Sonya Bader

Arjan Kortholt

Peter J M Van Haastert
email: van.haastert@rug.nl

SEVEN DICTYOSTELIUM PHOSPHODIESTERASES DEGRADE THREE POOLS OF cAMP AND cGMP

Keywords: cAMP, cGMP, PDE, Dictyostelium discoideum, chemotaxis, development DTT, dithiothreitol; IBMX, 3-Isobutyl-

The Dictyostelium genome uncovers seven cyclic nucleotide phosphodiesterases, of which six have been characterized previously and the seventh is characterized here. Three enzymes belong to the ubiquitous class I common in all eukaryotes, while four enzymes belong to the rare class II PDEs that are present in bacteria and lower eukaryotes. Since all Dictyostelium PDEs are now characterized we have calculated the contribution of each enzyme in the degradation of the three important pools of cyclic nucleotides: 1) extracellular cAMP that induces chemotaxis during aggregation and differentiation in slugs, 2) intracellular cAMP that mediates development, and 3) intracellular cGMP that mediates chemotaxis. It appears that each cyclic nucleotide pool is degraded by a combination of enzymes that have different affinities , allowing a broad range of substrate concentrations to be degraded with first order kinetics. Extracellular cAMP is degraded predominantly by the class II high affinity enzyme DdPDE1 and its close homolog DdPDE7, and in the multicellular stage also by the low affinity transmembrane class I enzyme DdPDE4. Intracellular cAMP is degraded by the DdPDE2, a class I enzyme regulated by histidin kinase/phosphorelay, and by the cAMP/cGMP-stimulated class II DdPDE6. Finally, basal intracellular cGMP is degraded predominantly by the high affinity class I DdPDE3, while the elevate d cGMP levels that arise after receptor stimulation are degraded predominantly by a cGMP-stimulated cGMPspecific class II DdPDE5. The analysis shows that the combination of enzymes is tuned to keep the concentration and lifetime of substrate within a functional range.

INTRODUCTION

The cyclic nucleotides cAMP and cGMP play a central role in diverse signal transduction processes in Dictyostelium discoideum. cAMP mediates chemotaxis during cell aggregation, and controls gene expression during development. cGMP regulates cytoskeletal organization affecting shape, stability and motility of single cells. The intracellular concentration of cAMP and cGMP is determined by the combined action of production and removal. Production depends on the enzymatic activity of the adenylyl and guanylyl cyclases to form cAMP and cGMP, respectively. Removal of intracellular cAMP or cGMP depends on the activity of phosphodiesterases (PDE) that hydrolyze cAMP and cGMP, and on the ability of Dictyostelium discoideum cells to secrete cAMP. This extrusion mechanism is pivotal in the formation of extracellular cAMP waves, which is the chemotactic signal for cell directed movement during aggregation, mound formation and slug migration.

In Dictyostelium discoideum cAMP is produced by three adenylyl cyclases (see [START_REF] Saran | Adenylyl Cyclase G Is Activated By an Intramolecular Osmosensor[END_REF]:

ACA is a transmembrane enzyme that is regulated by G-protein coupled receptors [START_REF] Pitt | Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development[END_REF], ACB encoded by the acrA gene [3], is a soluble adenylyl cyc lase with high basal activity [START_REF] Meima | Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation[END_REF], while ACG is an osmosensitive adenylyl cyclase [START_REF] Saran | Adenylyl Cyclase G Is Activated By an Intramolecular Osmosensor[END_REF]. cGMP synthesis is mediated by two guanylyl cyclases, the transmembrane GCA and the soluble sGC that are both regulated by G-protein coupled receptor pathways (see [START_REF] Roelofs | Characterization of two unusual guanylyl cyclases from dictyostelium[END_REF]). For the degradation of cAMP and cGMP six different phosphodiesterases have been reported and characterized in Dictyostelium discoideum. These PDEs represent two classes that exhibit distinct differences in the amino acid sequence of the putative catalytic domains, namely class I, which is ubiquitous in mammals, and class II that predominantly occurs in bacteria and lower eukaryotes (see Figure 1).

We will follow the nomenclature of Dictyostelium PDEs that has been proposed by Bosgraaf et al [START_REF] Bosgraaf | Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium[END_REF] and will give original or alternative names on first mentioning. DdPDE1 (PdsA) is a class II dual specificity PDE that degrades extracellular cAMP and cGMP. The enzyme can be secreted in the medium, or exposed on the cell surface. Cells without DdPDE1 (pdsA-null strain UK7) reveal that DdPDE1 is the main PDE that degrades extracellular cAMP during cell aggregation and is thereby essential for shaping cAMP waves. UK7 cells fail to aggregate, but can be rescued by adding exogenous phosphodiesterase activity [START_REF] Barra | pdsA, a gene involved in the production of active phosphodiesterase during starvation of Dictyostelium discoideum amoebae[END_REF][START_REF] Faure | Rescue of a Dic tyostelium discoideum mutant defective in cyclic nucleotide phosphodiesterase[END_REF][START_REF] Faure | The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development[END_REF][START_REF] Franke | The expression of two transcripts of the phosphodiesterase gene during the development of Dictyostelium discoideum[END_REF][START_REF] Lacombe | Molecular cloning and developmental expression of the cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum[END_REF][START_REF] Podgorski | The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum utilizes alternate promoters and splicing for the synthesis of multiple mRNAs[END_REF][START_REF] Sucgang | Null mutations of the Dictyostelium cyclic nucleotide phosphodiesterase gene block chemotactic cell movement in developing aggregates[END_REF][START_REF] Weening | Contrasting activities of the aggregative and late PDSA promoters in Dictyostelium development[END_REF][START_REF] Wu | The phosphodiesterase secreted by prestalk cells is necessary for Dictyostelium morphogenesis[END_REF][START_REF] Hall | The role of the cyclic nucleotide phosphodiesterase of Dictyostelium discoideum during growth, aggregation, and morphogenesis: overexpression and localization studies with the separate promoters of the pde[END_REF][START_REF] Yeh | Independent regulation of the extracellular cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation by exogenous cyclic AMP in Dictyostelium discoideum[END_REF]. DdPDE2 (RegA) encodes a cAMP-specific PDE localized in the cytosol of the cell where it degrades intracellular cAMP. The protein harbors a class I PDE catalytic domain and a response regulator domain, which is phosphorylated and activated by histidine kinases via the phospho-relay protein RdeA [START_REF] Thomason | The RdeA-RegA system, a eukaryotic phospho-relay controlling cAMP breakdown[END_REF]. The DdPDE2 knock-out cell strain regA - develops into very small aggregates and shows defects in spore formation. Additionally DdPDE2 has been implicated in suppression of pseudopodium formation during chemotaxis [START_REF] Meima | Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation[END_REF][START_REF] Kim | A novel adenylyl cyclase detected in rapidly developing mutants of Dictyostelium[END_REF][START_REF] Laub | A molecular network that produces spontaneous oscillations in excitable cells of Dictyostelium[END_REF][START_REF] Loomis | Histidine kinases in signal transduction pathways of eukaryotes[END_REF][START_REF] Mohanty | Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium[END_REF][START_REF] Shaulsky | Developmental signal transduction pathways uncovered by genetic suppressors[END_REF][START_REF] Wang | A two-component histidine kinase gene that functions in Dictyostelium development[END_REF][25][26]. DdPDE3 is a cGMP-specific class I PDE that consists of a catalytic domain without regulatory domains, and is constitutively active. The ddpde3 -null cell strain shows a moderate phenotype with increased basal cGMP levels, but only a small effect on cAMPstimulated cGMP levels compared to wild-type cells. [START_REF] Kuwayama | Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium[END_REF]. DdPDE4 is a type I cAMPspecific enzyme with two transmembrane segments and an extracellular catalytic domain.

The protein is expressed mainly in the multicellular stage where it is involved in the degradation of extracellular cAMP. The ddpde4 -null strain shows normal cell aggregation, but is defective in multicellular development [START_REF] Bader | DdPDE4, a Novel cAMP-specific Phosphodiesterase at the Surface of Dictyostelium Cells[END_REF]. DdPDE5 (GbpA or PDED) is characterized as a cGMP-stimulated cGMP-specific class II PDE. The ddpde5 -cell strain exhibits a phenotype with strongly elevated levels of cGMP, both basal levels and especially after stimulation with cAMP. The biochemical phenotype of this ddpde5 -null cell is similar to what is observed in the mutant NP368, a Streamer F cell strain [START_REF] Bosgraaf | Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium[END_REF][START_REF] Saran | cAMP signaling in Dictyostelium. Complexity of cAMP synthesis, degradation and detection[END_REF][START_REF] Goldberg | Identification of four candidate cGMP targets in Dictyostelium[END_REF]. Finally, DdPDE6 (GbpB or PDEE) is a dual specificity class II PDE that degrades predominantly intracellular cAMP and to a minor extend cGMP [START_REF] Bosgraaf | Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium[END_REF][START_REF] Goldberg | Identification of four candidate cGMP targets in Dictyostelium[END_REF][START_REF] Meima | Characterization of a cAMPstimulated cAMP phosphodiesterase in Dictyostelium discoideum[END_REF][START_REF] Bosgraaf | A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium[END_REF].

As mentioned above, Dictyostelium cells possess two cyclic nucleotide phosphodiesterases on their cell surface, the cAMP/cGMP-nonspecific DdPDE1 and the cAMP-specific DdPDE4. In an attempt to characterize DdPDE4 we used the cell line UK7 which has a deletion of the gene encoding DdPDE1. As reported, UK7 cells have very low cell surface phosphodiesterase activity [START_REF] Sucgang | Null mutations of the Dictyostelium cyclic nucleotide phosphodiesterase gene block chemotactic cell movement in developing aggregates[END_REF], but surprisingly we observed that the majority of this residual activity is not DdPDE4, but resembles DdPDE1 in respect to catalytic properties and sensitivity to inhibitors. Upon completion of the Dictyostelium genome-sequencing project, we recognized the presence of a new PDE, that we name DdPDE7 for its order of identification. Here we demonstrate that DdPDE7 is a close homologue of DdPDE1 in respect to amino acid sequence and enzymatic properties, and is responsible for the residual activity in UK7 cells .

To understand the function of phosphodiesterases in the regulation of intracellular and extracellular cAMP and cGMP levels it is essential to be confident that all PDEs in Dictyostelium have been identified and characterized. Extensive searches of the now completed Dictyostelium genome for type I or type II PDEs do not provide evidence for additional cyclic nucleotide phosphodiesterases. Searches for homologues of bacterial type III PDEs [START_REF] Richter | 5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism[END_REF] have not yielded any potential candidate for this type of phosphodiesterase in Dictyostelium, suggesting that most likely all cyclic nucleotide phosphodiesterases have now been characterized in Dictyostelium. We have used the kinetic properties of all enzymes to determine the contribution of each enzyme in the degradation of the three pools of cyclic nucleotides.

EXPERIMENTAL PROCEDURES

Construction of the over expressor

To express the full length protein, a DNA fragment was amplified by PCR using the primers PDE7F: GAAGATCTAAAATGAAATATTTAATTTTAATTTTAATATTTTTTATTG and PDE7R: GAACTAGTTAAACAAATTGGTACACCTTGGTTTG. The BglII/SpeI fragment was cloned in the BglII-SpeI site of a modified MB74GFP vector that contained a hygromycin-instead of a neomycin-resistance cassette [START_REF] Bosgraaf | RasGEF-containing proteins GbpC and GbpD have differential effects on cell polarity and chemotaxis in Dictyostelium[END_REF], resulting in the plasmid pDM115-PDE7-GFP. Finally UK7 Dictyostelium cells [START_REF] Sucgang | Null mutations of the Dictyostelium cyclic nucleotide phosphodiesterase gene block chemotactic cell movement in developing aggregates[END_REF] were transfected with 5 µg plasmid. Hygromicin-resistant cells were analyzed; we will refer to this over expressor as DdPDE7 OE .

Cluster analysis

Multiple sequence alignments were constructed using the CLUSTAL W program [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF], followed by manual optimization. Distance matrices were constructed from the alignments with the PROTDIST program of the PHYLIP package, which uses the Dayhoff PAM 001 matrix for the calculation of evolutionary distances (Phylip 3.5, J. Felsenstein, 1993 [36]). Phylogenetic trees were generated by using the FITCH program of the PHYLIP package, with 1000 bootstrap replications to assess the reliability of the nodes.

Culture and incubation conditions

AX3, UK7, UK7/regA -and DdPDE7 OE strains were grown on HG5 medium (14.3 gr/l Oxoid Pepton, 7.15 gr/l Yeast extract, 1.36 gr/l Na 2 HPO 4 .12H 2 O, 0.49 gr/l KH 2 PO 4 , 10.0 gr/l glucose) either on 9 cm dish or in shaking culture at 22 o C. The over expressor DdPDE7 OE strain was grown in HG5 medium in the presence of 10 µg /ml Hygromicin B.UK7/regA - cells were kindly provided by David Trayner and Rob Kay; the strain (HM1020) was made by disruption of the regA gene in UK7 cells using the vector as described for generating regA - cells [START_REF] Thomason | An intersection of the cAMP/PKA and two-component signal transduction systems in Dictyostelium[END_REF].

Phosphodiesterase assays

Dictyostelium cells were starved in 10 mM KH 2 PO 4 /Na 2 HPO 4 , pH 6.5 (PB) for 5 hours at a density of 10 7 cells/ml. Cells were centrifuged for 3 min at 300 g, and the supernatant was saved as medium. The cells were washed with PDE lysis buffer (20 mM HEPES/NaOH, pH 7.0, 1 mM EGTA) resuspended in PDE lysis buffer at a density of 10 8 cells/ml. The PDE assay mixture (final concentrations) contained 20 mM HEPES/NaOH, pH 7.0, 1 mM EGTA, 5 mM MgCl 2 , 10 nM [ 3 H]cAMP or 10 nM [ 3 H]cGMP as substrate, and 30 µl of cells or extracellular medium in a total volume of 100 µl. The assay mixture was incubated at room temperature for 30 minutes and the reaction was terminated by boiling for 1 minute. The product was dephosphorylated by calf intestine phosphatase (1 unit of enzyme in 50 µl of CIP buffer incubated for 1 hour at 37 °C). Finally, 150 µl of DOWEX AG1X2 was added to remove remaining substrate. After 10 minutes of incubation under regular mixing, samples were centrifuged for 2 minutes at 14000 g, and the radioactivity in 200 µl of the supernatant was determined.

Calculation of degradation of three cyclic nucleotide pools

Using the measured kinetic constants of cAMP and cGMP degradation by the seven

The equations of the enzymes with enzymatic constants of table I are:

The allosteric enzymes PDE1and PDE7:

n n n MAX S K S V v + = 0.5
The Michaelis-Menten enzymes PDE2, PDE3 and PDE4:

S K S V v M MAX + =
The substrate-activated enzymes PDE5 and PDE6:

      + + + + + = S K S S K K S K S S K S V v L M A A H M A

MAX

In these equations MAX V is the maximal velocity of the enzyme, S is the substrate concentration, M K is the Michaelis-Menten constant, The half life of the substrate was calculated with α v S t 6 . 0 5 .

=

, where t 0.5 is the half life of substrate in minutes, S is the substrate concentration in µM, v is the calculated rate of degradation in pmol/min/10 7 cells, and α is the volume of 10 7 cells in µl. The proportionality constant 0.6 is the average between ln2 (0.69, for first order degradation at S<<Km) and 0.5 (for zero order degradation at S>>Km). The cell density during aggregation is 10 8 cells/ml yielding α = 100 µl for cell surface enzymes during aggregation. The volume of a cell is about 630 um 3 [START_REF] Klein | Kinetics of fluid-phase pinocytosis in Dictyostelium discoideum amoebae[END_REF] yielding α = 6.3 µl for cytosolic enzymes. We assume that in slugs the volume between cells is in the same order as the volume of the smaller cells (α = 5 µl)

RESULTS

Deduced amino acid sequence of DdPDE7

The deduced DdPDE7 protein consists of 425 amino acids, of which the first 17 amino acids are predicted to form a signal peptide with probable cleavage between amino acids 17 and 18

(Figure 1A and2). The predicted catalytic domain starts at amino acid 22 till the C-terminus and belongs to the group of class II phosphodiesterases that are present in lower eukaryotes and bacteria, but not in higher eukaryotes. The homologous DdPDE1 is somewhat larger (452 amino acids), mainly at the N-terminus. The deduced amino acid sequence of DdPDE1 is also predicted to contain a signal sequence with probable cleavage site between amino acids 23 and 24, and a catalytic domain that starts at amino acid 52. The catalytic domains of DdPDE1 and DdPDE7 share 62% amino acid identity and 78% identical or conserved amino acids (Figure 2).

To further characterize the homology between DdPDE1 and DdPDE7, we investigated the genomic organization of the respective genes [39] at http://dictybase.org/.

DdPDE7 is encoded by BC5V2_0_00956 on chromosome 5, whereas DdPDE1 is encoded by the pdsA gene on chromosome 4. DdPDE7 lies two genes downstream of DdPDE4 in the opposite orientation. DdPDE7 has no introns in the coding region, whereas PDE1 has one intron in the coding region and two introns in non-coding regions. The upstream region of DdPDE7 containing the putative promoter region does not show a clear resemblance to the complex promoter region of PDE1 (data not shown).

Homology and cluster analysis of the catalytic domain

DdPDE7 belongs to the group of class II cyclic nucleotide phosphodiesterases, which are present in some bacteria and lower eukaryotes. Dictyostelium DdPDE1 also belongs to this class, as well as DdPDE5 and DdPDE6, although the catalytic domains of DdPDE5 and DdPDE6are shorter and resemble metallo-beta-lactamases. We made a multiple sequence alignment of the catalytic domains of these four Dictyostelium PDEs together with class II PDEs from other lower eukaryotes and bacteria, and performed a phylogenetic/protein cluster analysis (Figure 1B). The result demonstrates at least two groups of Type II cyclic nucleotide phosphodiesterases (bootstrap 95%): the originally defined type II phosphodiesterases [40],

and a group resembling metallo-beta-lactamases. Based on a bootstrap value of 94%, the type II phosphodiesterases may consist of two sub groups, one subgroup with DdPDE1, DdPDE7 and presently four enzymes from a few prokaryotes (all belonging to the class of gammaproteobacteria), and a subgroup of about ten enzymes from eukaryotes (all belonging to the division Ascomycota of the Fungi kingdom). Interestingly, the proteins of the metallobeta-lactamase type II phosphodiesterases all harbor a metallo-beta-lactamase domain and one or usually two cyclic nucleotide binding domains. Sequences are found in a few prokaryotes, and in the eukaryotes Tetrahymena thermophila and Dictyostelium discoideum.

For Dictyostelium discoideum DdPDE5 and DdPDE6, it has been demonstrated that the metallo-beta-lactamase domain possesses phosphodiesterase activity, which is activated by cAMP or cGMP binding to the cyc lic nucleotide binding domains [START_REF] Meima | Characterization of a cAMPstimulated cAMP phosphodiesterase in Dictyostelium discoideum[END_REF].

Catalytic activity of DdPDE7

Previously, only two phosphodiesterases, DdPDE1 and DdPDE4, were known in Dictyostelium that have their catalytic activity exposed on the cell surface (see [START_REF] Bader | DdPDE4, a Novel cAMP-specific Phosphodiesterase at the Surface of Dictyostelium Cells[END_REF]). With the recognition of DdPDE7 as a close homologue of DdPDE1, we have carefully analyzed our older data on the residual phosphodiesterase activity of the ddpde1-null cell line UK7.

DdPDE1 is a dual cAMP/cGMP phosphodiesterase that is strongly inhibited by DTT and insensitive to IBMX, whereas DdPDE4 is insensitive to DTT but inhibited by IBMX.

Five hour starved wild type cells have a very active phosphodiesterase activity on their surface (3000 fmol/min/10 7 cells at 10 nM cAMP), which is essentially completely inhibited by 10 mM DTT and insensitive to IBMX (Figure 3A). UK7 cells have a 50-fold reduction of surface phosphodiesterase activity (60 fmol/min/10 7 cells), implying that DdPDE1 contributes by far the majority of PDE activity on the surface of 5h starved Dictyostelium cells, as was discussed previously [START_REF] Sucgang | Null mutations of the Dictyostelium cyclic nucleotide phosphodiesterase gene block chemotactic cell movement in developing aggregates[END_REF]. The residual activity on UK7 cells is Copyright 2006 Biochemical Society inhibited only slightly by IBMX; the 15% inhibition (9 fmol/min/10 7 cells) most likely represents the IBMX-sensitive DdPDE4 that is known to be expressed at these low levels in 5h starved cells [START_REF] Bader | DdPDE4, a Novel cAMP-specific Phosphodiesterase at the Surface of Dictyostelium Cells[END_REF]. The residual activity of UK7 cells is inhibited 90% by DTT, suggesting that UK7 cells may express a homologue of DdPDE1.

To demonstrate that the DTT sensitive activity of UK7 cells is attributed to DdPDE7, we over expressed the ddpde7 gene in UK7 cells (Figure 3B). UK7 cells express the DTTsensitive phosphodiesterase activity on the cell surface and secreted in the medium in a ratio of 15:85. Over expression of DdPDE7 leads to a 2.4-fold increase of PDE activity, both in the medium and expressed on the surface at the same ratio as in control cells. The enhanced activity of UK7/DdPDE7 OE cells hydrolyzes both cAMP and cGMP, is sensitive to inhibition by DTT and not by IBMX (data not shown). These properties of UK7/DdPDE7 OE cells are essentially identical to those of UK7 cells. We conclude that DdPDE7 is the major phosphodiesterase activity of 5 h starved UK7 cells.

Kinetics of DdPDE7

Phosphodiesterase activity was measured with different concentrations of cAMP or cGMP using intact UK7 cells. The Eady-Hofstee plot (Figure 4) reveals a slight negative cooperativity as has been observed for DdPDE1 (see [START_REF] Van Haastert | Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum[END_REF] for kinetic data of DdPDE1). The inset shows a Hill plot, yielding a Hill coefficient of 0.79 for DdPDE7, compared to 0.8 for DdPDE1 [START_REF] Van Haastert | Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum[END_REF]. At subsaturating concentrations of 10 nM, UK7 cells hydrolyze cAMP about 1.6-fold faster than cGMP, indicating that DdPDE7 is a dual specificity enzyme, as has also been observed for DdPDE1. However, the affinity of DdPDE7 for cAMP is much lower than that of DdPDE1 (the apparent K M is 0.8 µM cAMP for DdPDE1, and 12.5 µM for DdPDE7).

Both DdPDE1 and DdPDE7 have a 2-3-fold lower affinity for cGMP compared to cAMP. In contrast, DdPDE7 appears to have a 2.6-fold higher V MAX for cGMP compared to cAMP, while DdPDE1 has a 1.5-fold lower V MAX for cGMP than for cAMP. Thus, both DdPDE1 and DdPDE7 are dual cAMP/cGMP specificity phosphodiesterases with negative cooperativity. However, the kinetic constants are very different for DdPDE1 and DdPDE7, especially the 15-fold lower cAMP affinity of DdPDE7 compared to DdPDE1.

DdPDE1, DdPDE4 and DdPDE7 activity during development

Extracellular cAMP is degraded by the IBMX-sensitive DdPDE4 and DTT-sensitive DdPDE1 and DdPDE7. We have previously determined total cell surface cAMP-PDE activity during development, and the proportion of this activity that is inhibited by IBMX and DTT [START_REF] Bader | DdPDE4, a Novel cAMP-specific Phosphodiesterase at the Surface of Dictyostelium Cells[END_REF]. The IBMX-sensitive activity is attributed to DdPDE4, while the DTT-sensitive activity is caused by DdPDE1 and DdPDE7. Since the latter two enzymes have nearly identical properties it is difficult to determine the activities of DdPDE1 and DdPDE7 during development of wild type cells. We used UK7/regA -cells that have deletions in both DdPDE1 and DdPDE2/RegA; due to the deletion of DdPDE1 all DTT-sensitive activity is due to DdPDE7, while deletion of DdPDE2 allows the aggregation-defective UK7 cell to develop up to the stage of tight aggregates with tip. We measured the hydrolysis of

Contribution of seven PDEs in the degradation of cAMP and cGMP pools.

In Dictyostelium three cyclic nucleotide pools are pivotal for the organism. Extracellular cAMP induces chemotaxis and differentiation, which are mediated by intracellular cGMP and cAMP, respectively. Since all Dictyostelium PDEs are now characterized we may calculate the contribution of each enzyme in the degradation of these three important pools of cyclic nucleotides. We assume that at different developmental stages each enzyme is expressed at different levels, but that the intrinsic kinetic properties do not change. This assumption implies that the sensitivity to inhibitors, substrate specificity, K M , K A and Hill coefficient are constant, while the V MAX is different depending on the developmental stages. First, we obtained the intrinsic kinetic constants K M , K A and Hill coefficient of all enzymes from Copyright 2006 Biochemical Society present and published data (see table I for data and references). Then, we used the location of the enzyme, the substrate specificity and the sensitivity to inhibitors to derive specific assay conditions to determine the V MAX for each enzyme (table II). These experiments were performed in wild type cells, except for DdPDE7, for which we have to rely on the measurements in UK7/regA -null cells, because in wild type cells we can not discriminate between DdPDE1 and DdPDE7. Subsequently, we used the observed values of K M , V MAX , and cooperativity (table I) to calculate the activity of each enzyme at different substrate concentrations (Figure 6). In this analysis we also calculated the half-life of cAMP or cGMP degradation at different substrate concentrations. To evaluate the outcome of these calculations, we used published data to estimate the cyclic nucleotide concentration of these three pools in vivo (indicated by the arrows in figure 6).

Extracellular cAMP is degraded by DdPDE1, DdPDE4 and DdPDE7. During cell aggregation, extracellular cAMP waves are propagated each 5 minutes with concentrations that oscillate between ~1 nM and ~1 µM [START_REF] Tomc Hik | Adenosine 3',5'-monophosphate waves in Dictyostelium discoideum: a demonstration by isotope dilution--fluorography[END_REF]. The half-life of 10 nM cAMP in a suspension of 10 8 cells/ml is calculated to be about 10s, as was observed experimentally [START_REF] Van Haastert | Excitation adaptation and deadaptation of the cAMP mediated cGMP response in Dictyostelium discoideum[END_REF]. The degradation of 10 nM cAMP is mediated largely by DdPDE1 (~97%), and only slightly by DdPDE4 (~0.3%) and DdPDE7 (~2.6%). At substrate concentrations above 1 µM the halflife of cAMP increases sharply, because the activity of DdPDE1 with its K M of 0.8 µM becomes saturated. The degradation of 1 µM cAMP is still mediated predominantly by DdPDE1 (~93%), and to some extent by DdPDE4 (~2%) and DdPDE7 (~5%).

In the multicellular stage the half-life of cAMP is calculated to be much shorter than during cell aggregation, because the cell density is much higher and the PDE activity per cell is only slightly lower than during aggregation. Degradation of low cAMP concentrations is again mainly mediated by DdPDE1 (~94%), while DdPDE4 (~5%) and DdPDE7 (~1%) have less contribution. Due to the small intercellular space the cAMP concentration in slugs may easily reach a concentration of 10 or 100 µM [START_REF] Saran | cAMP signaling in Dictyostelium. Complexity of cAMP synthesis, degradation and detection[END_REF][START_REF] Abe | A new class of rapidly developing mutants in Dictyostelium discoideum: Implications for cyclic AMP metabolism and cell differentiation[END_REF][START_REF] Merkle | Localization and levels of cyclic AMP during development of Dictyostelium discoideum[END_REF]. At these high cAMP concentrations DdPDE1 is completely saturated and degradation also depends on the enzymes with a lower affinity for cAMP, such as DdPDE4 and DdPDE7. Since DdPDE4, but not DdPDE7, is expressed at relatively high levels in slugs compared to single cells, DdPDE4 has a significant contribution of ~58% in the degradation of 10 µM cAMP in slugs. DdPDE7 contributes only ~2% and DdPDE1 the remaining 40%.

Intracellular cAMP is a second messenger that transduces the extracellular cAMP signal to induce development. After stimulation, intracellular cAMP may reach a concentration of 5 µM (25 pmol/10 7 cells [START_REF] Roos | Adenylate cyclase activity oscillations as signals for cell aggregation in Dictyostelium discoideum[END_REF][START_REF] Van Haastert | A method for studying cAMP-relay in Dictyostelium discoideum -the effect of temperature on cAMP-relay[END_REF]). Intracellular cAMP is degraded by the high affinity DdPDE2 and the low affinity DdPDE6. At low cAMP concentrations the halflife of cAMP is ~15s, and cAMP is degraded by both DdPDE2 (~40%) and DdPDE6 (~60%).

At a cAMP concentration of 10 µM, which is just above the maximal intracellular cAMP concentration, the low affinity DdPDE6 becomes more important and degrades about 85% of cAMP. At this high cAMP concentration, the half-life of cAMP is still only 15 seconds, and even at 100 µM cAMP degradation is still very fast.

Intracellular cGMP mediates chemotaxis and is degraded by DdPDE3, the cGMPstimulated DdPDE5, and possibly DdPDE6. After stimulation, intracellular cGMP may reach a concentration of 3 µM (15 pmol/10 7 cells; [START_REF] Van Haastert | Excitation adaptation and deadaptation of the cAMP mediated cGMP response in Dictyostelium discoideum[END_REF]). At a basal cGMP concentration of 0.03 µM, both the high affinity DdPDE3 (~40%) and the low affinity DdPDE5 (~60%) contribute in the degradation of cGMP. At elevated cGMP levels of 3 µM, DdPDE5 (~90%) becomes the predominant enzyme in the degradation of cGMP. At very high cGMP levels, which may occur after osmotic stimulation [START_REF] Oyama | cGMP accumulation induced by hypertonic stress in Dictyostelium discoideum[END_REF][START_REF] Kuwayama | Protection against osmotic stress by cGMP-mediated myosin phosphorylation[END_REF], DdPDE6 may become increasingly important in the degradation of intracellular cGMP.

DISCUSSION

The identification of all phosphodiesterases in Dictyostelium is very important for understanding signal transduction and development in Dictyostelium discoideum. The completed genome sequence [39] uncovers seven PDEs. In this paper we have characterized the last member, DdPDE7. The deduced amino acid sequence and the kinetic properties reveal that DdPDE7 is highly homologous to DdPDE1. Enzyme activity measurements reveal that both DdPDE1 and DdPDE7 are secreted into the medium and are attached to the cell surface. The deduced amino acid sequence reveals that both contain a signal sequence that is probable responsible for secretion. The mechanism for membrane association is not known

Dictyostelium

  enzymes it is possible to calculate the contribution of each enzyme in the degradation of extracellular cAMP (by DdPDE1, DdPDE4 and DdPDE7), intracellular cAMP (by DdPDE2 and DdPDE6), and intracellular cGMP (by DdPDE3, DdPDE5 and DdPDE6).

5 . 0 K

 50 is the apparent Michaelis-Menten constant, n is the Hill-coefficient, A K is the activation constant, and L M K and H M K are the Michaelis-Menten constant of the basal and activated enzyme, respectively.

[ 3 H

 3 ]cAMP by UK7/regA -cells in the absence and presence of 10 mM DTT to discriminate between the DTT-sensitive DdPDE1 and the DTT-insensitive DdPDE4. The results show a steady increase of cell surface PDE activity during development. During the first five hours of development nearly all PDE activity is DTT sensitive (i.e. attributed to DdPDE7). At later stages of development, this DTT-sensitive activity declines (figure 5A) and most of the PDE activity becomes sensitive to IBMX ([28] and data not shown). These results suggest that vegetative cells have low activity of DdPDE7, which increases about 3-fold during cell aggregation, and declines in the multicellular stage. In figure 5B we calculated the activity of the three enzymes by combining the present data on the activity of DdPDE7 in UK7/regA - cells with previously determined DdPDE1+DdPDE7 and DdPDE4 activity in wild-type cells, on the assumption that wild -type and UK7/regA -cells express the same amount of DdPDE7 activity. DdPDE1 represents by far the most prominent activity during development at a substrate concentration of 10 nM. Maximal DdPDE7 activity is about 75 fmol/min/10 7 cells during cell aggregation, compared to about 3000 fmol/min/10 7 cells for DdPDE1, both measured at 10 nM cAMP.

forFigure 1 .

 1 Figure 1. The phosphodiesterases family of Dictyostelium. (A), Seven PDEs have been recognized and identified in the completed genome of Dictyostelium. The enzymes belong to the class I enzymes prevalent in higher eukaryotes, or to the class II enzymes that are present in lower eukaryotes and some bacteria. Based on bootstrap data of phylogenetic cluster analysis of type II enzymes, the type II enzymes are recognized as catalytic phosphodiesterases or catalytic metallo-beta-lactamases. The localization and activity of the seven enzymes are regulated by signal sequences, trans-membrane segments, a response regulator domain, or cyclic nucleotide-binding domains. (B), cluster analysis of the catalytic domains of Type II enzymes from prokaryotes and lower eukaryotes. All nodes have bootstrap values above 70%, except for the nodes indicated by the small white ovals. Bootstrap values of the inner nodes are indicated. These bootstrap values and the length of

Figure 2 .

 2 Figure 2. Alignment of DdPDE1 and DdPDE7. The deduced amino acid sequence of DdPDE1 and DdPDE2 . Underlined are the predicted signal sequences with cleavage between amino acid 17 and 18 for DdPDE1 and 23 and 24 for DdPDE7. A potential hydrophobic transmembrane segment is indicated in small italic letters. The asterisk indicates a potential GPI anchor.

Figure 3 .

 3 Figure 3. Initial characterization of DdPDE7. (A), Inhibition by IBMX and DTT of phosphodiesterase activity at the surface of 5 hours starved wild-type AX3 cells and UK7 cells with a deletion of DdPDE1. The activity in the absence of inhibitors is set at 100 % (2950 fmol/min/10 7 cells in AX3 and 61 fmol/min/10 7 cells in UK7). The results show that the residual PDE activity exposed on UK7 cells is inhibited strongly by DTT and only partly by IBMX. (B), Over expression of DdPDE7 in UK7 cells reveal enhanced activity both on the cell surface and secreted in the medium.

Figure 4 .

 4 Figure 4. Kinetics of DdPDE7. Five hours starved UK7 cells were incubated with different concentrations of cAMP or cGMP. The hydrolysis was measured after 30 minutes and is expressed as Eady-Hofstee plot in the main figure and as Hill plot in the inset. The dotted lines were fitted for Michaelis-Menten kinetics using linear regression, while the straight lines were fitted for negative cooperativity using the Hill equation. The deduced kinetic constants for the Hill equation are for cAMP apparent K M =12.5 µm, V MAX =28pmol/min/10 7 cells, Hill coefficient = 0.79, and for cGMP apparent K M =36 µm, V MAX =72 pmol/min/10 7 cells, Hill coefficient = 0.9.

Figure 5 .

 5 Figure 5. DdPDE7 activity during development. (A), UK7/regA -cells were starved on plates for the times indicated, harvested, washed and assayed for cell surface cAMP hydrolysis using 10 nM [ 3 H]cAMP. Cell aggregation was observed at 6 hours of development, and tight aggregates and slugs were observed at 9 hours; development was arrested at this stage. (B), Activity of DdPDE1, DdPDE4 and DdPDE7 during development, deduced from panel A for DdPDE7, and from figure 6 in Bader et al [28] for DdPDE1 and DdPDE4.

Figure 6 .

 6 Figure 6. Contribution of the seven phosphodiesterases to the hydrolysis of three cyclic nucleotide pools. (A), extracellular cAMP during aggregation; (B), extracellular cAMP in slugs; (C), intracellular cAMP during aggregation; (D), intracellular cGMP during aggregation. The activity of each enzyme was calculated at different substrate concentrations using the equations presented in the section Experimental Procedures and the kinetic constants as presented in table I. From these activities we calculated the half time of degradation of the indicated substrate concentration, and the relative contribution of the participating enzymes in the degradation of substrate. The arrows indicate the range of substrate concentrations that have been measured in vivo during cell stimulation.

Biochemical Journal Immediate Publication. Published on 16 Oct 2006 as manuscript BJ20061153

Biochemical Journal Immediate Publication. Published on 16 Oct 2006 as manuscript BJ20061153

Biochemical Journal Immediate Publication. Published on 16 Oct 2006 as manuscript BJ20061153

ACKNOWLEDGMENTS.

We are indebted to all of the teams involved in the Dictyostelium sequencing projects. UK7/regA -null cells were kindly provided by David Trayner and Rob Kay (MRC Cambridge).

Source of data: The intrinsic kinetic properties K M , K A and Hill coefficient were obtained from present and published data, DdPDE1, [START_REF] Van Haastert | Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum[END_REF]; DdPDE2 [START_REF] Thomason | An intersection of the cAMP/PKA and two-component signal transduction systems in Dictyostelium[END_REF] and our own measurements;

]; DdPDE3, [START_REF] Kuwayama | Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium[END_REF]; DdPDE4, [START_REF] Bader | DdPDE4, a Novel cAMP-specific Phosphodiesterase at the Surface of Dictyostelium Cells[END_REF]; DdPDE5, [START_REF] Bosgraaf | Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium[END_REF][START_REF] Meima | the catalytic domain suggest two main groups, metallo-beta-lactamases and phosphodiesterases; the latter may consist of two subgroups. The species abbreviations, gene identifiers, and taxonomy are: Dh, Debaryomyces hansenii, gi|50424793; Eukaryota; Fungi. Cal, Candida albicans, gi|7548341; Eukaryota; Fungi. Kl, Kluyveromyces lactis, gi|50307193; Eukaryota; Fungi. Eg, Eremothecium gossypii, gi|44984787; Eukaryota; Fungi. Cg, Candida glabrata, gi|49526440; Eukaryota; Fungi. Sc, Saccharomyces cerevisiae, gi|6321189; Eukaryota; Fungi. Yl, Yarrowia lipolytica, gi|50554561; Eukaryota; Fungi. Nc, Neurospora crassa, gi|85086599; Eukaryota; Fungi. Sp, Schizosaccharomyces pombe, gi|3581909; Eukaryota; Fungi. Dd, Dictyostelium discoideum[END_REF]; DdPDE6, [START_REF] Bosgraaf | Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium[END_REF][START_REF] Meima | Characterization of a cAMPstimulated cAMP phosphodiesterase in Dictyostelium discoideum[END_REF]; DdPDE7, this report.

The V MAX was determined using enzyme-specific assays as indicated in table II.

*, These enzymes are activated by their substrate. In PDE5 this is shown to be mediated by reduction of the K M , while the V MAX remains the same; for DdPDE6 we assume the same kinetic mechanism. For each enzyme, two K M values are presented, of the high affinity and low affinity form, respectively. The concentrations of inhibitors used are 0.8 mM IBMX or 5 mM DTT.

#, the activity is measured in the absence and presence of IBMX; the difference of these activities is attributed to the enzyme indicated. -------------------------


Arnold H Van Der Luit

Marianne Budde

Shuraila Zerp

Wendy Caan

Jeffrey B Klarenbeek

Marcel Verheij

Wim J Van Blitterswijk
email: w.v.blitterswijk@nki.nl

expression with consequent sphingomyelin-and cholesterol-deficiency in lipid rafts

Keywords: sphingomyelin synthase, SM, sphingomyelin, exoSM, exogenous SM, C12 apoptosis resistance, edelfosine, lipid raft, sphingomyelin synthase, cholesterol, endocytosis 2

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The synthetic alkyl-lysophospholipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3phosphocholine (ALP; Et-18-OCH 3 ; Edelfosine) is the prototype of a group of new clinically used anti-cancer agents [START_REF] Mollinedo | Selective induction of apoptosis in cancer cells by the ether lipid ET-18-OCH 3 (Edelfosine): molecular structure requirements, cellular uptake, and protection by Bcl-2 and Bcl-X(L)[END_REF][START_REF] Ruiter | Alkyl-lysophospholipids activate the SAPK/JNK pathway and enhance radiation-induced apoptosis[END_REF][START_REF] Ruiter | Alkyllysophospholipids as anticancer agents and enhancers of radiation-induced apoptosis[END_REF][START_REF] Ruiter | Anti-cancer alkyl-lysophospholipids inhibit the phosphatidylinositol 3kinase-Akt/PKB survival pathway[END_REF]. These unnatural phospholipids with one single alkyl chain readily insert into the plasma membrane lipid bilayer, accumulate because they are poorly degradable by lipases [START_REF] Van Blitterswijk | Accumulation of an alkyl lysophospholipid in tumor cell membranes affects membrane fluidity and tumor cell invasion[END_REF], and disturb lipid metabolism in the cell. For example, ALP inhibits the activity of CTP:phosphocholine cytidylyltransferase, the ratedetermining enzyme for phosphatidylcholine (PC) biosynthesis [START_REF] Boggs | Lysophosphatidylcholine and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine inhibit the CDPcholine pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase step[END_REF]. We reported that this cytidylyltransferase inhibition is a direct trigger for apoptosis in S49 lymphoma and HeLa carcinoma cells [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF][START_REF] Van Der Luit | Different modes of internalization of apoptotic alkyl-lysophospholipid and cellrescuing lysophosphatidylcholine[END_REF]. Since cytidylyltransferase is active inside the cell (ER and nucleus [START_REF] Kent | CTP:phosphocholine cytidylyltransferase[END_REF]), ALP needs to be internalized to inhibit the enzyme. We found that ALP preferentially accumulates in a detergent-resistant sphingolipid-and cholesterol-rich membrane fraction, representative for microdomains known as lipid rafts [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF]. ALP is then internalized by lipid raft-and dynamin-dependent, clathrin-independent endocytosis [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF][START_REF] Van Der Luit | Different modes of internalization of apoptotic alkyl-lysophospholipid and cellrescuing lysophosphatidylcholine[END_REF]. In an ALP-resistant cell variant, S49 AR , selected by continuous culturing of S49 cells in the presence of ALP, we found no raft-dependent ALP internalization and, consequently, no disturbance of the PC metabolism and no initiation of apoptosis [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF]. The mechanism behind this uptake failure and consequent cellular resistance to ALP is unknown.

As a first approach to elucidate why lipid rafts in S49 AR cells are unable to mediate internalization of ALP, we questioned if the rafts in these resistant cells have a different lipid composition compared to the parental S49 cells. This question is the more relevant since we found that artificial breakdown of plasma membrane sphingomyelin (SM) by an exogenous (bacterial) sphingomyelinase, or extraction of cholesterol prevented ALP internalization and apoptosis [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF]. We therefore focused on the analysis of the sphingolipids, as these are major raft constituents that, in association with cholesterol, determine the detergent insolubility [START_REF] Van Meer | Sphingolipid transport: rafts and translocators[END_REF]. It is well known that SM and cholesterol show tight mutual interaction [START_REF] Van Blitterswijk | Quantitative contributions of cholesterol and the individual classes of phospholipids and their degree of fatty acid (un)saturation to membrane fluidity measured by fluorescence polarization[END_REF][START_REF] Slotte | Sphingomyelin-cholesterol interactions in biological and model membranes[END_REF] and that cellular SM levels therefore co-regulate cholesterol homeostasis [START_REF] Slotte | Sphingomyelin-cholesterol interactions in biological and model membranes[END_REF][START_REF] Ridgway | Interaction between metabolism and intracellular distribution of cholesterol and sphingomyelin[END_REF]. Conversely, whether changes in membrane cholesterol may also affect the levels and/or synthesis of SM is less clear [START_REF] Ridgway | Interaction between metabolism and intracellular distribution of cholesterol and sphingomyelin[END_REF]. To disturb cholesterol established inhibitor of Niemann-Pick C (NPC1) protein-mediated vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane [START_REF] Liscum | The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one[END_REF][START_REF] Lange | Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles[END_REF][START_REF] Vruchte | Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport[END_REF][START_REF] Garver | Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae[END_REF].

We report here that the ALP-resistant S49 AR cells are unable to synthesize SM through downregulated sphingomyelin synthase (SMS1) expression. This lack of SM is especially apparent in the detergent-resistant membrane fraction together with a decrease in cholesterol. Conversely, disturbance of cholesterol homeostasis, or siRNA-induced downregulation of SMS1 directly, prevented SM synthesis in parental S49 cells. We show in either cell variant that the lack of SM production is directly linked to abrogated ALP uptake and apoptosis resistance. were from Merck (Darmstadt, Germany).

Cells and culture conditions

Mouse S49.1 lymphoma cells (S49) were cultivated in Dulbecco's modified Eagle's medium (Gibco-Invitrogen), supplemented with 8% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin at 37°C and 5% CO 2 . ALP-resistant S49 AR cells were obtained by two selection rounds of growth of S49 cells in the presence of 15 µM ALP (Et-18-OCH 3 ) for 72 hours, followed by plating in semi-solid medium and isolation of colonies of surviving cells [START_REF] Smets | Signaling steps in apoptosis by ether lipids[END_REF]. The selected S49 AR clone could be grown continuously in 15 µM ALP with a doubling time of 12 h, similar to that of the parental S49 cells. S49 AR cells maintained resistance by treatment with ALP every two weeks. All experiments with S49 AR cells were performed with cells grown without the selection agent for at least three days.

Loading of S49 AR cells with exogenous C12:0-sphingomyelin was performed by injecting the ethanol-dissolved lipid into the complete culture medium (final concentration 20 µM C12:0-SM; 0.02% ethanol) and culturing the cells in this medium for three days.

PCR and retroviral transduction

In order to amplify SMS1 and SMS2 by reverse transcriptase PCR, the following primers For retroviral gene transduction, cDNAs were cloned into the retroviral vector pRETRO-SUPER [START_REF] Brummelkamp | Stable suppression of tumorigenicity by virus-mediated RNA interference[END_REF]. Empty vector or vector containing the siRNA oligonucleotide directed to SMS1 were transfected into the 293T human embryonic kidney cell-derived packaging line Phoenix Ampho, using FuGene-6 transfection reagent, according to instructions of the manufacturer (Roche Molecular Biochemicals, Mannheim, Germany).

Transfected cells were selected with 1 µg/ml puromycin (Invitrogen Life Technologies (Breda, The Netherlands). Virus-containing supernatants were harvested after 2-5 days and stored at -80 °C until further use. S49 cells were seeded on dishes coated with RetroNectin (Roche Molecular Biochemicals) and transduced with 1 ml of virus-6 containing supernatant per 0.5 × 10 6 cells. Supernatants were removed after overnight incubation, and cells were cultured in fresh medium. Transduced cells were selected from 48 h after transduction for growth in the presence of 200 µg/ml puromycin.

ALP uptake and apoptosis assay

Cells were grown to a density of 1 x 10 6 /ml and ALP (Et-18-OCH 3 ; Edelfosine) was added in the effective apoptotic concentration of 15 µM, supplemented with 0.2 µCi 3 H-ALP/ml. At given time points, samples were taken, put on ice for 2 minutes and then washed three times with ice-cold PBS. Samples were lysed in 0.1 N NaOH prior to liquid scintillation counting.

Apoptosis was induced with ALP (15 µM) or FasL (500 ng/ml) for 6h. Cells were washed in PBS and lysed overnight at 4°C in 0.1% (w/v) sodium citrate, 0.1% (v/v) Triton X-100 and 50 µg/mL propidium iodide, according to Nicoletti et al. [START_REF] Nicoletti | A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry[END_REF].

Fluorescence intensity of propidium iodide-stained nuclei was determined on a FACScan (Becton Dickinson), and data were analyzed using Lysis software.

Lipid analysis

Cells at 1 x 10 6 cells/ml were incubated with [methyl- Mass sphingolipid analyses were conducted by liquid chromatography, electrospray tandem mass spectrometry as described by Merrill et al. [START_REF] Merrill | Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry[END_REF].

In vitro SM synthase assay SM synthesis in vitro was performed (1 hour, 37 o C) on a crude membrane preparation from HeLa cells, using C6-NBD-ceramide (60 nM) as a substrate [START_REF] Nikolova-Karakashian | Assays for the biosynthesis of sphingomyelin and ceramide phosphoethanolamine[END_REF]. To this end, cells were homogenized by 5x passing through a 27½ G needle. Nuclei were spun down at 300

x g, 10 minutes, and from the supernatant, crude membranes were obtained by centrifugation at 100,000 x g for 1 hour. Lipids were extracted and separated by TLC (see above).

Isolation of lipid rafts

A lipid raft fraction was prepared by detergent extraction of cells and sucrose gradient centrifugation, essentially as described [START_REF] Lisanti | Caveolae purification and glycosylphosphatidylinositol-linked protein sorting in polarized epithelia[END_REF]. In brief, 2 x 10 8 cells were solubilized into 1 ml of an ice-cold buffer consisting of 25 mM MES, 150 mM NaCl and 1% Triton X-100, and homogenized using a Dounce homogenizer. The extract was centrifuged on a discontinuous sucrose gradient at 39,000 rpm in a SW41 rotor for 20 hours at 4°C, followed by 11 × 1.0 ml fractions manual collection from the top of the gradient.

Results

S49 lymphoma cells made resistant to ALP (S49 AR cells) show a defect in the synthesis of sphingomyelin

Previously, we reported that the synthetic ether-lipid Et-18-OCH 3 (Edelfosine; ALP) induces apoptosis in S49 lymphoma cells, in a dose-and time-dependent fashion. The onset of apoptosis in these cells was relatively fast, and already apparent after 3 hours [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF].

An ALP-resistant variant cell line, S49 AR , generated by culturing S49 cells in the presence of ALP and continuous selection of surviving cells, did not undergo apoptosis by ALP. S49 AR cells were not derived from a single cell clone with a single genetic mutation, but is a population of cells in which the sensitivity to ALP is suppressed in a reversible manner: After 4-5 weeks of culturing in the absence of ALP, the cells gradually become resensitized to the lipid, and regain the phenotypic properties (to be described below) of the parental S49 cells (data not shown). We demonstrated that, in order to induce apoptosis, ALP needs to be internalized by endocytosis via lipid rafts [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF][START_REF] Van Der Luit | Different modes of internalization of apoptotic alkyl-lysophospholipid and cellrescuing lysophosphatidylcholine[END_REF]. This particular process was disrupted in the ALP-resistant S49 AR cells, although ALP still remained associated to the detergent-resistant raft fraction from these cells [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF].

Since the uptake of ALP could be prevented by treatment of cells with cholesterol sequestering agents or the breakdown of SM [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF], we addressed the question whether the difference in ALP sensitivity of the cells could be related to a different lipid composition of their lipid rafts, which may impede internalization. We focused on analyzing the sphingolipids, as being major raft constituents [START_REF] Van Meer | Sphingolipid transport: rafts and translocators[END_REF]. Importantly, we found that S49 AR cells were deficient in sphingomyelin (SM) (Figure 1; Table 1). Radiolabeling of the cells with the SM precursors [ 14 C]choline or [ 14 C]serine, followed by lipid analysis by 2D-TLC separation, revealed two typical SM spots in S49 cells, which were absent in S49 AR cells (Figure 1A). These two SM spots correspond respectively to a (major) pool with relatively short acyl chains (16 C-atoms) and a (minor) SM pool with long acyl chains (predominantly C24:1) and containing a C16-dihydro-(sphinganine) species (Table 1) [START_REF] Ramstedt | Analysis of natural and synthetic sphingomyelins using high-performance thin-layer chromatography[END_REF]. No other conspicuous differences in lipid profile between the two cell lines were seen on these 2-D TLC plates.

The lack of SM synthesis in S49 AR cells was also reflected in SM mass levels in these cells, as determined by mass spectrometry (Table 1). S49 cells contain mainly the C16:0-SM species, which is reduced to about 20% in the S49 AR cells. Given the almost complete lack of SM synthesis in S49 AR cells (see also Figures 1B, 2 and 3), this residual mass SM has most likely been taken up by the cells directly from the fetal calf serum in the culture medium, as this serum is also rich in C16:0-SM (Table 1).

To visualize the synthesis of other sphingolipids in the cells, we radiolabeled the cells with a different metabolic precursor, [1-3 H-]sphingosine. Figure 1B shows the timedependent synthesis of sphingolipids in the two cell lines. It is clear that, during the 4 h time period, S49 AR cells were unable to synthesize SM from sphingosine, while S49 cells were able to do so (predominantly the SM pool with C16 acyl-length), evidently via the intermediate ceramide. No profound differences in the synthesis of glucosylceramide (GlcCer) or lactosylceramide (LacCer) were seen between the two cell lines. The spot of phosphatidylethanolamine (PE) represents a metabolite from sphingolipid degradation, via sphingosine-1-phosphate lyase-mediated phosphoethanolamine production, as shown and discussed before [START_REF] Hanada | Sphingolipids are essential for the growth of Chinese hamster ovary cells[END_REF].

We next isolated the detergent-resistant lipid raft fractions from [ 3 H]sphingosinelabeled cells, using sucrose density-gradient centrifugation. Figure 2 shows that, in S49 cells, SM is concentrated in the lipid raft fractions (3 to 5), as expected, whereas SM is lacking in the raft fractions from S49 AR cells. Again, no clear differences in the other lipids were seen between the two cell lines. GlcCer, LacCer and ceramide were normally present in the raft fractions of both cell lines. Taken together, the data indicate that ALPresistant S49 AR cells show a specific defect in the synthesis of the lipid raft component SM, but not of other (sphingo)lipids. Furthermore, given the type of SM precursors used, and the unimpeded production of the precursor ceramide (Figures 1B and2), it is apparent that the defect in SM production lies in the final biosynthetic step, catalyzed by SM synthase (SMS).

S49 AR cells do not express SMS, whereas S49 cells only express SMS1

In order to investigate the mechanism underlying the defective SM synthesis in S49 AR cells, we analyzed the expression of SMS1 and SMS2, the two recently identified isozymes capable of executing the final enzymatic step of SM synthesis [START_REF] Huitema | Identification of a family of animal sphingomyelin synthases[END_REF][START_REF] Yamaoka | Expression cloning of a human cDNA restoring sphingomyelin synthesis and cell growth in sphingomyelin synthase defective lymphoid cells[END_REF]. RT-PCR analysis revealed SMS1 expression in S49 but not in S49 AR cells (Figure 3A). SMS2 was not detectable at all in either cell type (transcripts were not visible after amplification up to 45 cycles of PCR). This result was verified by real-time PCR: Compared to S49 cells, SMS1 expression in S49 AR cells was about 50-fold reduced (Figure 3B). SMS2 was not expressed in either cell type (no detectable transcript after 50 PCR cycles, whereas it was readily detected in cDNA made from mouse embryo cells, as a positive control; data not shown). Thus, the lack of SMS expression in S49 AR cells directly explains the defect in SM synthesis in the S49 AR cells.

Downregulation of SMS1 expression by siRNA blocks SM synthesis and prevents

ALP uptake and apoptosis induction in S49 cells

We previously demonstrated that S49 AR cells are resistant to ALP because, contrary to S49 cells, they are unable to internalize ALP via lipid rafts [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF]. Since S49 AR cells lack SMS expression and SM synthesis, we first tried to introduce the SMS1 gene into S49 AR cells. This, however, resulted in loss of viability of SMS1-transfected cells (both in S49 and in S49 AR cells; data not shown). We next downregulated SMS1 expression in S49 cells by stable transduction of small interfering RNA (siRNA) specific for SMS1, yielding so-called S49 siSMS1 cells, to see if reduced SM synthesis would lead to impaired ALP internalization, like in the S49 AR cells. SMS1 mRNA levels in S49 siSMS1 cells were 6-fold reduced, as revealed by real-time PCR (Figure 3B). Figure 3C confirms that these SMS1-downregulated cells show significantly reduced SM synthesis, down to 10% of mock-transduced S49 control cells, whereas little or no changes were seen in the synthesis of the other sphingolipids. Thus, by inhibiting SMS1 gene expression specifically, we can block the synthesis of SM in S49 cells to a similar low level as in S49 AR cells.

We next tested if the inhibition of SM synthesis would lead to reduced ALP uptake. Figure 4A shows that, indeed, the time-dependent uptake of 3 H-ALP by S49 siSMS1 cells was decreased to a similar extent as observed in S49 AR cells, whereas S49 and S49 mock cells showed the same high uptake kinetics as previously reported [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF]. The synthesis of SM is, therefore, essential for ALP internalization in these cells.

The production of SM can also be inhibited indirectly by preventing the synthesis of the SMS1 substrate ceramide, using myriocin/ISP-1, an inhibitor of serinepalmitoyltransferase [START_REF] Miyake | Serine palmitoyltransferase is the primary target of a sphingosine-like immunosuppressant, ISP-1/myriocin[END_REF], the initiating enzyme of the sphingolipid biosynthetic pathway.

This inhibitor (at 5 µM, 5 h) was found to decrease the incorporation of 14 C-serine precursor into 14 C-SM of S49 cells by 63%. Figure 4B shows that myriocin/ISP-1 inhibited the uptake of 3 H-ALP by S49 cells depending on the time of preincubation (1h or 16 h) with this inhibitor, to values that were intermediate between those of S49 and S49 AR cells.

We next tested if the reduced uptake of ALP in SMS1-downregulated S49 siSMS1 cells would abrogate apoptosis induction in these cells, like in the S49 AR cells. Figure 4C shows that, indeed, the level of apoptosis induced by ALP in these SMS1-deficient cells dropped from 55% (S49 and S49 mock cells) to 15%. The sphingolipid synthesis inhibitor myriocin/ISP-1 (5 µM, preincubated for 2h) inhibited ALP-induced apoptosis in S49 cells, measured at 6 h, by 31 ±6 % (n=4) (data not shown in figure). We note, however, that the inhibitor tended to be apoptotic by itself at longer preincubation times.

To answer the question if the reduced ALP uptake in the SMS1-deficient was due to the lack of SMS activity or the lack of SM as such in plasma membrane rafts, we loaded the S49 AR cells with exogenous SM. In our experience, the most efficient way to do this was to let the cells take up lauroyl-SM (C12:0-SM, which worked better than SM with longer acyl chains) via the serum lipoproteins in the culture medium. To this end, C12:0-SM was injected into the complete culture medium at 20 µM final concentration (see 'Experimental'). After three days of culturing in this medium, cells were washed and their lipid rafts were isolated. Figure 5A shows that the exogenous C12:0-SM (exoSM)

was incorporated in the S49 AR detergent-resistant raft (R) fractions to at least similar (or higher) proportions as the endogenous C16:0-SM in rafts from S49 cells. However, this exoSM loading of S49 AR cells did not result in more 3 H-ALP uptake by these cells (Figure 5B), nor did it affect the resistance of these cells to ALP-induced apoptosis (Figure 5C).

Collectively, these data strongly suggest that SM synthesis, rather than a mere SM accumulation in lipid rafts at the plasma membrane, is required for ALP internalization to induce apoptosis.

SM deficiency in lipid rafts is accompanied by reduced cholesterol content

Paradoxically, we demonstrated in previous work [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF][START_REF] Van Der Luit | Different modes of internalization of apoptotic alkyl-lysophospholipid and cellrescuing lysophosphatidylcholine[END_REF] that raft SM hydrolysis by exogenous (bacterial) sphingomyelinase prevented ALP internalization and apoptosis induction. The same happened when lipid rafts were disrupted by cholesterol sequestering agents such as methyl-β-cyclodextrin. Furthermore, it is well known that SM tightly associates with cholesterol [START_REF] Van Blitterswijk | Quantitative contributions of cholesterol and the individual classes of phospholipids and their degree of fatty acid (un)saturation to membrane fluidity measured by fluorescence polarization[END_REF][START_REF] Slotte | Sphingomyelin-cholesterol interactions in biological and model membranes[END_REF], which favors lipid raft formation and causes detergent insolubility of these membrane microdomains [START_REF] Brown | Structure and function of sphingolipid-and cholesterol-rich membrane rafts[END_REF]. We therefore questioned if the lack of SM synthesis in S49 AR cells would be accompanied by decreased cholesterol content in the lipid raft fraction. To answer this question, we incubated S49 and S49 AR cells with exogenous [ 3 H]cholesterol overnight to label the various subcellular compartments to equilibrium, and then determined the distribution of [ 3 H]cholesterol among the sucrose gradient fractions of Triton-solubilized cell membranes. Figure 6A shows that that radiolabeled cholesterol is enriched in the detergent-insoluble raft fractions, and that cholesterol partitioning to these raft fractions is higher in the S49 cells than in the S49 AR cells.

Thus, detergent-insoluble lipid rafts from ALP-resistant S49 AR cells are not only deficient in SM but have also reduced cholesterol content. These results are consistent with the notion that the levels of SM and cholesterol in lipid rafts are mutually interdependent, conceivably due to the well known tight physical interaction between these two lipids [START_REF] Van Blitterswijk | Quantitative contributions of cholesterol and the individual classes of phospholipids and their degree of fatty acid (un)saturation to membrane fluidity measured by fluorescence polarization[END_REF][START_REF] Slotte | Sphingomyelin-cholesterol interactions in biological and model membranes[END_REF].

SM synthesis and apoptosis sensitivity depend on cholesterol homeostasis

SMS1 is located in the Golgi apparatus [START_REF] Huitema | Identification of a family of animal sphingomyelin synthases[END_REF] where its two enzymatic products, SM and diacylglycerol, play an important role in, respectively, new lipid raft formation and secretory vesicle formation at the trans-Golgi network for transport to the plasma membrane (see Discussion). SMS catalyses the production of SM and diacylglycerol from ceramide and PC, but can also catalyse the reverse reaction [START_REF] Van Helvoort | Conversion of diacylglycerol to phosphatidylcholine on the basolateral surface of epithelial (Madin-Darby canine kidney) cells. Evidence for the reverse action of a sphingomyelin synthase[END_REF], resulting in an equilibrium. Given the strong physical association of SM with cholesterol, one would expect that the availability of cholesterol at the site of SM synthesis influences this enzymatic equilibrium and stabilizes SM formation through its sequestration in a (transient) SM-cholesterol complex, thus facilitating/driving SM production and new lipid raft formation. We reasoned that, when cholesterol is deficient at this SMS1 location, SM and diacylglycerol can be more easily converted back into PC and ceramide, the net result being that SM production is inhibited. To test this hypothesis, we used U18666A to disturb NPC1-mediated vesicular transport of cholesterol out of the endosomal compartments [START_REF] Liscum | The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one[END_REF][START_REF] Lange | Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles[END_REF] to the trans-Golgi network and lipid rafts [START_REF] Garver | Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae[END_REF], and we determined SM production and cholesterol partitioning in the lipid raft fractions. So, S49 cells were allowed to take up and process exogenous [ 3 H]cholesterol in the presence or absence of U18666A. As shown in Figure 6B, cells that received U18666A showed less [ 3 H]cholesterol partitioning in the raft fractions than control cells. In fact, the altered cholesterol profile from the U18666A-treated cells looked very similar to that from the S49 AR cells (compare the graphs of Figures 6A and6B).

We next tested how this disturbance of cholesterol homeostasis affected SM synthesis. Figure 7A shows that the time-dependent synthesis of SM in S49 cells is inhibited by about 80% in the presence of U18666A. This inhibition of SM synthesis was indirect, via cholesterol, as confirmed by a control experiment shown in Figure 7B. In this experiment, we incubated crude cellular membranes with fluorescently NBD-labeled ceramide as a substrate for endogenous SMS. In this in vitro assay, U18666A did not inhibit SM synthesis, whereas D609, a known SMS inhibitor [START_REF] Luberto | Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?[END_REF][START_REF] Meng | Sphingomyelin synthase as a potential target for D609-induced apoptosis in U937 human monocytic leukemia cells[END_REF], indeed blocked SM production (Figure 7B). Collectively, the data suggest that SM synthesis is blocked indirectly through U18666A-mediated disturbance of intracellular cholesterol homeostasis.

Since U18666A not only disturbs cholesterol homeostasis, but abrogates SM synthesis concomitantly, we next tested if this inhibitor would prevent ALP uptake and apoptosis induction, like in the SMS1-deficient cells. Figure 8A shows the timedependent uptake of [ 3 H]ALP by S49 and S49 AR cells. In the presence of U18666A, the uptake by S49 cells decreased to the level of S49 AR cells. Furthermore, U18666A treatment dose-dependently blocked ALP-induced apoptosis in S49 cells, whereas the drug did not induce apoptosis by itself (Figure 8B). As an additional control, U18666A had no effect on the induction of apoptosis by an entirely different inducer, Fas ligand (FasL; Figure 8B), indicating that the drug specifically blocked the ALP uptake, but exerted no aspecific effect on apoptotic signaling steps.

We previously reported that ALP induces apoptosis primarily by raft-mediated endocytic uptake and subsequent inhibition of PC synthesis inside the cell [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF][START_REF] Van Der Luit | Different modes of internalization of apoptotic alkyl-lysophospholipid and cellrescuing lysophosphatidylcholine[END_REF]. Since U18666A appeared to compromise 'normal' lipid raft formation (both SM synthesis and cholesterol incorporation in the detergent-resistant raft fraction were inhibited), and inhibits ALP internalization, one would expect that U18666A also prevents PC inhibition. Figure 8C shows that indeed ALP-induced PC inhibition is alleviated by U18666A. Thus, it appears that U18666A, through disturbing cholesterol homeostasis and decreased SM synthesis, which disables cholesterol-SM association in lipid rafts, prevents ALP from reaching its intracellular target, cytidylyltransferase in the ER.

15

Discussion

In this paper, we have shown that S49 lymphoma cells express only one of the two known mammalian sphingomyelin synthase isotypes, SMS1, and that the activity of this enzyme is required for ALP internalization and ALP-induced apoptosis in these cells. To initiate apoptotic signaling, ALP has to be internalized by endocytosis via lipid rafts to inhibit the rate-determining enzyme, CTP:phosphocholine cytidylyltransferase, of PC synthesis in the ER [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF][START_REF] Van Der Luit | Different modes of internalization of apoptotic alkyl-lysophospholipid and cellrescuing lysophosphatidylcholine[END_REF]. In cells that were made resistant to ALP, S49 AR cells, this raftdependent process is blocked [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF]. We have demonstrated by using three different radiolabeled precursors, [ 14 C]choline, [ 14 C]serine and [ 3 H]sphingosine, that these ALPresistant S49 AR cells do not synthesize SM. The lack of SM in these cells, confirmed by mass spectrometry, was due to absence of SMS expression as determined by PCR.

Furthermore, siRNA-induced downregulation of SMS1 expression in the parental S49 cells prevented SM production, ALP uptake and apoptosis induction, like in the S49 AR cells. Similar results were obtained when the S49 cells were treated with U18666A, which disturbs cholesterol homeostasis and in this way inhibits SM synthesis indirectly.

That this inhibition by U18666A is indeed indirect, was verified by the fact that the compound did not affected SMS activity in a cell-free system (isolated membranes), whereas D609, a known SMS inhibitor [START_REF] Luberto | Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?[END_REF][START_REF] Meng | Sphingomyelin synthase as a potential target for D609-induced apoptosis in U937 human monocytic leukemia cells[END_REF], blocked this in vitro activity. D609 (at 30 µg/ml) also blocked the in vivo synthesis of SM (but notably inhibited the biosynthesis of other lipids to some moderate extent as well) and prevented ALP-induced apoptosis in S49 cells (data not shown). Indirect inhibition of SM production by myriocin/ISP-1, an inhibitor of serine palmitoyltranseferase, an early enzymatic step in sphingolipid synthesis, likewise inhibited ALP uptake and apoptosis induction in these cells. The results obtained by gene silencing of SMS1 could thus be mimicked by pharmacological inhibition of the enzyme directly, or of the entire biosynthetic pathway of sphingolipids.

How precisely S49 cells become resistant to ALP upon continuous culturing in the presence of this ether-lipid remains unknown. Our results suggest a mechanism that includes transcriptional downregulation of SMS1. Possible involvement of stress mediators such as heat shock proteins or lipid (ALP?)-sensitive transcription factors may be considered in future studies. To escape ALP toxicity, the cells apparently shut off their raft-mediated endocytic uptake, by downregulating the synthesis of the raft keycomponent SM (but not of other sphingolipids; Figures 1B and2). This SM synthesis occurs in the Golgi, the location of SMS1 [START_REF] Huitema | Identification of a family of animal sphingomyelin synthases[END_REF], and must somehow be linked to endocytic uptake of ALP at a spatially distant site, the plasma membrane. An appealing physicochemical linker of these two spatio-temporal processes is the dynamic, and at any time transient (SM containing) lipid raft that is newly assembled in the trans-Golgi network, is subject to vesicular trafficking to the plasma membrane [START_REF] Van Meer | Sphingolipid transport: rafts and translocators[END_REF][START_REF] Ikonen | Roles of lipid rafts in membrane transport[END_REF] and from there mediates ALP internalization through endosomes [START_REF] Van Der Luit | Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[END_REF][START_REF] Van Der Luit | Different modes of internalization of apoptotic alkyl-lysophospholipid and cellrescuing lysophosphatidylcholine[END_REF][START_REF] Ikonen | Roles of lipid rafts in membrane transport[END_REF]. In this concept, ALP internalization by raft-dependent endocytosis may thus be viewed as part of a constitutive SMS1-dependent membrane recycling process.

In the Golgi apparatus, SMS1 and its two enzymatic products, SM and diacylglycerol, play a major role in microdomain (raft) formation and vesicle biogenesis at the trans-Golgi network [START_REF] Ridgway | Interaction between metabolism and intracellular distribution of cholesterol and sphingomyelin[END_REF][START_REF] Bankaitis | Lipids and the exocytotic machinery of eukariotic cells[END_REF][START_REF] Van Lint | Protein kinase D: an intracellular traffic regulator on the move[END_REF][START_REF] Lev | Lipid homeostasis and Golgi secretory function[END_REF], respectively. Diacylglycerol represents a major cue for protein kinase D (PKD1) recruitment to trans-Golgi membranes [START_REF] Baron | Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane[END_REF]. This PKD1 regulates the fission of transport vesicles on their way to the plasma membrane [START_REF] Bankaitis | Lipids and the exocytotic machinery of eukariotic cells[END_REF].

Inhibition of SMS1-mediated diacylglycerol production was shown to block this process [START_REF] Baron | Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane[END_REF]. In the Golgi, newly synthesized SM is assumed to seek association with cholesterol in nascent lipid rafts [START_REF] Van Meer | Sphingolipid transport: rafts and translocators[END_REF][START_REF] Simons | How cells handle cholesterol[END_REF]. Cholesterol partitioning in new rafts was shown to occur before it reaches the plasma membrane [START_REF] Heino | Dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface[END_REF] and to be required for the formation of secretory vesicles from the trans-Golgi network [START_REF] Ikonen | Roles of lipid rafts in membrane transport[END_REF][START_REF] Wang | Cholesterol is required for the formation of regulated and constitutive secretory vesicles from the trans-Golgi network[END_REF]. Availability of cholesterol at this site has been shown to be regulated by the NPC1 protein [START_REF] Garver | Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae[END_REF]. In NPC1-knockout cells, cholesterol transport from endosomal compartments to the trans-Golgi network is blocked, and this defect can, in normal cells, be mimicked by the compound U18666A [START_REF] Liscum | The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one[END_REF][START_REF] Lange | Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles[END_REF]. Treatment of S49 cells with this inhibitor of cholesterol homeostasis indeed reduced cholesterol distribution in lipid rafts and inhibited SM production indirectly. The latter may be tentatively explained by insufficient stabilization of newly synthesized SM through lack of cholesterol at this site (see also reasoning in Results section). In turn, we speculate that this deficient SM synthesis with consequent lack of SM-cholesterol assembly in new rafts might be a causal link to inhibition of ALP uptake and apoptosis induction, like in the ALP-resistant S49 AR cells.

Blockade of constitutive SM synthesis (and of concomitant production of diacylglycerol), as we effectuated in various ways (siRNA of SMS1, myriocin/ISP-1, D609) is likely to inhibit anterograde vesicular trafficking, as noted [START_REF] Bankaitis | Lipids and the exocytotic machinery of eukariotic cells[END_REF][START_REF] Lev | Lipid homeostasis and Golgi secretory function[END_REF]. The mere accumulation of SM in plasma membrane rafts, by supplying SM-deficient S49 AR cells with exogenous SM, failed to facilitate ALP internalization and apopotosis induction (Figure 5). Likewise, loading of the plasma membrane with exogenous (cyclodextrinformulated) cholesterol did not facilitate ALP uptake and apoptosis (see ref. [START_REF] Smets | Signaling steps in apoptosis by ether lipids[END_REF]; and data not shown). Our results are therefore consistent with the possibility that constitutive biosynthesis of SM, in concert with recruited (endogenous) cholesterol, directs the assembly of SM-containing lipid rafts at the trans-Golgi network and further translocation to the plasma membrane, and that this dynamic and constitutive process is essential for the raft-dependent internalization of ALP. Collectively, these results support the idea that ALP internalization is carried by the retrograde route of constitutive raftvesicular cycling, that may exist between the trans-Golgi, the plasma membrane and the endosomal compartments [START_REF] Van Meer | Sphingolipid transport: rafts and translocators[END_REF][START_REF] Ikonen | Roles of lipid rafts in membrane transport[END_REF][START_REF] Hoekstra | Lipid trafficking and sorting: how cholesterol is filling gaps[END_REF][START_REF] Van Blitterswijk | Ceramide: second messenger or modulator of membrane structure and dynamics?[END_REF][START_REF] Nabi | Caveolae/raft dependent endocytosis[END_REF][START_REF] Sannerud | Retrograde traffic in the biosynthetic-secretory route: pathways and machinery[END_REF]. This hypothesis and the role of SMS1 in this particular recycling process and ALP sensitivity needs to be further verified in future studies. b C20:0, C22:0, C24:0, C26:0 and C26:1 in amounts less than 1.9 and 0.8 pmol/cell x 10 6

--------------------------------------------------------------------------------------------------- (pmol/
for S49 and S49 AR cells, respectively, and less than 60.1 pmol/mg protein for fetal calf serum. Triton-X100-insoluble lipid rafts were isolated on sucrose density gradients, and lipids were extracted and separated by TLC (see "Experimental"). T, total lysate extract.

LEGENDS TO FIGURES

Positions of lipids are indicated as in Figure 1B. spots (cf Fig. 1A; see text). (B) D609 (50 µg/ml), but not U18666A (2 µg/ml), inhibits the synthesis of (NBD-conjugated) SM, when added to crude membranes with NBDceramide as a substrate (see "Experimental").

  -octadecyl-2-O-methyl-rac-glycero-3phosphocholine) and U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one) were purchased from BioMol (Plymouth Meeting, PA). [ 3 H]Et-18-OCH 3 ( 3 H-ALP; 58 Ci/mmol), was synthesized by Moravek Biochemicals (Brea, CA). [Methyl-14 C]choline chloride (58 mCi/mmol), L-[3-14 C]serine (54 mCi/mmol) and [1α,2α(n)-3 H]cholesterol (49 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). [1-3 H]sphingosine was synthesized by Mr. Piet Weber at DSM (Delft, The Netherlands). N-Lauroyl-D-erythro-sphingosylphosphorylcholine (Lauroyl sphingomyelin; C12:0-SM) was from Avanti Polar Lipids, Inc. (Alabaster, AL). C6-NBD-ceramide (N-[6-[(7-nitro-benz-2-oxa-1,3-diazo-4-yl)amino]caproyl]-ceramide) was from Molecular Probes, Inc. (Invitrogen Life Technologies, Breda, The Netherlands). D609 (tricyclodecan-9-yl-xanthogenate) was from Calbiochem-Novabiochem GmbH (Schwalbach, Germany). Myriocin/ISP-1 was from Sigma. Soluble recombinant human Fas ligand (FasL; APO-1L) was from Alexis (Leiden, The Netherlands). Reagents for lipid extraction and subsequent analyses, as well as Silica 60 TLC plates (20 x 20 cm)

  ----------------------------------------------------------------------------------------------0-DH, sphingomyelin containing an N-palmitoyl-dihydrosphingosine moiety.

Figure 1 ALP

 1 Figure 1ALP-resistant S49 AR cells are devoid of sphingomyelin synthesis

Figure 2

 2 Figure 2Comparison of sphingolipid distribution of detergent-resistant lipid raft fractions isolated from S49 and S49 AR cells

Figure 3

 3 Figure 3Expression of SMS1 and SMS2 in S49 and S49 AR cells

Figure 4

 4 Figure 4 Inhibition of SM synthesis, through siRNA-induced SMS1 silencing (S49 siSMS1 cells) or myriocin/ISP-1 treatment (S49 cells) abrogates ALP uptake and apoptosis induction, similar to S49 AR cells

Figure 5 Exogenous

 5 Figure 5Exogenous SM incorporates in lipid rafts of S49 AR cells but does not facilitate ALP uptake and apoptosis induction in these cells

Figure 6

 6 Figure 6 Cholesterol levels are decreased in lipid raft fractions from S49 AR cells and U18666A-treated S49 cells

Figure 7

 7 Figure 7 Disturbance of cholesterol homeostasis by U18666A indirectly inhibits SM biosynthesis

Figure 8 U18666A

 8 Figure 8 U18666A inhibits ALP uptake and ALP-induced apoptosis, and alleviates ALP-mediated inhibition of PC synthesis

Table 1 Sphingomyelin mass levels in S49 and S49 AR cells and in the fetal calf serum used for cell culturing

 1

	Sphingomyelins in lipid extracts were assayed by liquid chromatography, electrospray
	tandem mass spectrometry [21]. Data are means of triplicates ± S.D.
	________________________________________________________________________
	SM type	S49 cells	S49 AR cells	Fetal calf serum
	-

Biochemical Journal Immediate Publication. Published on 19 Oct 2006 as manuscript BJ20061178 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 19 Oct 2006 as manuscript BJ20061178

Acknowledgements

We thank Alfred Merrill, Elaine Wang and Samuel Kelley for conducting the sphingomyelin analyses by mass spectrometry using the Sphingolipid Analysis Core funded by NIH GM069338 as part of the Lipid MAPS Consortium. We thank Jannie Borst, Nullin Divecha, Jon Halstead and Menno van Lummel for useful discussions.

Cathelijne Stortelers is thanked for advice on real-time PCR. This study was funded by the Dutch Cancer Society, grant NKI 2005-3377.