Ceramide Kinase Profiling by Mass Spectrometry ... - ACS Publications

Ceramide kinase (CERK) produces ceramide-1-phosphate, a novel bioactive lipid. Using mass-spectrometry, we identified S340 as a phosphorylation site ...
0 downloads 0 Views 3MB Size
Download PDF
Ceramide Kinase Profiling by Mass Spectrometry Reveals a Conserved Phosphorylation Pattern Downstream of the Catalytic Site Wei-Qiang Chen,† Christine Graf,‡ David Zimmel,§ Philipp Rovina,| Kurt Krapfenbauer,⊥ Markus Jaritz,# Peter J. Parker,¶ Gert Lubec,† and Fre´de´ric Bornancin*,O Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, A-1090, Vienna, Austria, Novartis Institutes for BioMedical Research (NIBR), Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria, Protein Phosphorylation Laboratory, London Research Institute, Cancer Research U.K., London, WC2A 3PX, United Kingdom, and the Division of Cancer Studies King’s College London, New Hunt’s House, Guy’s Hospital, St Thomas Street, London SE1 1UL, United Kingdom Received August 27, 2009

Ceramide kinase (CERK) is essential for production of ceramide-1-phosphate (C1P), a bioactive lipid whose formation critically modulates ceramide levels. To explore how CERK is regulated, we used insect cell-expressed, recombinant hCERK and searched for post-translational modifications, using massspectrometry techniques. This led to identification of two phosphorylated serine residues, at positions 340 and 408. Point mutations preventing phosphorylation at either of these sites did not lead to detectable changes in subcellular localization or activity. However, preventing phosphorylation at S340 resulted in CERK instability as revealed by the behavior of the S340A mutant protein under various assay conditions in vitro. Phosphorylation of a cognate serine residue in sphingosine kinases was previously shown to be important. Therefore, phosphorylation within a conserved “regulation loop” downstream of the catalytic domain emerges as a new paradigm for regulation of kinases of the diacylglycerol kinase family. This “regulation loop” is reminiscent of the “activation loop” that controls AGC protein kinases, being a similar distance from the critical ATP binding site determinants in the primary sequence. Keywords: phosphorylation • ceramide • kinase • activation loop • glycine-rich loop • LC-MS

Introduction Ceramide kinase (CERK) belongs to the diacyglycerol kinase (DAGK) family of lipid kinases. It is so far the only activity known to produce ceramide-1-phosphate (C1P).1 However, studies with CERK deficient (Cerk-/-) mice have shown that another route for production of C1P must exist, at least in mammals.2,3 The latter pathway, which is still unknown, does not appear to require the related CERK-like protein (CERKL);4,5 it may rather rely on a type D-sphingomyelinase.2 In any case C1P appears to stem from different origins, part of it only being * To whom correspondence should be addressed. Present address: Novartis Pharma AG, Forum 1, CH-4056 Basle, Switzerland. Phone: +41 61 32 43136. Fax: +41 61 32 40562. E-mail: [email protected]. † Medical University of Vienna. ‡ NIBR - Present address: AFFiRiS AG, Viehmarktgasse 2A, A-1030 Vienna, Austria. § NIBR - Present address: Charite´ - University Medicine Berlin and German Rheumatism Research Center, Charite´platz 1, D-10117, Berlin, Germany. | NIBR - Present address: Roche Diagnostics GmbH, Engelhorngasse 3, A-1211 Vienna, Austria. ⊥ NIBR - Present address: EPMA headquarters, avenue des Mimosas, B-1150 Brussels, Belgium. # NIBR - Present address: Research Institute of Molecular Pathology, A-1030 Vienna, Austria. ¶ Cancer Research UK and King’s College, U.K. O NIBR - Present address: Novartis Pharma AG, Forum 1, Novartis Campus, CH-4056 Basle

420 Journal of Proteome Research 2010, 9, 420–429 Published on Web 11/09/2009

produced by CERK. Thus, it may not be surprising that signaling properties discovered for C1P, such as the effect on cell proliferation and cell survival6 or the control of cytosolic phospholipase A2 activity,7 could be recapitulated neither by knocking down the CERK gene3 nor by using a CERK inhibitor.8 In a recent study we have shown that C1P produced by CERK is rapidly metabolized,2 thus placing CERK at center stage for the regulation of ceramide levels. In fact, CERK-mediated phosphorylation of ceramide appears to play a dual role, enabling the production of a signaling lipid (C1P) at the expense of another signaling lipid with opposite function (ceramide itself). The latter feature in particular may be uniquely associated to CERK in case the alternative C1P producing activities do not proceed via phosphorylation of ceramide. In this context, it has become increasingly important to clarify the function of CERK-mediated production of C1P and to understand how it is regulated. In their original study on the identification of CERK, Sugiura et al. noticed an increased expression of CERK upon treatment with phorbol esters, leading them to propose a possible regulation of CERK by protein kinase C (PKC).1 Subsequently, Chalfant and co-workers reported on the stimulation of CERK activity upon treatment of A549 carcinoma cells with interleukin-1 beta, or with the calcium ionophore A23187;7 consistently, we observed a 20% increase of recombinant CERK activity upon 10.1021/pr900763z

 2010 American Chemical Society

Ceramide Kinase Profiling by Mass Spectrometry IL-1 treatment of COS-1 cells (F. Bornancin, unpublished). More recently, Mitra et al. reported that CERK is permissive for activation of extracellular-regulated kinases (ERK) 1/2 and Protein Kinase B by epidermal growth factor in A549 cells.9 Treatment with the calcium ionophore A23187 was also reported to increase CERK activity in RBL-2H3 cells10 or CERKoverexpressing CHO cells11 whereas the calmodulin antagonist W-7 was reported to decrease CERK activity in RBL-2H3 cells overexpressing CERK.12 In addition, recent work from our laboratory has shown that calcium and serum are potent stimulators of CERK in primary bone-marrow derived macrophages.2 Collectively these observations suggest that posttranslational modifications of CERKsphosphorylation in particularsmay take place and regulate CERK function. In addition, there is precedence showing that the related sphingosine kinases 1 and 2 are regulated by phosphorylation.13,14 In the present work, we profiled the post-translational modifications of insect-cell produced and purified human CERK, after one-dimensional gel electrophoresis and subsequent multienzyme digestion followed by MALDI-TOF/TOF and nano-LC-ESI-MS/MS analysis of visualized bands. This led to detection of two phosphorylation sites at serines 340 (S340) and 408 (S408) in hCERK. The identification, description and preliminary characterization of these phosphorylated sites as well as that of a possible additional phosphorylation site at serine 427 (S427) is presented.

Experimental Procedures CERK Expression and Purification. Recombinant full-length hCERK was obtained from baculovirus-infected Sf9 cells as a glutathione-S-transferase fusion using described methodologies; purified CERK was stored frozen at -80 °C.8 Both LC-MS analysis and coomassie-stained SDS gels showed the expected molecular weight and a purity of about 95%. Confirmation studies were performed using another source of GST-CERK, purchased from Abcam. In-Gel Digestion. Protein bands were manually excised and placed into 0.5 mL low-bind Eppendorf tubes. In-gel digestion and sample preparation for mass spectrometric analysis was performed as described before.15 Gel plugs were washed several times with 10 mM ammonium bicarbonate and with 50% acetonitrile in 10 mM ammonium bicarbonate. After addition of 100% acetonitrile to shrink the gel, gel plugs were dried in a Speedvac Concentrator 5301 (Eppendorf). The dried gel pieces were reswollen in digestion buffer (5 mM β-octylglucoside (OG) in 10 mM ammonium bicarbonate) containing 40 ng/µL trypsin (sequencing grade; Promega) and incubated for 1.5 h at 30 °C. Chymotrypsin digestion were performed by addition of 25 mM ammonium bicarbonate containing 25 ng/µL chymotrypsin (sequencing grade; Roche diagnostic) and incubated for 1.5 h at 30 °C. Proteinase K digests were performed by addition of 5 mM ammonium bicarbonate containing 50 ng/µL of proteinase K (Sigma) and incubated for 1 h at 37 °C. Peptide extraction was performed with 20 µL of 1% formic acid in 5 mM OG for 30 min, and subsequently 20 µL 0.1% formic acid for 30 min and 20 µL 0.1% formic acid in 20% acetonitrile for 30 min. Extracted fractions were pooled for analysis using nanoLC-ESI-(CID/ETD)-MS/MS Phosphatase Treatment. Phosphatase treatmentsto check for phosphorylation eventsswas carried out as described previously.16 The excised, destained, and dried protein bands were incubated in a solution of 0.5 µL of alkaline phosphatase in the presence of 100 mM ammonium bicarbonate for 1 h at

research articles 37 °C, resulting in dephosphorylation. The protein bands were subsequently washed in 100 mM ammonium bicarbonate, shrunken in acetonitrile, dried in speed vacuum, digested using corresponding enzymes, extracted, and analyzed using nanoLC-ESI-(CID/ETD)-MS/MS. Analysis of Peptides by nano-LC-ESI-(CID/ETD)-MS/ MS. Forty microliters of extracted peptides were analyzed by high capacity ion trap (HCT). The HPLC used was an Ultimate 3000 system (Dionex Corporation) equipped with a PepMap100 C-18 trap column (300 µm × 5 mm) and PepMap100 C-18 analytic column (75 µm × 150 mm). The column was run with a flow rate of 300 nL/min using a two buffer-system (A ) 0.1% formic acid in water, B ) 0.08% formic acid in acetonitrile) according to the following procedure: 4% to 30% B from 0 to 105 min, 80% B from 105 to 110 min, and 4% B from 110 to 125 min. A HCT ultra ETDII PTM discover system (Bruker Daltonics) was used to record peptide spectra over the mass range of m/z 350-1500, and MS/MS spectra in information dependent data acquisition over the mass range of m/z 100-2800. Repeatedly, MS spectra were recorded followed by four data-dependent CID MS/MS spectra generated from four highest intensity precursor ions. On neutral loss peaks of precursor mass -32.66 and -48.99, ETD MS/MS spectra of the corresponding precursor was recorded. An active exclusion of 0.4 min after two spectra was used to detect low abundant peptides. The voltage between ion spray tip and spray shield was set to about 1500 V. Drying nitrogen gas was heated to 150 °C and the flow rate was 10 L/min. The collision energy was set automatically according to the mass and charge state of the peptides chosen for fragmentation. Multiple charged peptides were chosen for MS/MS experiments due to their good fragmentation characteristics. MS/MS spectra were interpreted and peak lists were generated by DataAnalysis 4.0 (Bruker Daltonics) and searched against Swissprot database using MASCOT 2.2.04. enzyme selected as used with three maximum missing cleavage sites, species limited to human, a mass tolerance of 0.2 Da for peptide tolerance, 0.5 Da for MS/MS tolerance, fixed modification of carbamidomethyl(C) and variable modification of methionine oxidation and phosphorylation (Tyr, Thr, and Ser). Positive protein identifications were based on a significant MOWSE scores. After protein identification, an error-tolerant search was done to detect unspecific cleavage and unassigned modifications. Protein identification and modification information returned from MASCOT were manually inspected and filtered to validate protein identification and modification. Plamid Constructs. The hCERK cDNA, corresponding to Genebank accession number AB079066, was obtained and subcloned in Gateway compatible entry vectors as described previously.17 Site directed mutagenesis was performed with the QuickChange Site Directed Mutagenesis system using primers designed according to the QuikChange Primer Design Program (Stratagene). All constructs were then transferred to pcDNA6.2DEST (Invitrogen) as C-terminally FLAG-tagged constructs or into pcDNA-DEST53 (Invitrogen) to express N-terminally fused GFP. COS Cells: Transfection, Microscopy, and Fractionation. COS-1 cells were obtained from DSMZ (ref ACC 63) and cultured in DMEM/10% FCS at 37 °C/5% CO2 in a humidified atmosphere. Cells were seeded at 105 cells/well in six-well plates. After 24 h, cells were transfected with 4 µg plasmid, using FuGENE 6 (Roche Molecular Biochemicals). Cells were incubated for 24 to 48 h following transfection. For harvest, cells Journal of Proteome Research • Vol. 9, No. 1, 2010 421

research articles were washed with ice-cold phosphate-buffered saline (PBS) and scraped into an appropriate lysis buffer (cf below). Fluorescence microscopy was performed as described previously17 on an inverted microscope (Axiovert 200 M, Zeiss) equipped with a high resolution microscopy camera (AxioCam MRc, Zeiss) as well as oil DIC objectives (Plan-Neofluar 40x/ 1.30 and Plan-Apochromat 63x/1.40). Subcellular fractionation was performed exactly as described in ref 18. Cell Labeling with 32P Orthophosphate. Ten-centimeter diameter dishes were seeded with 106 COS-1 cells, grown, and tranfected with 15 µg of pcDNA6.2-DEST plasmid encoding C-terminally FLAG tagged hCERK. Twenty-four hours after transfection, the medium was removed and cells were incubated with 7 mL of phosphate-free DMEM medium containing 10% dialyzed FCS. After 4 h the medium was discarded and replaced by 7 mL of medium of the same composition supplemented with 0.2 mCi 32P orthophosphate/ml. After an overnight incubation the medium was discarded, cells were rinsed twice with Tris Buffer Saline and used for FLAG taggedmediated immunoprecipitation according to the manufacturer’s instructions (FLAG- immunoprecipitation kit and EZview Red ANTI-FLAG M2 affinity gel # F2426 were from Sigma). To 6 mL of lysis buffer one tablet of COMPLETE Mini EDTA-free protease inhibitors (Roche Molecular Biochemicals) and 60 µL of HALT phosphatase inhibitor coktail (Pierce) were added. The same immunoprecipitation procedure was also used for purification of unlabeled C-terminally FLAG tagged hCERK. CERK Assays. For in vitro kinase assays, cells were scraped into lysis buffer (10 mM MOPS pH7.2; 2 mM EGTA, 150 mM KCl, 2% Triton X-100, 1 mM DTT, and protease inhibitors). The suspension was homogenized by 20 strokes in a PotterElvejhem homogenizer and used immediately. Kinase activity assays were performed exactly as described in ref 18, in total volumes of 100 µL composed of: 880 µM C8-Ceramide; 1 mM Cardiolipin; 1.5% β-Octylglucoside; 0.2 mM diethylenetriaminepentaacetic acid; 20 mM MOPS pH 7.2; 50 mM NaCl; 10 mM DTT; 2 mM EGTA; 3 mM CaCl2 and 500 µM [γ-32P]ATP (40-100 mCi/mmol). Assays were started by the addition of 20 µL of protein sample and vortexing, and allowed to proceed for 15 min at 30 °C. Reactions were stopped by adding 1 mL of freshly prepared CHCl3/CH3OH (1:1) and 420 µL of 1 M KCl in 20 mM MOPS pH 7.2. After vortexing 400 µL of organic phase were transferred into a new tube and 300 µL of 1 M KCl in 20 mM Mops pH 7.2 were added. Samples were vortexed again and spun down. Two-hundred microliters of the final organic phase were collected and counted directly. Cell-based CERK assays were performed as described previously.2 After 1 h incubation with 5 µM NBD-Cer, cells were rinsed with HBSS buffer supplemented with 10 mM EDTA. Lipids were then extracted with 200 µL methanol, 200 µL chloroform and 150 µL HBSS/EDTA. Samples were vortexed and spun in a table top centrifuge. The organic phase then taken and dried using a Thermo SPD Speed Vac. Dried samples (corresponding to total lipid extracts from an equivalent number of cells) were dissolved in 10 µL methanol/chloroform 1:1 and analyzed on Silica Gel 60 HPTLC plates (Merck) using butanol/acetic acid/water, 3:1:1 as the mobile phase. TLC plates were dried and imaged using Fuji film LAS3000 intelligent dark box in SYBR Green fluorescent light.

Results hCERK is a Phosphoprotein. To address whether the CERK protein is phosphorylated we produced recombinant FLAG422

Journal of Proteome Research • Vol. 9, No. 1, 2010

Chen et al.

Figure 1. hCERK is a phosphorylated protein. (A) hCERK-FLAG, immuno-precipitated following expression in COS cells labeled with 32P orthophosphate, was analyzed on SDS-PAGE followed by Western Blot using an anti-FLAG antibody (left) or followed by autoradiography (right). (B) COS-cell expressed and immunoprecipitated hCERK-FLAG as well as an E. coli expressed fusion protein made of N-terminal glutathione S-transferase fused to the Pleckstrin Homology domain of phospholipase C detlta one (PH-PLCδ1)18 were allowed to bind to a phosphorylated peptidebinding column (Qiagen PhosphoProtein Purification Kit, Cat. No. 37101). Flow-through fractions were collected (FT) as well as eluted fractions (EL) obtained according to the protocol provided by the manufacturer.

tagged human CERK in COS-1 cells labeled with 32P orthophosphate. The cell lysate was analyzed after immunoprecipitation with an anti-FLAG antibody followed by Western blotting and autoradiography. A radio-labeled band was identified that was also detected with an anti-FLAG antibody (Figure 1A). To confirm that CERK is a phosphoprotein we used an affinity purification procedure whereby phosphorylated peptides are bound to a matrix allowing for subsequent elution under competitive conditions. After expression of FLAG-tagged CERK in COS-1 cells followed by cell lysis, the whole extract was loaded onto a phospho trap column. CERK was quantitatively retained on the column and was eluted upon application of a competitor (Figure 1B). In contrast, the Pleckstrin Homology (PH) domain of phospholipase C delta (PLCδ), expressed and extracted from E. coli to serve as an unphosphorylated control protein, could not be retained on the matrix and was entirely recovered in the flow-through fraction (Figure 1B, lower panel). These experiments identify that recombinant hCERK is produced as a phosphoprotein when expressed in mammalian cells. Identification of Phosphorylated Serine Residues in Insect-Cell Expressed Recombinant hCERK. To identify the phosphorylated sites in hCERK we used recombinant, insectcell expressed, hCERK obtained and purified as described previously.8 The protein was run using one-dimensional SDSPAGE under reducing conditions. After staining and destaining, protein bands were excised and processed for protease digestion. Peptides were extracted and characterized by nano-LCESI-(CID/ETD)MS/MS as described in the Experimental section. Eighty-two percent coverage of the CERK peptidic sequence was achieved using trypsin, chymotrypsin, and proteinase K digestion, and 2 phosphorylation sites were identified, both at serine residues S340 and S408 (Table 1, Figure 2). Analysis before and after phosphatase treatment confirmed that the 80 Da increase, measured for the phosphoserine-containing peptides, can indeed be assigned to a phosphate group (Table 1). The two identified phosphorylated serine residues lie downstream of the catalytic domain, in a region that was already shown to contain important regulatory elements such as a calcium/calmodulin binding site12 and a critical cysteinyl motif.19,20 S340 is located between the CERK conserved domains 1 and 2 (CC1, CC2)20 whereas S408 lies within the first

research articles

Ceramide Kinase Profiling by Mass Spectrometry a

Table 1. hCERK Peptide Identification Using nano-LC-ESI-CID/ETD-MS start-endb

observed m/zc

005-017 006-015 021-029 022-032 034-050 051-066 051-068 078-084 078-090 098-105 105-115 116-126 132-143 132-147 134-140 153-177 159-172 178-187 182-200 189-212 213-231 224-232 230-246 247-259 260-275 261-281 283-298 283-302 292-305 305-310 311-317 312-320 318-342 318-342 349-355 367-387 368-387 371-388 388-401 388-401 389-396 406-424 406-424 407-420 407-424 407-425 408-420 413-424 435-453 436-453 438-454 458-463 464-473 492-514 500-516 517-523

699.4205 557.3033 501.8118 570.4269 856.9656 862.4756 955.087 443.259 537.3176 533.2801 779.3761 687.4572 671.4043 571.4041 425.2673 894.5561 757.4587 619.3566 1051.516 845.1383 668.4494 471.3051 941.4728 619.8445 833.4908 732.1233 897.9876 752.0989 614.6903 358.2011 415.2457 514.3113 943.481 727.917 435.2763 798.7384 1133.567 1062.055 814.9367 822.9296 424.2287 631.3786 658.0618 698.9389 946.5283 674.1047 613.8421 629.9334 800.7589 763.0867 1087.55 379.234 609.7939 873.1011 644.7177 468.2906

Mr (expt)d Mr (calc)e

1396.826 1112.592 1001.609 1138.839 1711.917 1722.937 1908.159 884.5034 1608.931 1064.546 1556.738 1372.9 1340.794 1711.191 848.52 2680.647 1512.903 1236.699 2101.018 2532.393 2002.326 940.5956 1880.931 1237.674 1664.967 2193.348 1793.961 2253.275 1841.049 714.3876 828.4768 1026.608 2827.421 2907.639 868.538 2393.193 2265.12 2122.096 1627.859 1643.845 846.4428 1891.114 1971.164 1395.863 1891.042 2019.292 1225.67 1257.852 2399.255 2286.238 2173.085 756.4534 1217.573 2616.282 1931.131 934.5666

deltaf

1396.735 0.0914 1112.623 -0.0309 1001.496 0.1126 1138.671 0.1683 1711.753 0.1641 1722.868 0.069 1907.984 0.1754 884.3738 0.1296 1608.743 0.1881 1064.508 0.0377 1556.69 0.0472 1372.738 0.1614 1340.724 0.0699 1710.993 0.1971 848.4796 0.0404 2680.516 0.1301 1512.804 0.0993 1236.587 0.1113 2100.913 0.1044 2532.247 0.1465 2002.157 0.1692 940.5706 0.0251 1880.913 0.0186 1237.586 0.0884 1664.856 0.1114 2193.15 0.1981 1793.888 0.073 2253.121 0.1544 1840.947 0.1019 714.4177 -0.03 828.3654 0.1115 1026.502 0.1058 2827.387 0.0347 2907.453 0.1861 868.3684 0.1697 2393.194 -0.0003 2265.026 0.0941 2121.968 0.1279 1627.727 0.132 1643.722 0.1228 846.4269 0.0159 1891.0163 0.0977 1970.983 0.1809 1395.663 0.1998 1891.016 0.0258 2019.111 0.181 1225.558 0.1122 1257.693 0.1594 2399.191 0.0638 2286.107 0.1313 2173.023 0.0619 756.3443 0.1092 1217.451 0.1226 2616.17 0.1118 1930.932 0.1993 934.5423 0.0244

missg

peptide sequence

0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0

T.GAAEPLQSVLWVK.Q G.AAEPLQSVLW.V R.CAVSLEPAR.A C.AVSLEPARALL.R R.WWRSPGPGAGAPGADAC.S C.SVPVSEIIAVEETDVH.G C.SVPVSEIIAVEETDVHGK.H K.MEKPYAF.T K.MEKPYAFTVHCVK.R W.KWAQVTFW.C F.WCPEEQLCHLW.L W.LQTLREMLEKL.T K.HLLVFINPFGGK.G K.HLLVFINPFGGKGQGK.R L.LVFINPF.G R.KVAPLFTLASITTDIIVTEHANQAK.E F.TLASITTDIIVTEH.A K.ETLYEINIDK.Y Y.EINIDKYDGIVCVGGDGMF.S Y.DGIVCVGGDGM*FSEVLHGLIGRTQ.R Q.RSAGVDQNHPRAVLVPSSL.R R.AVLVPSSLR.I S.SLRIGIIPAGSTDCVCY.S Y.STVGTSDAETSAL.H L.HIVVGDSLAMDVSSVH.H H.IVVGDSLAMDVSSVHHNSTLL.R R.YSVSLLGYGFYGDIIK.D R.YSVSLLGYGFYGDIIKDSEK.K G.FYGDIIKDSEKKRW.L R.WLGLAR.Y R.YDFSGLK.T Y.DFSGLKTFL.S K.TFLSHHCYEGTVSFLPAQHTVGSPR.D K.TFLSHHCYEGTVSFLPAQHTVGS*PR.D R.AGCFVCR.Q K.KALYGLEAAEDVEEWQVVCGK.F K.ALYGLEAAEDVEEWQVVCGK.F Y.GLEAAEDVEEWQVVCGKF.L K.FLAINATNMSCACR.R K.FLAINATNM*SCACR.R F.LAINATNM.S R.GLSPAAHLGDGSSDLILIR.K R.GLS*PAAHLGDGSSDLILIR.K G.LSPAAHLGDGSSDL.I G.LSPAAHLGDGSSDLILIR.K G.LSPAAHLGDGSSDLILIRK.C L.SPAAHLGDGSSDL.I H.LGDGSSDLILIR.K F.LIRHTNQQDQFDFTFVEVY.R L.IRHTNQQDQFDFTFVEVY.R R.HTNQQDQFDFTFVEVYR.V K.FQFTSK.H K.HMEDEDSDLK.E C.CTVSNSSWNCDGEVLHSPAIEVR.V W.NCDGEVLHSPAIEVRVH.C H.CQLVRLF.A

modification

scoreh

53 44 65 42 66 70 97 29 50 40 44 54 59 41 36 39 63 60 52 Oxidation (M199) 46 56 36 55 43 53 55 99 40 63 27 28 48 51 Phospho (S340) 43 36 47 93 56 97 Oxidation (M396) 100 44 51 Phospho (S408) 79 41 102 39 64 92 61 48 103 34 43 47 63 52

enzyme

Trypsin Chymotrypsin Trypsin Chymotrypsin Proteinase K Chymotrypsin Trypsin Trypsin Trypsin Chymotrypsin Chymotrypsin Chymotrypsin Trypsin Trypsin Chymotrypsin Trypsin Chymotrypsin Trypsin Chymotrypsin Chymotrypsin Chymotrypsin Trypsin Chymotrypsin Chymotrypsin Chymotrypsin Chymotrypsin Trypsin Trypsin Chymotrypsin Trypsin Trypsin Chymotrypsin Trypsin Trypsin Trypsin Trypsin Trypsin Chymotrypsin Trypsin Trypsin Chymotrypsin Trypsin Trypsin Chymotrypsin Trypsin Trypsin Chymotrypsin Trypsin Chymotrypsin Chymotrypsin Trypsin Trypsin Trypsin Trypsin Chymotrypsin Chymotrypsin

a Peptides from hCERK (swissprot accession no. Q8TCT0) were identified after multiple in-gel enzyme digestion and nano-LC-ESI-CID/ETD MS/MS analysis as detailed in the Materials and Methods. b Position of peptide sequence. c Measured mass to charge ratio in Thomson. d Measured mass in Dalton. e Theoretical mass in Dalton. f Error between measured and theoretical. g Number of miss cleveage site. h ion score of MASCOT search. The ion score criteria is higher than 25. The MS/MS result shown in this table provided data for 82% sequence coverage of hCERK. Methionine oxidation events are artifacts due to gel electrophoresis and subsequent sample handling.

part of the CC2 domain (Figure 3A). Both S340 and S408 are well conserved in animal CERK sequences (Figure 3B). To investigate the location of the two phosphorylation sites within

the protein fold we used a alignment of the C-terminal region of CERK which we previously performed based on the structure template of YegS, a recently crystallized lipid kinase from E. Journal of Proteome Research • Vol. 9, No. 1, 2010 423

research articles

Chen et al.

Figure 2. Phosphorylation of hCERK at S340 and S408. (A) CID-MS/MS spectrum of peptide (318TFLSHHCYEGTVSFLPAQHTVG(pS)PR342, m/z ) 727.917, 4+). (B) ETD-MS/MS spectrum of peptide (318TFLSHHCYEGTVSFLPAQHTVG(pS)PR342, m/z ) 727.917, 4+). (C) CIDMS/MS spectrum of peptide (318TFLSHHCYEGTVSFLPAQHTVGSPR342, m/z ) 943.481, 3+) after phosphatase treatment. (D) CID-MS/ MS spectrum of peptide (406GL(pS)PAAHLGDGSSDLILIR424, m/z ) 658.0618, 3+). (E) ETD-MS/MS spectrum of peptide (406GL(pS)PAAHLGDGSSDLILIR424, m/z ) 658.0618, 3+). (F) CID-MS/MS spectrum of peptide (406GLSPAAHLGDGSSDLILIR424, m/z ) 631.3786, 3+) after phosphatase treatment.

coli.19,21 According to this alignment, the sequence encompassing the S340 phosphorylation site would be part of a loop, surfacing between two strands (Figure 4A). The S408 site would also be located in a coiled structure, accessible for phosphorylation (Figure 4). The kinases involved in the phosphorylation of these two sites can be expected to be proline-dependent given that a conserved proline residue immediately follows the serine residue (Figure 4B). According to the Scansite prediction tool22 the cellcycle-dependent kinases (Cdc2, Cdk5) as well as the extracellularregulated kinase Erk1/2 are possible candidates. Given that coverage of the CERK protein sequence analyzed in the present work did not reach 100% (Table 1), some other phosphorylation sites in CERK may have been missed. One likely candidate is S427 which lies in the middle of the CC2 region (Figures 3A and 4B), within a calcium/calmodulin binding motif that was previously described in CERK.12 We have indeed evidence that preventing phosphorylation at S427 results in CERK inhibition (see below). S340 Phosphorylation Site: Conservation within the DAGK Family. Although not recognized previously, the S340 phosphosite in CERK appears to have a counterpart in all 424

Journal of Proteome Research • Vol. 9, No. 1, 2010

members of the DAGK family (Figure 5). In SPHK1 it is S225, the unique phosphorylated residue in this kinase whose function is to increase the kcat of the enzyme.13 In SPHK2 it is S351 whose phosphorylation appears to regulate agonistmediated enzyme activation.14 Other members of the DAGK family do also have an equivalent site although this site has not been reported to be phosphorylated yet. In hCERKL it is S409 and it is T579 in DAGKR. In the case of acylglycerol kinase, there is a glutamic acid residue instead that might act as a phosphorylation surrogate. S340 as well as the equivalent site in DAGK kinase family members lies some 140 amino acid (e.g., 142 amino acids for CERK) downstream of the glycine-rich loop that determines the nucleotide binding site of these kinases (Figure 5).23 This is reminiscent of the “activation loop” which was described in the growth factor/insulin stimulated AGC family of protein kinases (reviewed in ref 24): the phosphorylated residue(s) in the activation loop of AGC kinases lies within a similar distance from the glycine-rich loop (e.g., 146 amino acids for PKCR25). Phosphorylation in the activation loop acts as a switch to allow for activation. Available data obtained with SPHK1 and SPHK2

Ceramide Kinase Profiling by Mass Spectrometry

research articles

Figure 3. Localization and conservation of the S340 and S408 phosphorylation sites. (A) Sequence of hCERK with the various domains defined to date.20 The phosphorylated serine residues are boxed in black. (B) Conservation of the phosphorylation sites in CERK from different species: NP_073603 (Homo sapiens), NP_001128333 (Rattus norvegicus), NP_663450 (Mus musculus), NP_001026511 (Gallus gallus), NP_001086037 (Xenopus laevis), NP_001099056 (Danio rerio). Alignments and color code were made as described in ref 19.

Figure 4. Putative kinases and accessibility of the phosphorylation sites. (A) The C-terminal part of hCERK encompassing the CC1, the CC2 and stopping at the beginning of the CC3 domain (cf. Figure 3) is aligned to the corresponding region of the E. coli lipid kinase YegS. Secondary structures are shown as arrows (strand) and rectangles (helix). Structure assignments in YegS arise from the X-ray structure21 and are colored as follow: green (helix), yellow (exposed strand), brown (buried strand). For CERK, secondary structure assignments are predictions only, obtained as described.19 S340 and S408 phosphosites are denoted with a red star; the hypothetical phosphosite at S427 is denoted with a black star. (B) Sequence surrounding the S340 and S408 sites showing that CERK kinases are likely to be proline-directed. Journal of Proteome Research • Vol. 9, No. 1, 2010 425

research articles

Chen et al.

Figure 5. Conservation of the S340 phosphorylation site in the DAGK family. Alignments and color code were made as described in.19 They display, for representative members of the DAGK family, the glycine-rich loop of the catalytic domain and the regulation loop containing a described or a putative phosphorylation site (equivalent to S340 in CERK). The number of aminoacid residues between the underlined G residue of the glycine-rich loop and the underlined phosphorylation site in the regulation loop is boxed on the left.

also indicate that phosphorylation in this region regulates enzyme function13,14 and first evidence is presented below for a role of phosphorylation of S340 in stabilizing active CERK. Therefore, it may be hypothesized that a “regulation loop” exists in kinases of the DAGK family that shares some relationship with the “activation loop” of the protein kinases. Cellular Activity and Subcellular Localization of Phosphoserine Site Point Mutant Proteins. We examined the result of point mutations of the S340 and S408 phosphorylation sites for their impact on cellular CERK activity and localization. Alanine substitutions (to mimic the unphosphorylated state) and aspartic substitutions (to partially mimic the phosphorylated state) were both tested. None of the point mutation did, on its own, impair enzymatic activity (Figure 6A) as seen from the ability of these mutants to phosphorylate an exogenously added cell permeable short chain fluorescent ceramide, which enables the specific monitoring of production of C1P by CERK.2 Similarly, none of the point mutations did induce detectable changes in the subcellular localization pattern of a GFP-CERK fusion protein, typified by prominent Golgi complex and vesicular labeling in COS-1 cells17 (Figure 6B). Subcellular fractionation was further performed for the S340A and S340D mutant proteins and, consistently, this did not reveal appreciable changes compared with WT CERK (Figure 6C). These observations collectively suggest that phosphorylation on either S340 or S408 has no gross effect on localization and activity of hCERK unless some compensatory phosphorylation at (an) alternative site(s) has taken place. Alternatively, subtle changes may have occurred which our experiments failed to detect. Phosphorylation at S340 in the “Regulation Loop” Stabilizes Active CERK in Vitro. Given the conservation of the CERK S340 site in DAGK family members and because of similarity of this locus with the activation loop site of AGC protein kinases, we have addressed the impact of its phosphorylation further. CERK activity in the S340A mutant protein was investigated in vitro on whole cell Triton X100-lysates, after 426

Journal of Proteome Research • Vol. 9, No. 1, 2010

Figure 6. Cellular activity and localization of the S340 and S408 CERK mutant proteins. (A) COS-1 cells overexpressing WT CERKFLAG, the catalytically deficient G198D CERK-FLAG mutant protein or the phosphorylation site mutants, were treated with 5 µM NBD-Cer for 1 h. Lipids were extracted and analyzed on thin-layer chromatography; std., NBC-Cer + NBD-C1P standards. A corresponding anti-FLAG Western Blot is shown on the right. (B) Subcellular localization of GFP-tagged CERK phosphorylation site mutant proteins observed by fluorescence microscopy by comparison to GFP-CERK WT and GFP-CERK G198D. (C) Comparative subcellular fractionation of COS-1 cells after expression of CERK-FLAG WT, S340A or S340D proteins, analyzed by Western Blot using anti-FLAG detection.

transient expression on COS-1 cells and using a validated radioassay procedure.26 Under standard conditions18 and if the

research articles

Ceramide Kinase Profiling by Mass Spectrometry

Figure 7. In vitro activity of the S340A and S340D CERK mutant proteins. (A) Activity of CERK mutant proteins relative to WT CERK, measured in the octylglucoside-based mixed micelle assay after extraction in Triton X-100; mean of 9 determinations obtained from 3 independent experiments ( SD. (B) Relative activities measured in the octylglucoside-based mixed micelle assay after extraction in octylglucoside; mean of 2 determinations ( SD. (C) Residual activity measured as in (A) after 30 min-incubation of CERK samples at 30 °C in a Triton X-100 containing extraction buffer; mean of 2 determinations ( SD.

assay was performed immediately after cell lysis, there was only a limited s albeit statistically significant s decrease (15%) in activity in the S340A mutant protein compared with the WT enzyme (Figure 7A). Previously, in an attempt to streamline extraction and assay of CERK using a single buffer, we had evaluated octylglucoside as an alternative to Triton X-100 for harvesting COS cells; however, Triton X-100sor Triton X-11418swas significantly better for maintaining active CERK throughout the extraction procedure (C. Graf and F. Bornancin, unpublished results). When octylglucoside was used instead of Triton X-100 for cell lysis, this dramatically reduced activity measured in the S340A mutant protein which reached only 15% of WT activity (Figure 7B). Increased sensitivity of the S340A CERK protein to the detergent used for cell lysis suggested a weaker stability of this mutant protein. To address stability more directly we extracted CERK using the Triton X-100-based standard procedure but submitted the samples to a 30 minincubation at 30 °C before measurement of residual CERK activity. At the end of the procedure, WT CERK had retained 30% of its initial activity, in agreement with previous work.18 Under the same conditions, residual activity in the S340A mutant protein did not exceed 8% of its initial activity whereas an intermediate recovery of 16% was observed for the S340D mutant protein (Figure 7C). Altogether these results strongly suggest that phosphorylation of the S340 residue in the “regulation loop” of CERK impacts stability of the active enzyme conformation. S427 in the Calcium/Calmodulin Domain of CERK: Another Putative Phospho-Regulatory Site. S427 lies in a previously identified 1-8-14 type B calcium/calmodulin binding motif,1,12 located in the middle of the CC2 domain.20 This serine residue is well conserved among CERK from various

species (Figure 8A). The net positive charge of calcium/ calmodulin motifs is important for function.27 Accordingly, phosphorylation at S427 in hCERK should be expected to quench the positive charge of its calcium/calmodulin binding motif and, as a result, impair function. Our proteolytic study followed by MS analysis failed to detect a peptide surrounding this serine residue and, therefore, whether this serine residue may become phosphorylated in hCERK is not known. To get insight, we prepared the hCERK S427A and S427D mutant proteins and measured their in vitro activity under standard conditions (cf previous paragraph). Activity in the S427A mutant protein amounted to only 30% of that of WT CERK whereas the S427D mutant protein was more active, displaying 50% of WT CERK activity (Figure 8B). Thus, although not detected in our MS/MS analysis, the S427 residue may serve as a phosphoacceptor site in CERK thereby enhancing CERK activity. Protein kinases A and G might be candidates for phosphorylation of S427, based on the consensus motif found in their phosphorylated substrates.

Discussion A combination of radio-labeling, affinity purification, proteolytic digestions, and mass spectrometry (nano-LC-ESI-MS/ MS) was implemented to search for post-translational modificationsofmammaliancell-andinsectcell-expressed,recombinant hCERK. This led to identification of two phosphorylated residues at S340 and S408 thus providing first evidence that CERK is a phosphoprotein (Figures 1 and 2, Table 1). The identification of phosphorylated S340 and S408 in hCERK provides an unprecedented opportunity to understand how CERK is regulated. In particular, defining the kinase(s) Journal of Proteome Research • Vol. 9, No. 1, 2010 427

research articles

Figure 8. S427 is a putative phosphorylated site. (A) Conservation of S427 in CERK from different species: NP_073603 (Homo sapiens), NP_001128333 (Rattus norvegicus), NP_663450 (Mus musculus), NP_001026511 (Gallus gallus), NP_001086037 (Xenopus laevis), NP_001099056 (Danio rerio). Alignments and color code were made as described in ref 19. (B) In vitro activity of S427A and S427D CERK mutants proteins relative to WT CERK, measured in the octylglucoside-based mixed micelle assay after extraction in Triton X-100; mean of 9 determinations obtained from 3 independent experiments ( SD.

responsible for phosphorylation of CERK can be expected to shed light on the mechanisms involved. These kinases are very likely proline-directed (Figure 4B). Among this type of kinases are those known to play a role in cell proliferation (e.g., ERKs) or cell-cycle control (e.g., CDKs); validation of such candidates would be consistent with the reported function of C1P in the regulation of cellular proliferation and survival.28 Phosphorylation of CERK by these kinases might also account for the steep regulation of CERK by serum.2 Clearly, further experiments are required to test these hypotheses. The phosphorylated residues S340 and S408 are well conserved in CERK from different species. Both are located downstream of the catalytic site, in a region that has been shown to have a regulatory role. S408, in the first half of the CC2 domain, precedes a calcium/calmodulin domain that was previously shown to be responsible for the Ca2+-sensitivity of CERK.12 In the CC1-CC2 interdomain region, S340 is followed by a conserved cysteinyl motif which is critical for CERK activity,19 for CERK to exit the nucleus as well as for correct addressing of CERK in the cytoplasm (e.g., association with the Golgi complex).20 The S340 site is remarkable for additional reasons. First, this site appears to have a counterpart in every kinase of the DAGK family with the exception of AGK (Figure 5); the case of AGK is interesting because in this protein the serine residue is substituted by a glutamic residue that provides a constitutive negative charge. Second, akin to the phosphorylation site in the activation loop of AGC protein kinases, S340 lies within 140-150 aminoacid downstream of the glycine-rich loop of the nucleotide binding site (Figure 5). This suggests the existence of a “regulation loop” in CERK which may, to 428

Journal of Proteome Research • Vol. 9, No. 1, 2010

Chen et al. some extent, bear similarity with the activation loop in AGC protein kinases. For SK1, SK2, and CERK, phosphorylation in the “regulation loop” is dispensable for basal activity and, therefore, phosphorylation in this region does not work as a switch for permissive activation as it does for AGC protein kinases.24 However, phosphorylation of SK1, SK2 and CERK at the regulatory loop site allowed for sustained activity: increased kcat for SK1,13 regulation of agonist-mediated enzyme activation for SK2,14 increased stability for CERK (Figure 7). Further experiments are necessary to understand if the impacts on these various readouts are in fact proceeding from a similar regulatory mechanism. It will also be valuable to explore if, as for AGC protein kinases, the regulation loop in CERK is preceded by a catalytic loop. Such catalytic determinants are still unknown in CERK. Definite evidence will require the side-by-side comparison of crystal structures. This field has rapidly progressed in recent years, as seen in the number of crystal structures of AGC protein kinases that have been elucidated together with the first structure of an E. coli homologue of the mammalian DAGKs,21 thus paving the way for future studies. In addition to S340 and S408, other phosphorylation sites may exist since 18% of the CERK sequence could not be analyzed in our work (Table 1). This may be due to the poor ability of some of the cleaved peptides to ionize in the electrospray source. One such site may be S427 within the calcium/calmodulin motif of the CC2 domain. Alanine or aspartic substitutions of S427 have provided indirect support for phosphorylation at this site; in fact, the mutagenesis study reported in Figure 8 suggests that phosphorylation of S427 would have a profound effect on CERK activity. However, direct evidence for phosphorylation of S427 remains to be demonstrated. Some other phosphorylation sites may have been missed due to low stoichiometry of the phosphorylation reaction. Our initial analysis had detected S55, S470 and S508 as possible additional phosphorylated sites in CERK. However, these sites were not confirmed subsequently. The S300 residue had been considered a possible important PKC phosphorylation site in CERK, because of conservation in SPHKs and because of increased CERK expression in response to phorbol esters.1 However, our MS/MS analysis did not identify S300 as a phosphorylation site (Table 1). Furthermore, purified CERK was not phosphorylated by various PKC isoforms in vitro (data not shown). In addition, we prepared a S300A CERK mutant protein and found that it was as active as WT CERK whereas a S300D mutant protein was significantly less active (not shown). Altogether these data do not support a positive regulation of CERK via PKC-mediated phosphorylation of S300. Regulation by PKC, if it occurs, may rather be indirect. In conclusion, this work provides first evidence that CERK is a phosphoprotein, with the identification of two phosphorylation sites in hCERK, at S340 and S408. The S340 site has a counterpart in other enzymes of the DAGK family and appears to serve as a common mean to regulate activity. Its localization within the primary sequence and its consensual regulatory role are reminiscent of the activation loop phosphorylation site in AGC protein kinases. The precise regulatory mechanism of phosphorylation at S340, the impact on CERK function of phosphorylation at S408 as well as the kinases responsible for phosphorylation of CERK, remain to be determined. Abbreviations: C1P, ceramide-1-phosphate; CERK, ceramide kinase; CID, collision induced dissociation; ETD, electron transfer dissociation; FCS, fetal calf serum; HCT, high capacity

research articles

Ceramide Kinase Profiling by Mass Spectrometry ion trap; LC-MS, liquid chromatography mass spectrometry; MS/MS, tandem mass spectrometry; NBD, N-[7-(4-nitrobenzo2-oxa-l,3-diazole)]; OG, β-octylglucoside; PH, Pleckstrin Homology; WT, wild-type.

References (1) Sugiura, M.; Kono, K.; Liu, H.; Shimizugawa, T.; Minekura, H.; Spiegel, S.; Kohama, T. Ceramide kinase, a novel lipid kinase: Molecular cloning and functional characterization. J. Biol. Chem. 2002, 277 (26), 23294–23300. (2) Boath, A.; Graf, C.; Lidome, E.; Ullrich, T.; Nussbaumer, P.; Bornancin, F. Regulation and traffic of ceramide 1-phosphate produced by ceramide kinase: Comparative analysis to glucosylceramide and sphingomyelin. J. Biol. Chem. 2008, 283 (13), 8517– 8526. (3) Graf, C.; Zemann, B.; Rovina, P.; Urtz, N.; Schanzer, A.; Reuschel, R.; Mechtcheriakova, D.; Muller, M.; Fischer, E.; Reichel, C.; Huber, S.; Dawson, J.; Meingassner, J. G.; Billich, A.; Niwa, S.; Badegruber, R.; Van Veldhoven, P. P.; Kinzel, B.; Baumruker, T.; Bornancin, F. Neutropenia with impaired immune response to Streptococcus pneumoniae in ceramide kinase-deficient mice. J. Immunol. 2008, 180 (5), 3457–3466. (4) Bornancin, F.; Mechtcheriakova, D.; Stora, S.; Graf, C.; Wlachos, A.; Devay, P.; Urtz, N.; Baumruker, T.; Billich, A. Characterization of a ceramide kinase-like protein. Biochim. Biophys. Acta - Mol. Cell Biol. Lipids 2005, 1687 (1-3), 31–43. (5) Graf, C.; Niwa, S.; Muller, M.; Kinzel, B.; Bornancin, F. Wild-type levels of ceramide and ceramide-1-phosphate in the retina of ceramide kinase-like-deficient mice. Biochem. Biophys. Res. Commun. 2008, 373 (1), 159–163. (6) Gomez-Munoz, A.; Duffy, P. A.; Martin, A.; O’Brien, L.; Byun, H.S.; Bittman, R. Short-chain ceramide-1-phosphates are novel stimulators of DNA synthesis and cell division: Antagonism by cellpermeable ceramides. Mol. Pharmacol. 1995, 47 (5), 883–889. (7) Pettus, B. J.; Bielawska, A.; Spiegel, S.; Roddy, P.; Hannun, Y. A.; Chalfant, C. E. Ceramide kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release. J. Biol. Chem. 2003, 278 (40), 38206–38213. (8) Graf, C.; Klumpp, M.; Habig, M.; Rovina, P.; Billich, A.; Baumruker, T.; Oberhauser, B.; Bornancin, F. Targeting ceramide metabolism with a potent and specific ceramide kinase inhibitor. Mol. Pharmacol. 2008, 74 (4), 925–932. (9) Mitra, P.; Maceyka, M.; Payne, S. G.; Lamour, N.; Milstien, S.; Chalfant, C. E. Ceramide kinase regulates growth and survival of A549 human lung adenocarcinoma cells. FEBS Lett. 2007, 581 (4), 735–740. (10) Mitsutake, S.; Kim, T.-J.; Inagaki, Y.; Kato, M.; Yamashita, T.; Igarashi, Y. Ceramide Kinase Is a Mediator of Calcium-dependent Degranulation in Mast Cells. J. Biol. Chem. 2004, 279 (17), 17570– 17577. (11) Shimizu, M.; Tada, E.; Makiyama, T.; Yasufuku, K.; Moriyama, Y.; Fujino, H.; Nakamura, H.; Murayama, T. Effects of ceramide, ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2alpha; additional role of ceramide-1phosphate in phosphorylation and Ca2+ signaling. Cell. Signalling 2009, 21 (3), 440–447. (12) Mitsutake, S.; Igarashi, Y. Calmodulin is involved in the Ca2+dependent activation of ceramide kinase as a calcium sensor. J. Biol. Chem. 2005, 280 (49), 40436–40441.

(13) Pitson, S. M.; Moretti, P. A. B.; Zebol, J. R.; Lynn, H. E.; Xia, P.; Vadas, M. A.; Wattenberg, B. W. Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation. EMBO J. 2003, 22 (20), 5491–5500. (14) Hait, N. C.; Bellamy, A.; Milstien, S.; Kordula, T.; Spiegel, S. Sphingosine Kinase Type 2 Activation by ERK-mediated Phosphorylation. J. Biol. Chem. 2007, 282 (16), 12058–12065. (15) Chen, W. Q.; Kang, S. U.; Lubec, G. Protein profiling by the combination of two independent mass spectrometry techniques. Nat. Protocols 2006, 1 (3), 1446–1452. (16) John, J. P. P.; Chen, W. Q.; Pollak, A.; Lubec, G. Mass Spectrometric Studies on Mouse Hippocampal Synapsins Ia, IIa, and IIb and Identification of a Novel Phosphorylation Site at Serine-546. J. Proteome Res. 2007, 6 (7), 2695–2710. (17) Carre, A.; Graf, C.; Stora, S.; Mechtcheriakova, D.; Csonga, R.; Urtz, N.; Billich, A.; Bornancin, F. Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain. Biochem. Biophys. Res. Commun. 2004, 324 (4), 1215–1219. (18) Rovina, P.; Jaritz, M.; Hofinger, S.; Graf, C.; Devay, P.; Billich, A.; Baumruker, T.; Bornancin, F. A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase. Biochem. J. 2006, 400 (2), 255–265. (19) Lidome, E.; Graf, C.; Jaritz, M.; Schanzer, A.; Rovina, P.; Nikolay, R.; Bornancin, F. A conserved cysteine motif essential for ceramide kinase function. Biochimie 2008, 90 (10), 1560–1565. (20) Rovina, P.; Schanzer, A.; Graf, C.; Mechtcheriakova, D.; Jaritz, M.; Bornancin, F. Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains. Biochim. Biophys. Acta - Mol. Cell Biol. Lipids 2009, 1791, 1023-1030. (21) Bakali, H, M. A.; Herman, M. D.; Johnson, K. A.; Kelly, A. A.; Wieslander, A.; Hallberg, B. M.; Nordlund, P. Crystal Structure of YegS, a Homologue to the Mammalian Diacylglycerol Kinases, Reveals a Novel Regulatory Metal Binding Site. J. Biol. Chem. 2007, 282 (27), 19644–19652. (22) Obenauer, J. C.; Cantley, L. C.; Yaffe, M. B. Scansite 2.0: proteomewide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res. 2003, 31 (13), 3635–3641. (23) Pitson, S. M.; Moretti, P. A. B.; Zebol, J. R.; Zareie, R.; Derian, C. K.; Darrow, A. L.; Qi, J.; D’Andrea, R. J.; Bagley, C. J.; Vadas, M. A.; Wattenberg, B. W. The Nucleotide-binding Site of Human Sphingosine Kinase 1. J. Biol. Chem. 2002, 277 (51), 49545–49553. (24) Parker, P. J.; Parkinson, S. J. AGC protein kinase phosphorylation and protein kinase C. Biochem. Soc. Trans. 2001, 29 (Pt 6), 860– 863. (25) Cazaubon, S.; Bornancin, F.; Parker, P. J. Threonine-497 is a critical site for permissive activation of protein kinase C alpha. Biochem. J. 1994, 301 (2), 443–448. (26) Bajjalieh, S.; Batchelor, R. Ceramide kinase. Methods Enzymol. 2000, 311, 207–215. (27) Rhoads, A. R.; Friedberg, F. Sequence motifs for calmodulin recognition. FASEB J. 1997, 11 (5), 331–340. (28) Gomez-Munoz, A. Ceramide 1-phosphate/ceramide, a switch between life and death. Biochim. Biophys. Acta - Biomembranes 2006, 1758 (12), 2049–2056.

PR900763Z

Journal of Proteome Research • Vol. 9, No. 1, 2010 429