It has been some 20 years since the initial discovery of ceramide 1-phosphate (C1P) and nearly a decade sinceceramide kinase (CERK) was cloned. Many studies have shown that C1P is important for membrane biologyand for the regulation of membrane-bound proteins, and the CERK enzyme has appeared to be tightlyregulated in order to control both ceramide levels and production of C1P. Furthermore, C1Pmade by CERK hasemerged as a genuine signalling entity. However, it represents only part of the C1P pool that is available in thecell, therefore suggesting that alternative unknown C1P-producing mechanisms may also play a role. Recenttechnological developments for measuring complex sphingolipids in biological samples, together with theavailability of Cerk-deficient animals as well as potent CERK inhibitors, have now provided new grounds forinvestigating C1P biology further. Here, we will review the current understanding of CERK and C1P in terms ofbiochemistry and functional implications, with particular attention to C1P produced by CERK.
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1. Introduction
Ceramide 1-phosphate (C1P) and ceramide phosphorylating activ-ities had long been recognized before the cloning of ceramide kinase(CERK). C1P was first identified as the product of a new kinase activitypresent in rat synaptic vesicles preparations [1]. Shortly afterwards,stimulated production of C1P was measured in the human promyelo-cytic leukemia cell line HL-60, in response to treatment with S. aureussphingomyelinase (SMase) [2]. In this latter study, only ceramide (Cer)produced by incubation of HL-60 cells with S. aureus SMase, but not Cerproduced with Rhodococcus endoglycoceramidase, could be metabo-lised into C1P, thus suggesting a selective path from sphingomyelin(SM) to C1P via phosphorylation of Cer. Subsequently, a Cer phosphory-lating activity was isolated and characterized from HL-60 microsomalmembranes [3]. Human neutrophils were also shown to produce C1P inresponse to SMase treatment [4] or phagocytosis of antibody-coatederythrocytes after priming with formylmethionylleucylphenylalanine(fMLP) [5]. Metabolic studies in the human A498 kidney carcinomaepithelial cell line showed that Cer phosphorylation occurs followingtreatment with S. aureus sphingomelinase or exposure to defined E. colistrains [6]. Similar studies in rat cerebellar granule cells demonstratedthat C1P can be produced using various precursors, e.g., SM, the GM1ganglioside or sphingosine (Sph) [7]; production of C1P fromSMwas byfar the dominant pathway and appeared to require differentiation of thegranule cells. Whether originating from SM or GM1 or Sph, C1P wasgenerated in parallel to Cer which suggested that a CERK activity ispresent at the plasma membrane (Cer released from SM hydrolysis) orat internal membranes (Cer produced from Sph recycling duringganglioside metabolism).
The CERK enzyme was finally cloned in 2002 [8] and, since then, itsinvestigation has confirmed the above-summarized pioneering obser-vations. In addition, evidence that CERK is not the unique enzymeresponsible for production of C1P has surfaced after finding significantresidual C1P levels in CERK-deficient cells [9]. However, CERKmay turnout to be a unique Cer kinase because acute phosphorylation of Cer wascompletely abolished in absence of CERK. Consistently, all studies so farindicate that there is no Cer kinase activity in a related CERK-like(CERKL) protein which was identified shortly after CERK [10–14].
2. Ceramide kinase (CERK)
2.1. The CERK protein
CERKwas cloned from a human leukemia cDNA library and amousebrain cDNA library [8]. Based on homology with the catalytic domain ofdiacylglycerol kinase (DAGK), the identification of CERK introduced anew subclass in the DAGK family, distinct from sphingosine kinases(SPHK). Primary sequence analysis (Fig. 1) identified a putativeN-myristoylation site on its NH2 terminus; modification of this sitewas confirmed in in vitro expression experiments [15]. CERK bears aPleckstrin Homology (PH) domain between residues 8 and 124 whichhas been analyzed in detail by homologymodeling [16] and in a numberof biochemical studies [15–19]. This domain is required for membranebinding in vitro, sub-cellular localization at membrane compartments,and enzymatic activity [15,18]. The modeled PH domain of CERKdisplays the typical PH domain superfold and appears to be stronglypolarized [16] (Fig. 1). Several positively charged residues wereidentified in an extended loop bridging the two beta strands 6 and 7that turned out to be essential for CERK localization and activity. In fact,the β6-β7 loop appears to contribute to stabilization of the PH domainand as a result, of the entire CERK protein. This was evidenced by theproperties of β6-β7 loop mutant proteins, which displayed an unstableconformation, subject to enhanced deactivation, proteolysis andaggregation [16] (Fig. 1). Mutagenesis of the N-terminal β-strand orwithin the C-terminal α-helix — both are necessary to stabilize the PHdomain fold — also led to severely compromised CERK proteins [16,17]
(Fig. 1). The binding of CERK to polyphosphoinositides (PPIn), via its PHdomainwas analyzed in two separate sudies [16,18]. Usingprotein-lipidoverlays, both studies showed a promiscuous binding of CERK to PPIn.An in vitro assay with lipids coated on a microtiter plate showed that,among PPIn, PtdIns (4,5)P2 is preferentially bound by the isolated PHdomain of CERK; however, this preference was almost abolished whenfull length CERK was analyzed [18]. Therefore, CERK might predomi-nantly associate with PtdIns (4,5)P2 containing membranes because ofabundance of PtdIns (4,5)P2 compared to other PPIn, rather than as aresult of specific binding. Further evidence that the PH domain of CERKserves structural and generalmembrane binding purposes rather than aprecise targeting function has come from a recent study comparingCERK with the related CERK-like (CERKL) protein [19]. CERKL alsocontains a PH domain [19] but, in contrast to CERK, CERKL does notlocalize to theGolgi complex [11,19]. Swapping the PHdomain of CERKLfor that of CERK did not bring about noticeable changes in thelocalization of CERKL, i.e., the chimeric CERKL protein was not inducedto bind the Golgi complex. In addition, analyzing the C-terminal regionof CERK by progressive deletion showed that sequences outside the PHdomain of CERK are also required for proper localization and activity ofthe CERK protein [19]. Like mutations in the PH domain, C-terminalmutations appeared to have impact on the protein structural stability(Fig. 1).
A conserved cysteinyl motif (CXXXCXXC) was identified downstreamof the catalytic domain, between the CC1 and CC2 subdomains [20](Fig. 1). This motif is required for CERK function, e.g., export out of thenucleus, localization at the Golgi complex and activity [19,20]. It isconserved in CERK from various species, but is absent in related enzymeslike CERKL. An equivalent motif occurs in radical-S-adenosylmethionineenzymes that are regulated by [4Fe.4S] complexes [21]. Further studiesarewarranted to understand how thismotif may regulate CERK function.
A calcium/calmodulin bindingmotif was identified in CERK, withinamino-acids 422-435 of the CC2 domain [8] (Fig. 1). Calmodulin wasshown to bind directly to this site, in a calcium-dependent manner[22]. Thus, calmodulin appears to function as a calcium sensor forCERK. Recently, it was proposed that the binding of calmodulin toCERK may be regulated by phosphorylation (at S427 in hCERK) [23].This latter mass spectrometry analysis also identified two additionalphosphorylation sites in hCERK, at S340 and at S408. Phosphorylationat S340 regulates catalytic activity [23]. This site has a counterpart inevery kinase of the DAGK family (with the exception of acylglycerolkinase which bears a glutamate residue instead that may mimicconstitutive phosphorylation). Furthermore, akin to the phosphory-lation site in the activation loop of AGC protein kinases, S340 lieswithin 140-150 aminoacid away from the glycine-rich loop of thenucleotide binding site therefore suggesting the existence in CERK of a“regulation loop” which may, at least to some extent, bear similaritywith the activation loop of AGC kinases [23] (Fig. 1). The consequenceof phosphorylation at S408 is still unknown. Kinases, responsible forphosphorylation of CERK, have not been identified yet. However,proline-directed kinases e.g., extracellularly-regulated kinases (ERKs)and cell-cycle-dependent kinases (CDKs) appear like candidates forphosphorylation of the S340/S408 sites whereas protein kinase A(PKA) might phosphorylate S427 [23].
The reported G412R mutation discovered in a Acd5 CERK mutantof Arabidopsis thaliana led to strongly reduced CERK activity [24].G412 is equivalent to G315 in hCERK which is in the CC1 region. Thesequence surrounding G315 was predicted with high confidence to bean helix, based on the X-ray structure assignments of the relatedE. coli lipid kinase YegS [20]. Therefore, it is possible that disruption ofthe putative G412-containing helix by substitution with an arginineresidue has led to loss of a structural feature of A. thaliana CERK that isimportant for function.
A mutation scan analysis of human chromosome 22 led to thediscovery of a single nucleotide polymorphism encoding the pointmutation V338G [25]. This valine residue is conserved betweenmouse
Fig. 1. The CERK protein. hCERK contains an N-terminal Pleckstrin Homology (PH) domain followed by a prototypic kinase domain which is found in all members of thediacylglycerol kinase (DAGK) family. Downstream of the kinase domain are three domains, named CC1 to CC3 because they are conserved only in CERK and CERKL (CERKL alsoknown as CERK-like is the closest homolog of CERK in the DAGK family). The N-terminal glycine residue of CERK is modified, probably by myristoylation. Human CERK was alsoshown to be constitutively phosphorylated at two positions, S340 and S408. The S340 site appears to have counterpart in other DAGK family members and, notably, is located in adistance from the ATP-binding site that is reminiscent of that of the activation loop phosphorylation site in the AGC family of protein kinases. Other data such as the presence of a keycysteinyl motif in the vicinity of S340 indicate that, akin to AGC protein kinases, CERK may harbor a “regulation loop” between CC1 and CC2 which is exposed to regulatory input.S408 as well as another likely phosphorylation site at S427, are localized in the CC2 domain; phosphorylation at the latter site might serve as a signal for modulating calcium/calmodulin-mediated activation of CERK. Nuclear export signals (NES) are located in the C-terminal half of CERK: one is in the CC3 domain, the other coincides with the cysteinylmotif in the regulation loop. A nuclear localization signal (NLS) was identified in the β6-β7 loop of the PH domain. This loop contains several positively charged amino-acid residuesthat contribute to a large extent to the strong electrostatic potential of the PH domain*. The PH domain is important for CERK to interact with the membrane. It bindspolyphosphoinositides (PPIn) within little specificity. The structure of CERK appears to require an intact PH domain as well as an intact C-terminal region since point mutations thatdestabilize the secondary structure, or limited deletions from both ends of the protein, severely compromise CERK localization and activity. *The modeled PH domain of CERK wasreproduced with permission. Original publication: P. Rovina, M. Jaritz, S. Hofinger, C. Graf, P. Devay, A. Billich, T. Baumruker, F. Bornancin, “A critical β6-β7 loop in the pleckstrinhomology domain of ceramide kinase”, Biochem. J. (2006) 400, 255-265.
and human sequences and is only 2 amino-acids away from theconserved regulatory phosphorylation site S340, i.e., it is in theregulation loop mentioned above. This mutation may therefore havefunctional consequences that remain to be determined.
2.2. The CERK enzyme
Cer phosphorylation by CERK occurs on the first position only,thereby yielding Cer 1-phosphate and not Cer 3-phosphate [1,3,26].CERK is a phosphotransferase that uses primarily ATP as thephosphate donor and works optimally at neutral pH [1,3,5]. Byanalogy with the nucleotide binding site in SPHK1 [27], G198 inhCERK was identified as a key residue for CERK activity; a G198Dmutant protein was completely devoid of activity [11,28] (Fig. 1).Submicromolar concentrations of calcium ions such as those found inactively secreting cells, strongly stimulate CERK activity. The discrep-ancy found in vitro, i.e., nanomolar concentrations of calcium weresufficient for activation of CERK in cell lysates [8] whereas micromolarconcentrations were required using immunoprecipitated protein [22],was clarified with the identification of calmodulin acting as a calciumsensor for CERK [22] (Fig. 1). Prior to cloning of CERK, the reportedenzymatic parameters of the purified CERK activities, using naturalCer (a mixture of stearoyl- and nervonyl-Cer species) in a mixedmicelle assay [29] in the presence of calcium, were in the range of 13-25 μM and 9-45 μM for the Michaelis-Menten constant for ATP andCer, respectively. All these properties turned out to be consistent withthose obtained with cell lysates containing recombinant CERK:
Optimal pH at 6.5, stimulation by calcium ions in the range of 0.1-500 μM, Km of 32 μM for ATP, and of 187 μM for Cer [8]. Calciumappears to regulate the velocity of the reaction without appreciableeffect on the affinity of the enzyme for Cer.
Substrate specificity for CERK has been characterized using variousassay conditions [26,30]. The ability of CERK to recognize Cer isstereospecific, with a clear preference for the D-erythro isomer.Although Cer species withN12 carbon chains are better substrates,short chain Cer species, including C2-Cer, can also be reacted if the assayconditions are met, a higher Vmax likely compensating the higher Km onthese short-chain species. The 4-5 trans double bond, the free hydrogenof the second amide group and the sphingoid chain are all necessaryfeatures for efficient phosphorylation of Cer by CERK.
In the cell, Cer is provided to CERK either by de novo synthesis inthe endoplasmic reticulum (ER) followed by Cer transport to the Golgicomplex [9,31,32], or as a result of SMase activation at the plasmamembrane level [2,4,7]. The Cer transfer protein may account, at leastin part, for Cer delivery to CERK [31,32]. However, the dependency onthis transfer protein seems to be more stringent for SM synthesis thanit is for C1P synthesis, therefore suggesting possible alternativepathways for providing Cer to CERK [9].
CERK activity is very sensitive to oxidation [20,29]. A cluster ofcysteine residues, important for CERK function, was described above(Fig. 1); whether these residues recapitulate CERK sensitivity tooxidation is currently unknown.
There is increasing interest in CERK as a drug target, which hasbeen triggered by the reported role of CERK in inflammation and
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growth control. As a result, several assays have been established tosearch for specific CERK inhibitors [33–36]. K1, an olefin isomer of thepreviously reported sphingosine kinase inhibitor F-12509A, wasshown to act as a non-competitive CERK inhibitor, resulting in partialinhibition of CERK and C1P production in cells at 100 μM [37,38]. Highthroughput screening assays using ATP-consumption as readout haveenabled the identification of potent CERK inhibitors [33,34]. The mostpotent is the diamino-benzothiazole derivative NVP-231, which isactive in the low nanomolar range (Ki=7.4 nM) on purified as well asintracellular CERK (Fig. 2). At 100 nM, NVP-231 has proved to bemorethan 80% efficacious for CERK inhibition in cell-based assays. Beyondthis concentration, off-target (e.g., anti-proliferative) effects mayoccur. For this purpose, NVP-995, a related compound bearing twomethoxy substituents, which is 170-fold less potent for CERKinhibition, has turned out to be a valuable control in cellular assays[34,36] (Fig. 2). NVP-231 (and NVP-995) may therefore help studyingCERK cellular biology. The usefulness of these compounds in vivo is,however, limited by a poor bioavailability together with a rapidclearance following i.v. administration [34].
2.3. CERK subcellular localization
Since the early days it has been known that solubilization of a Cerkinase from membranes requires prolonged treatments with acombination of detergent, salt and chelator [29]. This was subse-quently confirmed using recombinant CERK [8,16] thus establishingthat CERK is strongly associated with the particulate fraction.Consistently, other studies have indicated that CERK can localize tolipid rafts [28] and heavy membrane fractions [31].
In the initial studies using GFP-CERK ectopic expression in COS-1and HUVEC cells [15] as well as other cell types, e.g. A549, SW1353,CHO and HEK293 [16], CERK was found to be enriched in three majorcompartments: The Golgi complex, the plasma membrane andcytoplasmic vesicles (Fig. 3). Localization to the latter two compart-ments was stimulated by osmotic cell swelling, which is known toinvolve vesicular traffic [15] (Fig. 3). This was confirmed in asubsequent study using COS-7 cells [18] . Furthermore, the trafficking
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Fig. 2. CERK enzymatic inhibition. A, The diamino-benzothiazole compound NVP-231 is a pois substituted by twomethoxy groups, the resulting derivative (NVP-995) is 170-fold less potNVP-231, in a ceramide-competitive manner. This figure was reproduced with permission.Oberhauser B., F. Bornancin, Targeting ceramide metabolism with a potent and specific cer
of CERK between these compartments was severely impaired bytreatment with nocodazole but not by cytochalasine D, therebysuggesting a microtubule-orchestrated process [16]. When endoge-nous CERK was subsequently analyzed by immunofluorescence, itconfirmed the data obtained with ectopic expression, it showed trans-Golgi rather than cis-Golgi localization and it suggested additionalcompartments, e.g., endosomal/exosomal and mitochondria [31]. Thislatter study also showed that CERK does not significantly localize tothe endoplasmic reticulum. In keeping with this observation, a recentwork with various fluorescent Cer analogs showed that Cer needs tobe transported to the Golgi complex for phosphorylation by CERK [9].Altogether, the subcellular localization data for CERK are consistentwith the early discovery of a Cer phosphorylating activity enriched inthe membrane fraction of synaptic vesicles [1]. In addition, a recentstudy has shown that CERK can be released in cell culture medium,probably as a result of C1P-induced vesicular shedding [39].
The PH domain of CERK, when expressed independently, isstrongly retained in the nucleus [15,19]. In fact, the sequence 93-RRHR-96 in the β6-β7 loop of the PH domain in hCERK bears aputative nuclear localization signal (NLS) [19] (Fig. 1). Therefore, inaddition to stabilizing the structure of CERK (cf above), chargedresidues in the β6-β7 loop may regulate nuclear accumulation ofCERK. The effect of progressive deletion of the C-terminus of CERK onprotein localization has also suggested the existence of nuclear exportsignals (NES). Two NES were identified, a traditional NES 511-IEVRVHCQLVRL-522 as well as a class 2-NES 347-CRAGCFVC-354, thelatter coinciding with the CXXXCXXC motif described above [19],(Fig. 1). In conclusion, although CERK has been essentially identifiedas a cytoplasmic protein, identification of NLS as well as NES signals inCERK suggests that shuttling between cytoplasm and nucleus may befunctionally relevant.
2.4. CERK expression and regulation
CERK is ubiquitously expressed, with moderate to high levels inthymus, peripheral blood leukocytes, brain, heart, skeletal muscle,kidney, liver and small intestine [8,11]. CERK mRNA levels were
tent CERK inhibitor in vitro and in cell-based assays. When the benzamide aromatic ringent and, therefore, can serve as a control. B, in vitro assays showing inhibition of CERK byOriginal publication: C. Graf, M. Klumpp, M. Habig, P. Rovina, A. Billich, T. Baumruker,amide kinase inhibitor. Molecular Pharmacology 74 (2008) 925-932.
Fig. 3. CERK and C1P biology. CERK traffics between the trans-Golgi network and the plasma membrane (PM) and, as a result, can phosphorylate ceramide (Cer) delivered to theGolgi complex from de novo synthesis, or Cer released upon sphingomyelinase (SMase) activation. C1P produced by CERK can stimulate cPLA2 alpha (cPLA2α), which is a key pro-inflammatory enzyme responsible for prostanoid synthesis. C1P produced by CERK can also stimulate ERK and PKB, which both play a role in cell proliferation.Whether C1P producedby CERK inside the cell can be released in the extracellular space is unknown. C1P applied exogenously to cultured cells has multiple effects. It is pro-inflammatory by activatingcPLA2α. It has proliferative and pro-survival effects due to inhibition of acidic sphingomyelinase (A-SMase) and serine palmitoyl transferase (SPT) activities, and because it canstimulate phosphoinositide -3 kinase (PI3K) and protein kinase C (PKC)-dependent signalling pathways. The mechanism that senses C1P to evoke these intracellular effects iscurrently unknown, as is the physiological concentration of C1P in the extracellular space. C1P also has pro-migrating activity in macrophages that appear to be mediated through aninhibitory guanylnucleotide binding protein (Gi)-coupled receptor. CERK also regulates the immune response, T-cell polarization in particular, but the precise regulation andwhether it is C1P-driven remains to be clarified. The reported effects of short-chain C1P species, whose biophysical properties cannot be extrapolated to natural C1P species, are notsummarized in this figure. Part of this figure was published already and has been reproduced with permission. Original publication: A. Carré, C. Graf, S. Stora, D. Mechtcheriakova,R. Csonga, N. Urtz, A. Billich, F. Bornancin, Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain. Biochemical and Biophysical ResearchCommunications 324 (2004) 1215-1219.
reported to be high in hematopoietic cells, e.g., in the lymph node,spleen, and thymus [40]. Accordingly, CERK was shown to be highlyexpressed in resting CD4+ and CD8+ T cells as well as in CD19+ B cellsand, remarkably, its expression was strongly reduced followingactivation of these cells, suggesting a possible negative feedbackmechanism [40]. Recent work showed that CERK is down-regulatedfollowing TLR engagement in primary immune cells, which results inpart from a transcriptional repression mechanism targeting theproximal region of the Cerk promoter [41]. However, the identity ofthe repressor remains to be determined. Inhibition of CERK duringinnate cell activation was somewhat unexpected given that cytosolicphospholipase A2 alpha (cPLA2α) which is a known target of C1P(reviewed in 4.2. below), is activated under similar conditions [42].However, this apparent contradiction does not rule out a role for C1Pin cPLA2α regulation in innate cells. Turning off CERK might ensurethat cPLA2α time of residency at the membrane [43] is not prolongedafter activation. Not only CERK, but also glucosylceramide synthase(GCS) and to a lesser extent SM synthase (SMS) appear to beregulated during macrophage homeostasis [44]. In particular, regu-lation of CERK and GCS was found to occur in opposite directionsduring macrophage phenotypic differentiation, thereby evoking a“ceramide anabolic switch” [44]. Regulation of CERK during macro-phage differentiation of the human promyelocytic leukemia cell lineHL-60 was also reported [45].
Recent studies have provided some insights beyond the immunesystem. CERK mRNA levels were down-regulated in rat white adiposetissue in response to central leptin infusion [46]. Another study showedCERK mRNA and protein levels to be reduced during all-trans retinoicacid (ATRA)-induced differentiation of the human neuroblastoma cellline SH-SY5Y [47]. This work identified the responsible elements in theproximal CERK promoter region and showed they are required for theobserved effects. Furthermore, COUP-TF1was identified asan importanttranscription factor in ATRA-induced CERK mRNA inhibition. Anotherstudy identified a functional response element in the first intron ofmCerk, necessary for peroxisome proliferator activated receptor (PPAR)β-dependent regulation of mouse keratinocyte survival [48].
3. Ceramide 1-phosphate (C1P)
3.1. C1P properties
Many studies have illustrated the fusogenic properties of C1P. C1Pwas found to increase the rate and extent of fusion of liposomeswhose composition approached that of the neutrophil plasmamembrane; this was mimicked by treating the liposomes with spiderD-type SMase. The pro-fusogenic effect of C1Pwas detectable when aslittle as 0.45% of the membrane phospholipid composition was C1P[5]. In COS-1 cells stably expressing the FcγRIIA receptor, expression
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of CERK resulted in a higher liquid crystalline order of the membraneat sites of phagocytosis of antibody-coated erythrocytes, therebyfavoring membrane fusion [28].
At the synapse C1P is believed to promote secretion by increasingthe fusibility of vesicle membranes [1]. This is supported by theobservation that addition of C1P to liposomes shifts the rate andextent of their calcium-dependent fusion [5]. A role for CERK/C1P invesicle-driven processes is also supported by their reported ability tostimulate mast cell degranulation [49].
In a recent study based on “back exchange” at the plasmamembrane, of NBD-labeled Cer metabolites, exchange of NBD-C1Poccurred more rapidly and more extensively than for NBD-GC andNBD-SM, suggesting that C1P can stimulate either vesicular transportormembrane fission/fusion events [9]. In linewith these observations,a role for CERK/C1P in vesicle shedding was recently proposed [39].Altogether, C1P clearly appears to play a role in vesicle biology; thisnow awaits further mechanistic insight.
3.2. C1P delivery to cells
The effects of exogenously applied C1P have been analyzed in avariety of studies and shown to depend on C1P chain length, theconcentration used, the vehicle, and the cell line studied. The initialreports, on fibroblasts (Rat1 and Swiss 3T3), showed the propensity ofexogenously added C2-C1P (5 μM), C8-C1P (5 μM), C16-C1P (10 μM)or natural C1P (25 μM), to stimulate DNA synthesis [50–52]. Theconcentrations of C1P, effective to promote cell proliferation/survival,have varied significantly among studies. What looked like discrepan-cies at first glance could in fact be clarified when taking into accountthe solvent used to vehicle C1P, which was not always specified inearlier studies. For instance, concentrations as high as 25 μM C8-C1P,administered by the means of sonicated micelles [53], had growthpromoting effects while 1 μM of the same C1P species was toxic whenadministered with ethanol/dodecane [54]. In this latter study forinstance, C1P showed a prevailing toxic effect on COS-1 fibroblasts, atconcentrations normally used to stimulate cell growth, which wasunexpected. A subsequent study helped clarify this conundrum bycomparing an epithelial cell line (A549) to a fibroblast one (NIH3T3)[55]. This report showed that C1P, prepared in ethanol/dodecane, canstimulate cell growth at concentrations lower than 1 μM. Anti-proliferative effects of C1P showed up above 1 μM in both cell lines,but were limited in A549 (–37% proliferation at 10 µM, IC50N25 μM)compared to NIH-3T3 (–85% proliferation at 10 µM, IC50 ~3-5 μM). Infact, 0.2 μM C1P was sufficient to completely prevent apoptosis ofA549 induced by serum deprivation whereas at 1 μM there were still30% of NIH-3T3 undergoing apoptosis. Thus, the possibility todiscriminate between positive (at low concentration) versus negative(at high concentration) effects of C1P, appears to be cell-linedependent. This may result from differential Cer metabolism steadystates and sensitivity to apoptosis between cell lines. This may alsodepend on the intracellular delivery efficiency of C1P. C1P (10 μM inethanol/dodecane) was shown to have major effect on mitochondria,including signs for mitochondria-ER membrane fusion, in COS-1 cells[54]. Consistently, delivery of C1P to these compartments in A549 cellswas more efficient when done with ethanol/dodecane compared tovesicular delivery [56].
3.3. Measurement of C1P levels
Tandemmass spectrometry with electrospray ionization, combinedwith liquid chromatography (LC ESI-MS/MS) has become the standardfor measurement of sphingolipid levels in biological systems [57].However, it is only very recently that a reliable method for quantifica-tion of C1P has become available. In reports published before 2008, C1Pwas measured after using a strong alkaline treatment to cleaveinterfering glycerolipids. It was then recognized that, if this treatment
is not followed by a neutralization step, SM — which is present in highamounts compared to C1P — can be hydrolyzed into C1P, resulting ingross overestimation of C1P levels [9,58].
Specific LC-MS/MS protocols for C1P measurements have now beenpublished that should enable rapid progress [9,32,59–61]. For example,in themacrophage-like RAW264.7 tumor cell line, C1P levels in the lowpMol/μg DNA range have been reported, therefore similar to S1P levels[60]. However, levels can be expected to differ significantly in primarymacrophages [41]; they may also vary according to the macrophagedifferentiation and activation status [44]. In A549 cells, levels of C16-C1P, C24-C1P andC24:1-C1P have all been shown topartially dependonCERK, in contrast to C18-C1P, the level of which seems completelyindependent of CERK [32]. Getting to knowC1P levels in biologicalfluidsis eagerly awaited as it will impact directly our understanding of C1Pproduction and C1P signalling.
Now thatmeasurement of the complete sphingolipidome appears tobe at hand, one of the key analyses ahead should be that of thesubcellular compartmentalization of these lipids andhow it is regulated.This can be expected to add a new dimension to our understanding ofsphingolipid metabolism.
3.4. The C1P life cycle
The fate of C1P was examined in two pioneering studies pointingto existence of C1P phosphatase activities enriched in liver and brainplasma membrane fractions as well as synaptic vesicles [62,63].Whether there is a specific C1P phosphatase remains to be elucidated.In a recent study of bone marrow derived macrophages, the fate ofC1P after its production by CERK was specifically studied when serumor calcium — both essential for CERK activity — were removed afterinitial production of NBD-labeled C1P. This provided first evidencethat C1P made by CERK is short-lived, thereby satisfying an essentialcriterion for signalling function [9]. In contrast, another study showedthat exogenously added C18.1-C1P is stable once incorporated intocells [56]. Given the broad intracellular distribution of added C18.1-C1P[56] and the fact that this C1P species is not normally made by CERK[32], these findings do not give insight into the fate of the C1P poolspecifically produced by CERK. In fact, the comparative stability of thedifferent pools of C1P made in the cell is still unknown.
Whether natural C1P can be secreted remains to be determined.NBD-labeled C1P could be secreted after its formation in primarymouse macrophages [9]. However, this may be a consequence of theproperties of the NBD moiety, known to increase the exchange rate oflipids between membrane leaflets: once within the external leaflet, itcan be exchanged into the medium. Given the topology of CERK,natural C1P is expected to be found at the cytosolic leaflet of cellularmembranes; occurrence of C1P at the extracellular leaflet has notbeen reported yet. Another study, using 3H-labelled C6-Cer as theprecursor, failed to detect secretion of C1P [64].
4. CERK/C1P signalling
4.1. Growth control
A study on program cell death in plants provided first evidence fora role of CERK in dampening the pro-apoptotic effect of Cer [24]. Acd5mutants whose CERK activity was strongly impaired as a result of apoint mutation in the CC1 domain (see below) displayed increaseddisease following infection by P. syringae. In fact, during pathogenesisin WT plants, mRNA levels of Cerk (Acd5) were sharply increased,suggesting this might represent a mechanism for cell survival afterinfection. It will be interesting to explore if up-regulation of CERK isalso a mechanism used by other living species to fight an infection.Along these lines, the increased susceptibility to S. pneumoniaeobserved in mice lacking CERK [59] might have resulted from theirimpossibility to up-regulate CERK for controlling cell survival.
Two other studies have illustrated further the role of CERK ingrowth control. One study used RNA interference in A549 humanadenocarcinoma cells, to show that CERK is required downstream ofepidermal growth factor (EGF) signalling, for phosphorylation ofprotein kinase B (PKB/Akt) and ERK [55]. Another study showed thatCERK plays a role in PPARβ-dependent control of mouse keratinocytesurvival, based on a comparative analysis between WT and Cerk-deficient keratinocytes [48].
Many reports have characterized C1P signalling by using exogenousaddition of C1P (Fig. 3). Thirty μM natural C1P (sonicated in water)completely protected against macrophage apoptosis induced byremoval of M-CSF [65], which is known to be mediated by increasedproduction of Cer by acidic SMase. C1P competitively inhibited aSMasein macrophage homogenates with a Ki of 14 μM [66]. In the samemacrophagemodel, 30 μMnatural C1P (sonicated in water) was shownto activate nuclear factor-κB (NF-κB) and increase the production ofthe anti-apoptotic regulator Bcl-XL as well as that of the nitric oxideproducing enzyme iNOS, via stimulation of the phosphatidylinositol3-kinase (PI3-K)/PKB pathway [66,67]. Furthermore, the mammaliantarget of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K)pathway was shown to play a major role downstream of PI3-K/PKB[68]. In a study of otic vesicles in culture, C8-C1P at 25 μM (sonicated inwater) could stimulate phosphorylation of PKB/Akt and production ofproliferative cell nuclear antigen (PCNA) [53]. Other downstreamtargets of PKB, e.g., cyclin D1 and c-Myc, were also up-regulated byC1P as well as phosphorylation of ERK1/2 and Janus kinases (JNK),providing additional evidence for a growth-promoting role of C1P [69].Furthermore, protein kinase C-alpha (PKCα) was recently proposed tobe an upstream component involved in the proliferative effect of C1P inmacrophages [70]. In the rat alveolar macrophage line NR8383, whereapoptosis induced by serum withdrawal depends on de novo Cersynthesis, 15 μMnatural C1P (sonicated inwater) could efficiently blockapoptosis by inhibiting serine palmitoyltransferase, the rate limitingenzyme for Cer synthesis; PKB and NF-κB were also activated by C1P inthis macrophage model [71]. Of note, none of these signalling eventstriggered byC1P seem tobe receptor-mediated, in light of the absence ofeffect of pertussis toxin, a known disrupter of G-protein coupledreceptor (GPCR)-dependent Gi protein activation [72].
In conclusion, there may be various pathways to account for C1Peffects on cell growth. These involve either inhibition of enzymesresponsible for Cer production, or efficient conversion of Cer into C1Pby CERK, thus limiting or reversing the formation of laterallysegregated gel-like Cer-enriched domains, which may in turn inhibitpro-apoptotic signalling [73]. An additional mechanism might be viaS1P synthesis, which was shown to be stimulated by C2-C1P [74].However, given the peculiar properties of this short chain C1P analog[36], it remains unknown if this can be extrapolated to natural C1P.
4.2. Inflammation
An important step forward in the field of C1P signalling came withthe study showing that C1Pmediates cytokine signalling in eicosanoidsynthesis in the A549 human lung carcinoma cell line [75]. CERK wasdefined as an upstream regulator of arachidonic acid release regulatedby IL-1β and calcium ionophore. Subsequent work identified cPLA2αas the target of C1P for mediating these effects [76] (Fig. 3). C1P wasshown to increase the affinity of cPLA2α for phosphatidaylcholinevesicles in vitro [77] and to induce translocation of cPLA2α from thecytosol to the Golgi apparatus [76]. An effect of C1P on cPLA2α via aPKC-dependent phosphorylation mechanism was also proposed thatmay be cell-type dependent [78,79]. C1P had no effect on theMichaelis-Menten constant of cPLA2α , but induced a large increasein Vm, suggesting that C1P acts as an allosteric activator of cPLA2α[77]. C1P lowered the EC50 of calcium for cPLA2α by more than 6-fold[76]. C1P was found to bind to the β-groove of the calcium-dependentlipid binding domain of cPLA2α [80]. This site is distinct from that of
PtdIns(4,5)P2, which is a known activator of cPLA2α [77]. There-fore, the current understanding is that C1P, by binding the cationicβ-groove of cPLA2α, activates the enzyme by lowering its membranedissociation. After extensive in vitro characterization, this novelparadigm of cPLA2α translocation and activation was tested in thecell, showing that the C1P/cPLA2α interaction is required forproduction of eicosanoids in embryonic fibroblasts [43]. Furthermore,the formation of lipid droplets, a cellular mechanism known todepend on cPLA2α, was recently shown to depend on CERK [81].However, the understanding of cPLA2α regulation by C1P/CERK is stillincomplete. In particular, there are discrepancies between in vitro andcellular data on the one hand and in vivo data on the other hand. Forinstance, Cerk-deficient mice were not protected in arthritis modelswhere cPLA2α a is known to play a major role [59]. The measurementof reduced PGE2 levels in the bronchoalveolar fluid of Cerk-deficientmice compared to WT has provided preliminary in vivo evidence forthe C1P/cPLA2α activation paradigm [82]. Overriding cPLA2α activa-tionmechanismsmay develop alongwith the inflammatory processesand mask the contribution of C1P. The discrepancies found may alsoreflect distinct predominant pathways for cPLA2α activation indifferent cell types. For instance, the C1P/ cPLA2α paradigm wasestablished and validated using lung epithelial carcinoma cellswhereas little is know to what extent this activation pathwaycontributes to immune cell inflammatory responses. In this respect,there is emerging evidence that regulation of CERK by pro-inflammatory stimuli may be cell-type dependent [44].
4.3. Other functions
There is recent evidence for a PTX-sensitive GPCR-mediated effectof C1P on migration of murine macrophage-like RAW264.7 tumorcells [83]. The receptor identity is still unknown, but S1P does notcompete for binding. Signalling downstream of this putative receptormay go via ERK1/2 towards NF-κB [83]. Therefore, both a receptor-dependent and a receptor-independent pathway (see above) for C1Psignalling may converge to regulate NF-κB activation (Fig. 3).
The regulation of ion channels by C1P has also been reported. Inrat pituitary cells, C2-C1P and C8-C1P were shown to stimulatecalcium entry through voltage-operated calcium channels, in a PKC-dependent manner, by a mechanism that remains to be elucidated[84]. In Jurkat T-cells, C8-C1P activated a store-operated calciumchannel to release calcium from the endoplasmic reticulum intothe cytoplasm, also by a mechanism unknown yet [85]. Conversion ofSM to C1P by a type-D SMase facilitated opening of the voltage-activated potassium channel Kv2.1 whereas conversion to Cer by atype-C SMase inhibited the opening, effects that may be accounted forby differential changes in the mechanical properties of the mem-brane surrounding the voltage sensor of this channel according to thesphingolipid generated [86].
Other effects reported for C1P include inhibition of lecithin-cholesterol acyltransferase, probably by regulating the binding ofphosphatidylcholine to the active site of the enyzme [87]. C1P wasalso reported to be a potent inhibitor of the protein phosphatases PP1and PP2A [75].
5. CERK/C1P biology
5.1. Role in cancer
Microarray data from 1581 tumor samples showed a significanthigher expression of CERK in estrogen receptor (ER)-negative breastcancer tumors compared to ER-positive ones. Within the ER negativegroup, the highest expression of CERK also correlated with the worseprognosis [88].
In the prostatic cancer cell line PC-3, CERK was shown to play acritical role for IL-6 secretion in a methanandamide-induced and
1006 F. Bornancin / Cellular Signalling 23 (2011) 999–1008
permissive way [89]. Reducing IL-6 can be expected to dampen theinflammation milieu with consequences on tumor development.Therefore, based on these findings, defining the role of CERK incannabinoid induced inflammation warrants further study. Theimpact of CERK on the viability of this cell line, however, needs tobe clarified.
A recent work with hepatoma cell lines used optimized irradiationto induce a so-called “hormeotic response” followed by a proteomicanalysis to identify regulated proteins [90]. CERKwas amongst the up-regulated proteins identified. Knock-down of CERK in the twohepatoma cell lines used (Mahlavu and HepG3) significantlyincreased the susceptibility to apoptosis induced by UV irradiation,even at low dose. Therefore, CERK may act as an apoptosis-evasionprotein in hepatoma cell lines.
Activation of apoptosis is a key mechanism involved in tumor cellkilling by current anti-cancer therapies and when it is defective thisleads to therapeutic resistance. Inhibiting CERK might enhanceefficacy of anti-cancer therapies e.g., cytotoxic drugs, radiation andimmunotherapy. This hypothesis was tested in a recent cell-basedmechanistic study using a combination of tamoxifen and NVP-231 forincreasing Cer levels [34].
In conclusion, there is emerging evidence associating CERKexpression and cancer, reminiscent of what has already beenextensively shown for SPHK1. Probably CERK expression will impacton the so called S1P/Cer rheostat both because CERK regulates Cerlevels and because it produces C1P which appears to have signallingproperties on its own, distinct but somewhat overlapping those of S1P(Fig. 4).
5.2. Role in the nervous system
CERK was originally identified as an activity that co-purified with ratbrain synaptic vesicle preparations, leading to propose that phosphory-lation of Cer may be associated with neurotransmitter release [1]. Sincethen, C1P has indeed been shown to act as a fusogenic lipid (describedabove) and some reports have suggested its function in neurotransmitterrelease. For example, sub-micromolar concentrations of C8-C1P, addedexogenously to rat adrenal pheochromocytoma (PC12) cells couldstimulate dopamine release [91].
Drosophila CERK was recently shown to be important for photore-ceptor homeostasis. Dcerk mutants with strongly repressed dCERKexpression were unresponsive to light and displayed severely down-regulated NORPA, a phospholipase C (PLC) β homolog. This was shownto be the result of increased Cer levels in dcerk mutant flies. IncreasedCer impacted PtdIns(4,5)P2 partitioning into the membrane, therebypreventing PLC association and leading to its degradation [92]. Targeteddisruption of mouse CERK was also shown to result in increased retinalCer levels [13]. Whether this predisposes mouse photoreceptor cells to
S1P Cer CERKC1PS1P
Cer
Fig. 4. CERK and the sphingolipid rheostat. S1P and Cer have opposite effects on cellproliferation and survival. If CERK is expressed and activated, it may impact the S1P/Cerrheostat because C1P is produced, which has proliferative/pro-survival effects, andbecause Cer itself is used up. The extent of depletion of Cer levels may be influenced bythe turnover rate of C1P.
activity-dependent degeneration, as it does in drosophila, warrantsfurther study.
CERK activity is high in the brain [8,11,30] and, particularly, inthe cerebellum [30]. Metabolic studies in differentiated cerebellargranule cells showed that Cer produced from SM hydrolysis at theplasma membrane is efficiently phosphorylated into C1P [7]. Recentfindings showed that the Purkinje cell layer may be even moreenriched in CERK than granule neurones [93]. Purkinje cells have arole in motor coordination as well as learning related behaviours.Absence of CERK did not perturb coordination and motor skillacquisition as revealed in a study comparing WT and Cerk-deficientmice [93]. In contrast, mice lacking CERK displayed abnormalemotional behaviour. These findings suggest that CERK may play arole in higher brain functions.
5.3. Role in the immune system
In COS-1 cells stably expressing the FcγRIIA receptor, expressionof CERK resulted in an increased phagocytic index together withincreased CERK activity which translocated to lipid rafts afterchallenge with antibody-coated erythrocytes; this did not occurwhen the catalytically-deficient G198D CERK mutant was usedinstead of WT CERK [28]. Furthermore, in lipid rafts, CERK wasshown to co-localize with the transient receptor channel 1 proteinand to promote periphagosomal calcium signalling [94]. In highlydifferentiated neutrophils, metabolic labeling studies have shownthere is little conversion of Cer into SM and almost no glycolipidformation [95]; in contrast, conversion to C1P is important [64].Similar data were obtained by employing NBD-labeled Cer as asubstrate [34]. High levels of CERK activity were reported in freshlyisolated human and mouse neutrophils. CERK was hardly stimulatedby fMLP alone [5,64] but priming with fMLP enabled potent CERKstimulation by opsonized erythrocytes [5]. By contrast, both non-opsonized and opsonized zymosan particles down-regulated CERKactivity in human neutrophils [44].
The participation of CERK and C1P to mast cell biology wasproposed based on experiments that analyzed the degranulationprocess in the rat basophilic leukemia line RBL-2H3 [49] . However,these findings could not be reproduced in primary mouse mast cells.Also, mast cells isolated from Cerk-deficient mice performed similarlyto wild-type when analyzed for mast cell activation parametersincluding degranulation [59]. In fact, a recent study has suggested thatCERK might modulate mild and chronic forms of mast cell activationrather than potent and acute forms [96]. This keeps in with thediscussion above on the regulation of cPLA2α by CERK/C1P.
In vivo-modeling studies of inflammatory responses inCerk-deficientmice have started providing somehints on the possible role of CERK, butthe current understanding is still very limited. InCerk-deficientmice themost striking feature was the reduced neutrophil numbers in blood andspleen compartments [59]. Neutropenia didnot seem tooccur as a resultof neutrophil sequestration or decreased bone marrow production. Inlight of the increased Cer levels resulting from CERK ablation [59],neutropeniawashypothesized to proceed through increasedneutrophilapoptosis, which in turn would explain why Cerk-deficient animalsfailed to survive an infection with S. pneumoniae. However, the reasonsfor the increased mortality are not completely understood. Besides acompromised neutropenic status in Cerk-deficient animals, the immuneresponse to S. pneumoniae may have failed due to poor control ofinflammatory responses. The latter hypothesis has gained support in thelight of recent findings that showed a role for CERK in the production ofTH2 cytokines [82]. If CERK polarizes towards TH2 responses, its absencemay, on the contrary, shift the response in the other direction (TH1).Because this absence is “permanent” in knock-out animals, there is nodynamic feedback mechanism whereby CERK would first be down-regulated and then be re-synthesized again to allow for a controlledresponse.
The first decade of ceramide kinase (CERK) has witnessedsignificant progress at biochemical and cellular levels. In addition,preliminary notions have emerged how CERK may regulate cellfunction. CERK is clearly implicated in vesicular biology and, as such,CERK appears to be strictly controlled in differentiated cells foradjusting biological responses. The interaction of C1P with cytosolicphospholipase A2 alpha has been analyzed in detail, paving the wayfor the study of novel C1P-interacting proteins that are yet to beidentified. A number of signalling intermediates have been reportedto mediate the extracellular action of ceramide 1-phosphate (C1P). Akey point to be elucidated is the primary mechanism used by C1P foreliciting these effects. C1Pmight act as an allostericmodulator capableof modifying the membrane where it is generated or inserted in and,thereby, be able to regulate membrane bound or embedded proteins.Whether C1P is also able to act as a genuine primary signal and bind tospecific receptors at the membrane, akin to sphingosine 1-phosphate(S1P) binding to S1P receptors, remains to be established. Gettingfurther insight will require measurement of C1P levels both in cellsand in biological fluids. Such data will also be valuable to obtain in thecontext of CERK-deficiency, to help elucidate the alternative C1P-generating activities which have remained elusive. Finally, no specificC1P phosphatase has been reported yet, in contrast to the known S1Pspecific phosphatases and the S1P lyase that control S1P levels.Because C1P produced by CERK appears to be a short lived species, thisimplies a control mechanism that remains to be discovered.
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