is broadly expressed. Top, PCR amplification products of a 528-bp 3'-fragment of the human MuLK cDNA amplified from a normalized panel of human cDNAs. Lanes correspond to the tissue indicated in the graph below. MuLK was expressed in all tissues surveyed. Bottom, quantification of PCR products reveals that MuLK expression was highest in the neuroendocrine tissues pancreas and brain. Net band intensities from three experiments were averaged. Error bars represent the S.D.

 
MuLK Is a Multi-substrate Lipid Kinase—To determine whether MuLK is a bona fide lipid kinase and test its substrate specificity, we analyzed the ability of recombinant MuLK to phosphorylate various lipids in vitro using an assay in which substrates were presented in detergent-lipid mixed micelles. Purified recombinant MuLK-FLAG phosphorylated 1,2-dioleoyl-sn-glycerol as well as monoacylglycerol. It also demonstrated significant activity toward ceramide but not sphingosine (Fig. 5A). This result indicates that MuLK can phosphorylate a number of neutral lipid species but does exhibit some substrate selectivity. We have indirect evidence that MuLK does not phosphorylate soluble polyols, because up to 20% (v/v) glycerol or ethylene glycol had no effect on its ceramide kinase activity (not shown). MuLK phosphorylated sn-1-oleoylglycerol and 1-AG to a similar extent. It showed little activity toward 2-AG when the assay was conducted using Triton X-100 mixed micelles (Fig. 4A). However, when kinase selectivity was assessed using {beta}-octylglucoside mixed micelles, a greater relative activity toward 2-AG was detected (not shown). Despite these observations, we cannot be certain that 2-AG is a bona fide substrate, because preparations of 2-AG readily isomerize to 1-AG (19).



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  		FIG. 5.
MuLK phosphorylates ceramide, diacylglycerol, 1-AG and 2AG, but not sphingosine. A, MuLK activity toward the indicated substrate (DOG, dioleoylglycerol; MOG, monooleoylglycerol; 1-AG; 2-AG; Cer, ceramide; and Sph, sphingosine); 0.5 mM (clear bars) or 5 mM (hatched bars) was assayed in Triton X-100 micelles as described under "Experimental Procedures." Data shown are the average of duplicates plus/minus the range and are representative of two experiments. MuLK phosphorylated all substrates tested with the exception of sphingosine. B, MuLK activity toward 50 µM DG (dioleoylglycerol) in Triton X-100 micelles was evaluated in the presence or absence of 2 mM of the indicated additional lipid. Data shown are the average of duplicates ± the range and are representative of two experiments. DGK, DG kinase.

 
To confirm that the activity measured was not due to contaminating E. coli DG kinase, an unrelated recombinant protein, SV2AN, purified in the same way as recombinant MuLK-FLAG, was assayed for lipid kinase activity. It exhibited minimal activity, indicating that MuLK was responsible for the majority of the kinase activity measured (not shown).

MuLK DG kinase activity was inhibited 86–94% by the presence of 40-fold higher concentrations of alternate substrates (Fig. 5B). Assuming only one active site on MuLK, together with the finding that MuLK phosphorylates ceramide, monoacylglycerol, and diacylglycerol, this inhibition suggests that the substrates of MuLK act as competitive inhibitors of one another. Interestingly, sphingosine also inhibited DG kinase activity (Fig. 5B), even though it was not a substrate for the enzyme. Both ceramide and DG kinase activities were inhibited by sphingosine in a dose-dependent manner (not shown).

MuLK Demonstrates Similar Affinities for Ceramide and DG but a Higher Vmax toward DG—To analyze further the substrate specificity of MuLK, we measured the apparent Km and Vmax toward ceramide and DG in two different detergent-based assays. Using the {beta}-octylglucoside-based assay used previously to assay ceramide kinase activity (13), we found a trend toward a slightly lower Km for ceramide and a consistently higher Vmax for DG (not shown). In a Triton X-100-based assay, the apparent Km for ceramide (34 µM) was again slightly lower but similar to that for DG (45 µM), suggesting that the enzyme binds both lipids with roughly equal affinity. Similar Km values were determined for dipalmitoylglycerol in TX-100 (not shown), suggesting that acyl chain length does not have significant effects on substrate affinity. In this assay the Vmax for dioleoylglycerol (159 nmol/min/mg) was 4–5 times the rate measured for ceramide (37 nmol/min/mg) (Fig. 6A).



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  		FIG. 6.
MuLK demonstrates similar affinity for ceramide and DG and a higher Vmax toward DG. A, MuLK activity toward various concentrations of DG (dioleoylglycerol) (circles) or long chain ceramides (squares) in TX-100 micelles was determined as described under "Experimental Procedures." Data shown are the average of duplicates plus/minus the range and are representative of three experiments. Kinetic parameters were generated using Prism Graph Pad. B and C, surface dilution analysis of MuLK activity toward 0.8 mM long chain ceramides (B) or 2 mM dioleoylglycerol (C) was determined in Triton X-100 micelles containing cardiolipin at one-tenth the total concentration of substrate, as described under "Experimental Procedures." Kinetic parameters were generated using Prism Graph Pad. Data shown are the average of duplicates plus/minus the range and are representative of two experiments. DGK, DG kinase.

 
We also performed assays in which we varied the mol % of the substrate in Triton X-100 micelles. Data derived in this manner can be compared with a surface dilution kinetic model (20). We found that ceramide kinase activity conformed to such a model, yielding a Km of 3.78 mol % and a Vmax of 68 nmol/min/mg protein (Fig. 6B). By contrast, the Km for dioleoylglycerol was independent of the surface concentrations tested, suggesting that it was too low to measure (Fig. 6C). These results were not an aberration of the assay system, because surface dilution kinetic parameters for a DG kinase activity from T cells has been determined using similar conditions.2 These results highlight the sensitivity of MuLK to substrate presentation and suggest that the affinities and kinetics measured in vitro using recombinant enzyme may differ from those of the enzyme in vivo.

MuLK Activity Is Modulated by Surface Charge and the Ionic Environment—Kinases require a counter ion for ATP, typically Mg2. In some cases kinase activity is also modulated by those same ions directly. DG kinases from E. coli (21) and pig brain (22) are both stimulated by magnesium in excess of ATP concentrations. We examined the Mg2+ requirement for the DG and ceramide kinase activities of MuLK and found that it stimulated MuLK kinase activity maximally when included at a 5–10 molar excess over ATP (not shown).

All reported endogenous ceramide kinase activities, as well as the recently identified ceramide-specific kinase, CERK, are stimulated by Ca2+ (7, 11, 23, 24). Type I DG kinases are also activated by Ca2+ (6). To determine whether MuLK is regulated by Ca2+, we compared MuLK activity in the presence of 1 mM EGTA (which buffers Ca2+at ~10–9 M) to its activity in the presence of 1 mM free Ca2+. We found that the effects of Ca2+ varied with the concentrations of Mg2+ in the assay (Fig. 7). At low Mg2+/ATP ratios (0.5:1), Ca2+ stimulated the phosphorylation of both DG and ceramide 2–9-fold. By contrast, when Mg2+ was in excess (5:1 Mg/ATP), Ca2+ inhibited kinase activity 2–5-fold.



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  		FIG. 7.
MuLK activity is modulated by cations and anionic lipids. Shown is MuLK activity assayed in varying concentrations of cardiolipin, Ca2+, and Mg2+. MuLK DG and ceramide kinase activities were evaluated in solutions containing 1 mM (2 mol %) dioleoylglycerol (A and B) or 1 mM (2 mol %) long chain ceramides (C and D) and either 0.1 or 1 mM cardiolipin in the presence or absence of 1 mM free Ca2+. The assays were initiated with the addition of 1 mM ATP and either 0.5 mM (A and C) or 5 mM (B and D) Mg2+. Data shown are the average of duplicate determinations plus/minus the range and are representative of two experiments.

 
Cardiolipin has been reported to be an activator of E. coli DG kinase (9), and for that reason it was included in the assay. We found that MuLK required the presence of cardiolipin in detergent micelles for ceramide kinase activity (not shown). Moreover, the stimulatory effect of cardiolipin was concentration-dependent for both ceramide and DG kinase activity and was most dramatic at low Ca2+ concentrations (Fig. 7). Together these findings indicate that MuLK activity is influenced by the ionic environment and by the charge of the lipid surface.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have described a novel lipid kinase that differs from known lipid kinases not only in sequence but also by demonstrating a broad substrate preference. Some DG kinases also phosphorylate monoacylglycerols (25), but little to no detectible ceramide kinase activity has been reported for any of the isoforms (26, 27).3 Analogously, the ceramide kinase CERK displays no activity toward DG (7). The only other lipid kinase with the broad substrate specificity of MuLK is E. coli DG kinase (9) (for review, see Ref. 28). Therefore, the broad substrate selectivity of MuLK appears to be unique among eukaryotic lipid kinases. MuLK orthologs were found in many eukaryotes ranging from plants to human, and it is expressed in all of the human tissues surveyed. This finding suggests that MuLK provides an important function that is required by all cells. We found no MuLK homologs in yeast, however, consistent with its function being subsumed by another enzyme in lower eukaryotes.

MuLK Activity Demonstrates Complex Regulation—The effects of lipids, detergents, and ions on MuLK activity suggest that its action in vivo is likely to be under complex regulation, a feature MuLK shares with other lipid kinases. The requirement for, and stimulatory effect of, cardiolipin parallels the effects of anionic lipids on E. coli DG kinase (21, 29) and eukaryotic DG kinases {alpha} (30, 31) and {xi} (32) and contrasts with the inhibitory effects on other isoforms (10, 27, 32, 33). The cationic lipid sphingosine, which exhibits both stimulatory and inhibitory effects on different DG kinase isoforms (34–37), potently inhibits MuLK activity. We originally hypothesized that sphingosine acts as a competitive inhibitor of MuLK; however, inhibition was not stereospecific and was sensitive to the concentration of cardiolipin in the assay (not shown), suggesting that the inhibitory action of sphingosine is more complex and may include charge neutralization at the lipid (substrate) surface.

We found that Ca2+ could either stimulate or inhibit MuLK activity, depending on the concentrations of Mg2+ and cardiolipin in the reaction. Similar reciprocal magnesium-dependent effects have been reported for phosphatidylinositol 3-kinase (37), and the effects of calcium on DG kinase {alpha} vary with reaction conditions and are phosphatidylserine-dependent (33). The inhibitory effect of Ca2+ could reflect surface charge neutralization of cardiolipin, which, in turn, decreases enzyme-membrane association. Consistent with the conditional effects of Ca2+, MuLK lacks a consensus calcium-binding domain like the EF-hand domains present in type I DG kinases. On the other hand, an alignment of MuLK with DG kinases {alpha}, , and {xi} (the latter two lack EF-hand domains and are regulated by Ca2+ in a complex fashion) (32) reveals that all four proteins contain two conserved aspartates in their carboxyl-terminal domains that are important to the stimulatory effect of Ca2+ on DG kinase {alpha} (30). Therefore, MuLK may share part of a novel motif that mediates the effects of Ca2+ on these lipid kinases.

Is MuLK a Chameleon Kinase?—The activity of MuLK in vitro was greatest toward DG, suggesting that it may act primarily as a DG kinase in vivo. On the other hand, given that detergents, lipids, and ionic conditions critically affect MuLK activity, it is likely that both substrate preference and the amount of lipid phosphate generated in vivo may vary from those measured in vitro. This feature is not unique to MuLK; for example, E. coli DG kinase expressed in mammalian cells causes large increases in ceramide 1-phosphate levels rather than phosphatidic acid (38). The more pronounced effect of cardiolipin on ceramide (versus DG) kinase activity suggests that the product(s) of MuLK could change with alterations in its lipid environment.

In addition to the phosphorylation of DG and ceramide, our results indicate that MuLK also demonstrates measurable phosphorylation of sn-1-monoacylglycerols. Given that we did not optimize the assay for that substrate, it raises the possibility that lysophosphatidic acids, which are ligands for G protein-coupled receptors (2, 3), may have a source other than phospholipases A1/A2 or lysophospholipase D (39). Furthermore, MuLK phosphorylation of 2-AG, an endogenous cannabinoid, raises the possibility that MuLK could act in that signaling pathway as well. Finally, the finding that MuLK activity is inhibited by sphingosine indicates that MuLK represents another point of cross-talk between glycerol- and sphingosinebased lipid signaling pathways (14).

Given its broad substrate selectivity and modulation by membrane components and ionic environment, MuLK could function as a "chameleon" kinase whose action depends on cellular location and conditions. Future studies of the action of MuLK in vivo will provide important clues to the role of this unique enzyme in cellular function.


   FOOTNOTES
 
* This work was supported by National Institute on Drug Abuse Grant R21 DA14954 (to S. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by National Institutes of Health Training Grant T32 GM07750. Back

§ To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington, Box 357280, Seattle, WA 98195. Tel.: 206-616-2962; Fax: 206-685-3822; E-mail: bajjalie{at}u.washington.edu.

1 The abbreviations used are: DG, diacylglycerol, 1-AG, 1-arachidonylglycerol; 2-AG, 2-arachidonylglycerol; CERK, ceramide kinase; GFP, green fluorescent protein; EGFP, enhanced GFP; MOPS, 4-morpholinepropanesulfonic acid. Back

2 D. Waggoner, unpublished observations. Back

3 H. Kanoh, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Ken Custer for generating the MuLK-FLAG construct and for help throughout the project, the Biochemistry Department at the University of Washington for use of the PhosphorImager, and Ken Custer and Dr. Joe Beavo for reviewing the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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