Julia Leidner

Lysann Palkowitsch

Uta Marienfeld

Dietmar Fischer

Ralf Marienfeld
email: ralf.marienfeld@uni-ulm.de

Identification of lysine residues critical for the transcriptional activity and polyubiquitination of the NF-B family member RelB

Keywords: co-factors, NF-B, RelB, ubiquitination, gene expression, protein stability

Identification of lysine residues critical

Introduction

The transcription factor NF-B plays an essential role for the regulation of immune and inflammatory responses, development and cancer. The basis for the pleiotropic effects of this transcription factor is the functional divergence of the different NF-B dimers, composed of the subunits RelA, RelB, c-Rel, NF-B1/p50, and NF-B2/p52. Inactive NF-B is restrained in the cytoplasm of resting cells by the members of the I B family which become site-specific phosphorylated upon cell stimulation by the multi-subunit IkappaB-kinase-complex -composed of the kinases IKK1 and IKK2, and the adaptor protein NEMO-and subsequently degraded by the proteasome [START_REF] Hayden | Signaling to NF-kappaB[END_REF][START_REF] Ghosh | Missing pieces in the NF-kappaB puzzle[END_REF]. Free NF-B translocates to the nucleus where it supports the expression of various NF-B target genes. Besides this canonical NF-B signaling pathway, an alternative IKK1-dependent pathway was described which is characterized by the activation of RelB-p52 heterodimers and the expression of a specific subset of NF-B target genes [START_REF] Senftleben | Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway[END_REF]. In addition to its role in the alternative NF-B pathway, RelB is also linked to various other functions in the NF-B system. For example, RelB is part of a second wave of the NF-B response, either supporting the expression of a defined set of genes or terminating the expression of another set of genes [START_REF] Saccani | Modulation of NF-kappaB activity by exchange of dimers[END_REF]. Thus, RelB acts either as an activator or a repressor of NF-B target gene expression by mainly unknown mechanisms. One feasible model is the recruitment of co-activators or co-repressors based on specific post-translational modifications of RelB. Indeed, an interaction of RelB with the Daxx-DNMT1 co-repressor complex has recently been described [START_REF] Croxton | Daxx represses expression of a subset of antiapoptotic genes regulated by nuclear factor-kappaB[END_REF]. Post-translational modifications like phosphorylation, ubiquitination or acetylation are crucial for the diverse functions of the NF-B transcription factors [START_REF] Chen | Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB[END_REF][START_REF] Zhong | The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1[END_REF][8]. For instance, the recruitment of the co-activator CBP to the NF-B subunit RelA depends on either site-specific phosphorylation or acetylation of RelA [START_REF] Chen | Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB[END_REF][START_REF] Zhong | The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1[END_REF]. In contrast, the molecular mechanisms underlying the ambivalent functions of RelB are currently unknown. In addition to acetylation and phosphorylation steps, growing evidence point to a prominent role of non-proteasomal ubiquitination for the regulation of transcription factors. The transcriptional activity of the trancription factor c-myc, for instance, is distinctively augmented by the ubiquitination-mediated recruitment of the coactivator p300 [9]. The HectH9 mediated ubiquitination of c-myc occurs in a NLS spanning region at various lysine-residues and is crucial for the expression of c-myc target genes, the interaction with p300 and induction of cell proliferation. Here, we aimed to determine whether NF-B family members, especially RelB, are also subject of an ubiquitination-based regulatory mechanism. Recently, we reported that the NF-B family member RelB is degraded during the initiation phase of the T cell activation via the proteasomal pathway [10]. The RelB-proteolysis is regulated by a specific phosphorylation at Thr 84 and Ser 552 of RelB. Here, we demonstrate that the poly-ubiquitination of the NF-B subunit RelB is not only a required trigger for its proteolysis, but also an important signal to enhance its transactivation potential. Yet, the nuclear localization or the DNA-binding activity of RelB remained unaltered upon polyubiquitination. Remarkably, the poly-ubiquitination of RelB is highly variable, taking place at several lysine residues throughout the RelB-protein. Yet, we identified lysine residues at positions 273, 274, 305 and 308 in RelB, which are critical for both, the basal transcriptional activity and the ubiquitination-induced increase in the activity of RelB. Conclusively, we demonstrate here that the ubiquitination of NF-B subunits might be a novel regulatory mechanism in the control of the NF-B dependent gene expression.

Experimental

Cells, reagents and antibodies

Antibodies for RelB, NEMO, IKK2, , I B and the HA-tag were purchased from Santa Cruz Biotechnology. An antibody specific for poly-ubiquitinated proteins was obtained from Biomol, and the anti-FLAG antibody and anti-FLAG-antibody coupled sepharose was from Sigma. 293 HEK cells were propagated in DMEM + 10%FCS containing streptomycin and penicillin. For the cultivation of the B cell lines Namalwa, 70Z/3 and S107 RPMI-media + 10% FCS including streptomycin and penicillin was used. PMA was purchased from Sigma-Aldirch, Ionomycin and MG132 were from Calbiochem.

Expression vectors and in vitro mutagenesis

The expression vectors encoding p50, p52, NIK, full-length Flag-RelB or the various carboxy-terminal RelB-deletion mutants were described previously [11,12]. The different Flag-RelB CT and Flag-RelB 240-320 vectors were cloned by inserting PCR-generated RelB fragments in frame into the EcoRI and BamHI restriction sites of the pFLag-CMV2 vector. The expression vectors for HA-ubiquitin, K48R-, K63R-, or K48/63R-ubiquitin have been described previously [13]. The NF-B-dependent luciferase reporter construct and the renilla luciferase reporter construct under the control of the ubiquitin-promoter have been described elsewhere [14]. For the in vitro mutagenesis the QuickChange in vitro mutagenesis kit was used following the manufacturers protocol. Sequences of the oligonucleotides used for cloning or for in vitro mutagenesis are available upon request.

In vivo ubiquitination analysis HEK 293 cells were transiently transfected with expression vectors coding for HA-ubiquitin and Flag-tagged RelB and lyzed after 48 hours in TNT-buffer (200 mM NaCl, 20 mM HEPES pH 7.6, 1 % Triton X-100, 1 mM DTT, 20 mM NaF, 20 mM Na-glycerophophate, 2 µM leupeptin, 1 mM PMSF) including 0.5 % SDS or in RIPA-buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP40, 0,5% Na-deoxycholat, 0,1% SDS, 20 mM NaF, 20 mM Naglycerophophate, 2 µM leupeptin, 1 mM PMSF) as indicated and were heated for 5 minutes at 95°C to ensure the denaturation of the cellular proteins. The resulting lysates were used for anti-Flag immunoprecipitations. The precipitates were washed extensively with TNT+0.5% SDS or RIPA-buffer and PBS, and were boiled for 5 minutes at 95°C prior to the separation of the precipitated proteins by standard SDS-PAGE. The resulting membrane was subjected to immunoblot analyses with antibodies specific for HA-tag, Flag-tag or RelB, as indicated.

Immunoprecipitation and immunoblot analysis

For immunoprecipitation of ectopically expressed or endogenous proteins 0.5-1 mg protein extract was incubated with the appropriate antibody for one hour on ice prior to the addition of 25 µl of 50% slurry of ProteinA-coupled Sepharose (Santa Cruz Biotechnology) and an additional incubation for one hour under agitation. The precipitates were subsequently washed with TNT+0.5%SDS or RIPA-buffer and PBS and subjected to immunoblot analysis. For anti-FLAG immunoprecipitations 10 µl anti-FLAG antibody coupled sepharose (Sigma) was used. Immunoblots were performed according to standard protocols. In brief, the proteins were separated by SDS-PAGE on 8-10% polyacryl amid gels, transferred on nitrocellulose membrane (Whatman) and the resulting membrane was blocked for one hour in TBS+Tween20 with 5% dry milk. Subsequently, the membrane was incubated o. n. with the appropriate primary antibody in TBS+Tween20 with 5% dry milk, washed extensively, incubated for one hour in the appropriate horse-raddish peroxidase coupled secondary antibody prior to the detection with ECL substrate (Pierce).

Transfection and Luciferase reporter assay

For the transfection of 293 HEK cells we used the CaPO 4 -method. Briefly, on the day before transfection the cells were plated with a density of 50 % on an appropriate tissue culture dish. On the day of transfection the DNA was incubated with a 0.2 molar CaCl 2 solution for five minutes prior to the addition of an appropriate volume of 2xHeBS (280 mM NaCl, 1.5 mM Na 2 HPO 4 , 50 mM HEPES, pH 7.04). After another five minutes of incubation the sample was added to the cells. For the electroporation of S107 pre B cells, 1x10 7 cells were mixed with DNA, incubated on ice for 5 minutes prior to a pulse with 250 V and 950 µF. The cells were subsequently transferred to a culture dish with the appropriate media and incubated for 24-48 hours. For determination of the luciferase activity, the cells were lyzed in TNT-buffer and the activity of the renilla luciferase as well as the firefly luciferase was measured according to the Dual-Glo luciferase assay protocol (Promega). For the calculation of the basal transcriptional activity the increase in activity obtained with wild-type RelB was set arbitrarily to 100%. For the calculation of the ubiquitin-induced augmentation the increase in fold induction in samples including ubiquitin was divided by the value without ubiquitin, the resulting value for wild type RelB was set arbitrarily to 100%. Both calculations are based on at least four independent experiments and the mean value and mean deviation are stated.

Results

Since RelB is able to act either as a positive or as a negative effector of the NF-Bdependent gene expression we set out to investigate whether a non-proteasomal ubiquitination might be involved in the regulation of RelB-functions. To determine whether RelB could be ubiquitinated in vivo, we established an in vivo ubiquitination assay using ectopical expression of HA-ubiquitin, FLAG-RelB or, as positive control, FLAG-NEMO in 293 HEK cells as indicated in Figure 1A. With the resulting whole cell extracts we performed two successive rounds of anti-Flag immunoprecipitations, using first mild and in the second round more stringent conditions including the addition of 0.5% SDS. An anti-HA signal was detected after both rounds of immunoprecipitation, implying a constitutive in vivo polyubiquitination of RelB under these experimental conditions. Consistent with the hypothesis that poly-ubiquitination alters the function of RelB, we observed a significant augmentation of the RelB-activity in NF-B reportergene assays using 293 HEK cells (Figure 1B) including either increasing amounts of an ubiquitin-expression vector alone or in combination with a RelB-encoding vector. Ectopical expression of HA-ubiquitin alone had no effect on the NF-B-dependent reporter gene expression. Similar results were obtained with S107 pre B cells which display a very low basal NF-B activity due to a loss of RelB-expression [15] and which are defective in various NF-B signal pathways (Figure 1C).

Next, we determined whether the observed positive effect of ubiquitin coexpression on the RelB-activity is based on the DNA-binding capability of RelB. A RelB-mutant with a serine to alanine substitution at position 368 (S368A-RelB) is defective in DNA-binding and heterodimer formation with other NF-B factors, like p50 or p52 [12]. Consistently, S368A-RelB was found to be inactive in NF-B reportergene assays (Figure 2A). Furthermore, in contrast to the situation using wild type RelB, we observed no positive effect on the activity of S368A-RelB upon addition of ubiquitin whereas the level of poly-ubiquitination was indistinguishable between wild type and S368A-RelB (Figures 2A, lanes 4+6, and 2B, lanes 5+6) suggesting that the DNA-binding and/or the heterodimer formation is necessary for the observed ubiquitin-effect on the activity of RelB.

To determine the molecular mechanism underlying the increased RelB-activity observed in the NF-B reporter gene assay, we analyzed the potential effect of polyubiquitination on nuclear localization and DNA-binding of RelB. As shown in Figure 3A, neither the basal NF-B DNA-binding activity in 293 HEK cells nor the binding of the ectopically expressed RelB was affected by the addition of HA-ubiquitin, suggesting that the ubiquitination-dependent enhancement of the RelB-activity is not caused by an increased DNA-binding of RelB. Moreover, the analysis of the subcellular distribution of the ectopically expressed Flag-RelB revealed no differences in the nuclear RelB levels regardless of the presence or absence of HA-ubiquitin (Figure 3B, lanes 10-12). In addition, also an ubiquitin-induced moderate increase of RelB-levels, which we occasionally observed, is most likely not the basis for the increased NF-B activity since the RelB-mediated NF-B-activity remained stable even with high amounts of RelB-expression vector (Figure 3C), probably due to the lack of sufficient amounts of endogenous p50 or p52 required for active RelBcomplexes.

RelB plays a key role in B cell biology and is highly expressed in different B cell lines [16][17][18]. An analysis of the poly-ubiquitination of the endogenous RelB in Namalwa B cells revealed a moderate basal RelB-poly-ubiquitination in unstimulated Namalwa cells (Figure 4A, lane 1). This moderate RelB-poly-ubiquitination remained stable upon stimulation with TNF (Figure 4A, lanes 4+5). In contrast, the stimulation with PMA+ionomycin induced a significant increase in RelB-poly-ubiquitination (Figure 4A, compare lanes 1+2). Previously, we reported a proteasomal-dependent degradation of RelB upon PMA+ionomycin stimulation of T cells. Indeed, we also observed a decrease of cellular RelB in PMA+ionomycinstimulated Namalwa B cells (Figure 4A, compare lanes 1,2 and 3), which correlated with the appearance of a faster migrating RelB signal and an increased poly-ubiquitination of RelB (Figure 4A). Consistent with our previously published results, the pretreatment of Namalwa cells with the proteasome-inhibitor MG132 led to a partial stabilization of the faster migrating RelB degradation products (Figure 4B, compare lanes 1-3 with lanes 4-6) which correlated with an increased RelB-poly-ubiquitination suggesting that the augmented RelB-polyubiquitination observed upon PMA+ionomycin stimulation is linked to a proteasomaldependent degradation of RelB.

Lipopolysaccharide has been demonstrated to induce the alternative NF-B pathway, which depends on the activity of RelB. Thus, we analyzed whether a LPS-stimulation for 8 or 24 hours had an effect on RelB-poly-ubiquitination levels. As shown in Figure 4C, we indeed observed an increased ubiquitin signal after RelB-immunoprecipitation. Yet, also the RelBlevels were augmented suggesting that there is no net increase in RelB-poly-ubiquitination by LPS-stimulation. To further analyze whether RelB-poly-ubiquitination might be rendered triggering the alternative NF-B signaling pathway, we performed an in vivo RelBubiquitination analysis without or with the addition of the protein kinase NIK, a crucial upstream component of the alternative NF-B pathway [START_REF] Senftleben | Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway[END_REF]. Again, the levels of RelB-polyubiquitination remained largely unaltered upon NIK-coexpression. However, we observed a slight change in the pattern of the RelB-poly-ubiquitination by NIK coexpression (Figure 4D, compare lanes 9 and 11).

Based on these results, the possibility remained that the RelB-poly-ubiquitination in HEK 293 cells is simply a part of the proteasomal degradation process of RelB. However, we observed no significant reduction of the half-life time of ectopically expressed RelB with or without the additional expression of HA-ubiquitin (Figure 5A). A site-specific phosphorylation often triggers the subsequent ubiquitination of a target protein, and the degradation of RelB is linked to its phosphorylation at Thr 84 and Ser 552 [10]. Yet, RelBmutants with either inactivating alanine substitutions (Flag-RelB-ST/A) or phospho-mimetic glutamic acid substitutions (-ST/E) at Thr 84 and Ser 552 revealed no significant differences regarding their in vivo poly-ubiquitination levels (Figure 5B). These results collectively imply that the basal poly-ubiquitination of RelB is not linked to the proteolysis of this transcription factor but might be involved in the regulation of the transcriptional activity of RelB.

The lysine in ubiquitin used for poly-ubiquitin chain conjugation determines the biological function of the poly-ubiquitination, with a Lys 48 -conjugated poly-ubiquitin chain subjecting the target protein to proteasomal degradation and a Lys 63 -conjugated poly-ubiquitin chain responsible for most non-proteasomal functions. Therefore, we next compared wild type HA-ubiquitin with HA-ubiquitin mutants harboring either a K48R-or a K63Rsubstitution in an in vivo RelB-poly-ubiquitination assay. Surprisingly, both ubiquitin mutants were efficiently attached to RelB in vivo (Figure 6A, lanes 3-5). Similar results were obtained with NEMO, another known ubiquitination target-protein, which we used as positive control (Figure 6C). In addition, both ubiquitin mutants, K48R-and K63R-ubiquitin, augmented the RelB-activity in NF-B reporter gene assays (Figure 6B). Surprisingly, an efficient RelBpoly-ubiquitination in HEK 293 cells was also observed with a HA-tagged ubiquitin-mutant harboring combined arginine substitutions of lysine 48 and 63 (Figure 6D) regardless of the presence or absence of MG132 suggesting that RelB-poly-ubiquitination involves unconventional poly-ubiquitin conjugation.

In order to map the RelB-domain(s) containing the ubiquitin-acceptor site(s) we performed in vivo ubiquitination assays with different carboxy-terminal Flag-tagged RelBdeletion mutants (for a schematic representation of the mutants used, see Figure 7A). Surprisingly, all Flag-RelB mutants used were poly-ubiquitinated using either wild type or K48/63R-ubiquitin (Figure 7B, upper panel, lanes 8-12 and 14-18; please note that the low poly-ubiquitination level of full length RelB is due to a lower expression level of this protein), although the poly-ubiquitination levels were lower in case of the RelB-deletion mutant spanning the amino acids 348-552 (RelB CT ). Thus, we concluded from these results that several ubiquitination acceptor-sites must exist which are located throughout the entire RelBprotein.

To define the lysine residues critical for the activity of RelB, we performed a mutational scanning analysis substituting the 21 lysine residues in the RelB-protein by arginine residues as indicated (Figure 8A). Subsequently, the various RelB-mutants with single or combined lysine to arginine substitutions were tested regarding their basal activity and ubiquitination-induced activity-increase in NF-B reporter gene assays (Figure 8B and8C). Here, only the mutants M7 (K220R), and more pronounced M9 (K273/274R) and M11 (K305/308R) displayed a significantly reduced basal activity (Figure 8B), which could not be fully restored by the addition of p50 or p52 expression vectors -with the exception of the M7-p50 combination (Figure 8D). Interestingly, the same three RelB-mutants showed also a less pronounced ubiquitin-induced activity-increase (Figure 8C), implicating a critical role of these lysine residues for the ubiquitination-based augmentation of RelB-activity. However, all of the lysines within M7, M9, and M11 are located in the Rel Homology Domain (RHD) required for dimerization and DNA-binding of RelB. To exclude the possibility that the substitution of Lys 220 , Lys 273/274 , or Lys 305/308 affect the DNA-binding of the M7-, M9-, and M11-mutants, we tested these RelB-mutants in a gelretardation assay with a NF-B-specific probe and included the M10-mutant as control. Yet, neither of the different RelB-variants tested revealed alterations in their DNA-binding capacity (Figure 9A, upper panel, lanes 3-7). Consistent with the high degree of variability of RelB-poly-ubiquitination observed with the different deletion mutants, the in vivo poly-ubiquitination of the M7-, M9-, M10-, and M11mutants remained unaltered regardless whether wild type HA-ubiquitin (Figure 9B, lanes [START_REF] Chen | Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB[END_REF][START_REF] Zhong | The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1[END_REF][8][9][10][11] or the HA-K48/63R-ubiquitin mutant (Figure 9B, lanes 12-17) was used. Indeed, a significant effect on the in vivo poly-ubiquitination was generally only detectable upon combined substitution of various lysine residues as shown for the substitution of all lysine residues in the carboxy-terminus of RelB (K386: Lys 386 , Lys 387 and Lys 389 ; K410: Lys 410 , Lys 413 , Lys 414 , Lys 415 , Figure 10A, lanes 7-10 and Figure 10B, lanes 2-4). Taken together, these results collectively suggest that among the various ubiquitination-target sites only the poly-ubiquitination at specific lysine residues of RelB mediate the positive effect on its transcriptional activity.

Discussion

The NF-B transcription factors regulate a wide variety of cellular processes, including inflammatory and immune responses [START_REF] Ghosh | Missing pieces in the NF-kappaB puzzle[END_REF]. Several of the control mechanisms ensuring the temporal and spatial activity of NF-B include post-translational modifications of the different NF-B subunits. For example, a tightly regulated recruitment of co-activators or corepressors -like CBP or HDAC1, respectively-depends on site-specific phosphorylations or acetylations of RelA [START_REF] Chen | Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB[END_REF][START_REF] Zhong | The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1[END_REF]. In contrast, the molecular mechanisms which determine whether RelB acts as either a gene-specific transcriptional activator or repressor are currently unknown. Here, we aimed to analyze whether a specific ubiquitination of RelB might be involved in the regulation of RelB-functions. Indeed, we observed a distinct in vivo polyubiquitination of RelB upon ectopic co-expression of Flag-RelB and HA-ubiquitin as well as in unstimulated Namalwa or 70Z/3 B cells. In addition, RelB-poly-ubiquitination was further increased by stimulation of the cells with PMA+ionomycin. However, this increase in RelBpoly-ubiquitination is most likely linked to the proteasomal degradation of RelB since we observed a correlation between an augmented RelB-poly-ubiquitination and a partial RelBstabilization upon pre-treatment with the proteasome inhibitor MG132. Proteasomal degradation of various NF-B subunits, like RelA/p65, RelB or NF-B1/p50 has been described previously [10,19,20]. In contrast, the unchanged half-life time of RelB upon ubiquitin coexpression (Figure 2), the constitutive RelB-poly-ubiquitination by K48Rubiquitin (Figure 5), and the normal poly-ubiquitination of the Ser 84 -and Thr 552 -mutants of RelB (Figure 2) pointed collectively to additional non-proteasomal functions of the basal RelB-poly-ubiquitination detected in unstimulated 293 HEK, 70Z/3 and Namalwa cells (Figures 1 and4). The increased RelB-activity observed by the addition of ubiquitin, as shown by reporter gene assays, suggests that the augmentation of the transactivational potential of RelB might be one of these additional non-proteasomal functions. As an ubiquitin-induced increase in nuclear expression or DNA-binding of RelB was not observed, we would like to speculate that poly-ubiquitination of RelB might affect its interaction with transcriptional co-activators or co-repressors similar to the reported ubiquitin-dependent interaction of c-Myc with p300 [9]. This notion is also supported by the fact that the ubiquitin-effect was not observed using the S368A-RelB mutant, which is incapable of interacting with p50 or p52 and thus incapable of interacting with DNA. Indeed, RelB has been shown to interact with the repressor-protein Daxx [START_REF] Saccani | Modulation of NF-kappaB activity by exchange of dimers[END_REF][START_REF] Croxton | Daxx represses expression of a subset of antiapoptotic genes regulated by nuclear factor-kappaB[END_REF]. However, whether the RelBpoly-ubiquitination causes a reduced Daxx-interaction of this transcription factor is currently unknown. The occasionally observed increased RelB levels contribute most likely not to the augmented RelB-activity as transfection of increasing amounts of a RelB-expression vector led to similar results in NF-B reporter assays probably due to low levels of the RelBinteraction partners p50 or p52. Usually, the Lys 48 -conjugated poly-ubiquitin chains assign the ubiquitinated protein for a proteasomal degradation, whereas the formation of Lys 63conjugated poly-ubiquitin chains renders the protein-interaction pattern of the target protein [21]. However, Lys 48 and Lys 63 are not the only lysine residues in ubiquitin used for conjugation. Furthermore, also mixed ubiquitin-conjugates have been identified [22]. Thus, the efficient RelB-poly-ubiquitination seen with K48/63R-ubiquitin double-mutant could either be caused by the formation of mixed ubiquitin conjugates, probably including endogenous ubiquitin moieties, or by lysine residues other then Lys 48 and Lys 63 [23]. NEMO seems to be another protein modified by differently conjugated poly-ubiquitin chains since we obtained similar results using NEMO regarding the poly-ubiquitination with K48R-and K63R-ubiquitin. Indeed, besides the Lys 63 -conjugated NEMO-poly-ubiquitination also a Lys 6conjugated NEMO-poly-ubiquitination has been described [23]. Mapping of the RelB domains containing the poly-ubiquitination sites revealed the existence of various polyubiquitination sites in all RelB-domains analyzed. Although the low specificity of protein ubiquitination has been reported for other ubiquitination targets, for instance c-Myc or MALT1 [9, 13], the high degree of redundancy of poly-ubiquitination target-sites in RelB is still surprising. However, the ubiquitination of the different lysine residues in RelB is not equally important since only the inactivation of Lys 220 (M7) and more pronounced of Lys 273/274 (M9) or Lys 305/308 (M11) had a profound effect on their basal activity as well as the ubiquitination-based activity increase, which cannot be explained by an affected DNAbinding. The specific role of Lys 273/274 (M9) and Lys 305/308 (M11) is furthermore highlighted by the fact that the activity of the M15-mutant with a complete inactivation of the lysine residues in the carboxy-terminal TAD of RelB remained unaltered. Thus, it is possible that the polyubiquitination at these particular lysine residues (Lys 273/274 (M9) or Lys 305/308 (M11)) affects the recruitment of transcriptional co-activators or the release of co-repressors of unknown identity. The co-activator CBP, known to interact with the NF-B-subunit RelA [START_REF] Zhong | The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1[END_REF], seems to be not responsible for the ubiquitin-effect as an interaction of CBP with RelB was not detectable regardless of the absence or presence of ubiquitin (data not shown). Interestingly, poly-ubiquitination could be a general regulatory mechanism to control the activity of nuclear NF-B, probably independent of the activation of the IKK-complex. Therefore, the reason for the reduced basal transcriptional activity of the M9 and M11 RelB-mutants might be the requirement of a constitutive but moderate poly-ubiquitination of RelB. Alternatively, also other post-translational modifications occurring at lysine residues, for instance an acetylation or the attachment of Sumo moieties could be involved. Indeed, we already observed an in vivo acetylation and sumoylation of RelB in 293 HEK cells (data not shown).

Conclusively, we demonstrate here that the NF-B subunit RelB is poly-ubiquitinated at various lysine residues in vivo and the inactivation of specific lysine residues in RelB leads to a decreased transcriptional activity of this transcription factor. However, several important questions remain to be answered in future experiments. First, it will be necessary to define the nature of poly-ubiquitin chain conjugation involved in RelB-poly-ubiquitination. Another important question pertain the identity of the ubiquitin ligases or deubiquitinases involved in the poly-ubiquitination of RelB. One feasible candidate for an E3-ligase involved in the proteolysis-linked RelB-poly-ubiquitination might be COMMD1, which mediates the ubiquitination and subsequent degradation of the NF-B family member RelA [24]. Furthermore, it will be interesting to explore the molecular mechanism by which the activity of RelB is altered, especially the identity of the recruited co-activators or released corepressors, like Daxx. Finally, we want to determine whether also other NF-B family members are modified by non-proteosomal ubiquitination. Indeed, we obtained similar results regarding an in vivo ubiquitination and augmentation of the transcriptional activity using the NF-B subunit RelA (J. L. and R.M., unpublished observations) suggesting that this kind of post-translational modification could be a general regulatory mechanism for the control of the NF-B system. Footnotes Abbreviations IB: immunoblot; IP: immunoprecipitation; Lys: lysine residue; LPS; lipopolysaccharide, PMA: Phorbol 12-Myristate 13-Acetate; TNF : tumor necrosis factor; Thr: threonine residue; Ser: serine residue; CT: carboxy terminus; EMSA: electrophoretic mobility shift assay.

Figure legends Figure 1: RelB poly-ubiquitination in vivo correlates with an augmentation of its transcriptional activity.

A, For an in vivo ubiquitination assay 293 HEK cells were transiently transfected with 1µg of either FLAG-RelB or HA-ubiquitin as indicated. The cells were lyzed in TNT-buffer and 90% of the cell lysates were subjected to an anti-FLAG IP. The resulting immunoprecipitates were boiled in TNT-buffer with 0.5% SDS. 50% of the sample was used for western blot analysis (IP 1, upper part) and 50% for a second anti-FLAG IP (IP 2, middle part). To control the expression of the proteins 10% of the cell lysates were subjected to an additional western blot (input, lower part). B, 293 HEK cells were transiently transfected with a NF-B dependent luciferase reporter construct together with a ubiquitin-promoter driven Renilla luciferase reporter either alone or in conjunction with increasing amounts of a HAubiquitin expression vector (200 ng and 400 ng), an expression vector for Flag-RelB (100 ng), or a combination of both expression vectors. The cells were incubated for 24 hours, lyzed in TNT buffer, and the dual luciferase analysis was performed as described under experimental procedures. C, A similar NF-B-dependent luciferase reporter analysis was performed with S107 pre B-cells transiently transfected by electroporation with expression vectors for RelB (5 µg) or HA-ubiquitin (5 and 10 µg) as indicated.

Figure 2: The ubiquitin-effect on RelB-activity depends on the DNA-binding potential of

RelB. A, NF-B luciferase reporter assay with HEK 293 cells expressing either HA-ubiquitin, wild type Flag-RelB or Flag-S368A-RelB alone or in combination, as indicated. Twenty-four hours post transfection, the cells were harvested and the activity of the firefly as well as the renilla luciferase was determined. B, In vivo ubiquitination analysis of wild type Flag-RelB or Flag-S368A-RelB. Ectopically expressed Flag-RelB was immunoprecipitated under denaturing conditions (RIPA-buffer) with anti-Flag antibodies and probed in a subsequent immunoblot for HA-ubiquitin (upper panel) and RelB (lower panel). A fraction of the samples was subjected to anti-HA and anti-RelB immunoblots to ensure an efficient expression of the different proteins (lower part).

Figure 3: DNA-binding and nuclear expression of RelB remains unaltered upon ubiquitin co-transfection.

A, Cells were transfected with expression vectors for RelB (1 µg) and HAubiquitin (0.5 and 1 µg) as indicated. After 48 hours, the cells were lyzed and the resulting whole cell extracts were subjected to gel retardation studies with either a B-specific (upper panel) or an Oct-specific oligonucleotide. To ensure the identity of the RelB-containing complex (I) supershift analysis was performed with antibodies specific for RelA or RelB using extracts from cells transfected with RelB and HA-ubiquitin (lanes 7+8). * and ** mark unspecific bands. Western blot analysis was performed to control for the expression of Flag-RelB and HA-ubiquitin (right part). B, For the determination of the nuclear localization of RelB, nuclear and cytoplasmic extracts from 293 HEK cells which ectopically express either RelB (lanes 4+10), increasing amounts of HA-ubiquitin (lanes 2+3 and 8+9) or both proteins (lanes 5+6 and 11+12) were generated and subjected to western blot analyses with the indicated antibodies. C, A NF-B-reporter assay was performed after transient transfection of 293 HEK cells with the indicated amounts of a Flag-RelB-expression vector.

Figure 4: Ubiquitination analysis of endogenous RelB B cell lines.

A, Namalwa B cells were either left untreated or were stimulated with PMA+ionomycin (P+I) or TNF for the indicated periods of time, lyzed with RIPA-buffer and the resulting whole cell extracts were subjected to an anti-RelB IP analysis. Anti-RelB and anti-I B immunoblots were additionally performed to control the efficiency of the stimulation. B, Namalwa cells were either left untreated or were pretreated with MG132 (25 µM) for 30 minutes prior to stimulation with PMA+ionomycin for the indicated time points. The cells were lyzed in RIPA buffer, the resulting whole cell extracts were used for anti-RelB IPs prior to immunoblot analysis with the indicated antibodies. C, 70Z/3 pre B cells were either left untreated or were stimulated with 1 µg/ml LPS for 8 or 24 hours prior to a lysis in RIPA buffer. The resulting whole cell extracts were subjected to an anti-RelB IP analysis. D, In vivo ubiquitination analysis with whole cell extracts from 293 cells transiently transfected with expression vectors for Flag-RelB, HA-ubiquitin without or with an additional NIK expression vector.

Figure 5: RelB stability remains unaltered by ubiquitin coexpression. A,

To analyze the impact of ubiquitin on the half-life of RelB, cells ectopically expressing either FLAG-RelB alone (lanes 1-3) or in conjunction with HA-ubiquitin (lanes 4-6) were either left untreated or were treated with cycloheximide (50 ng/ml) for the indicated times prior to cell lysis. The resulting whole cell extracts were used for western blot analyses with the indicated antibodies. B, In vivo ubiquitination assay with either wild type FLAG-RelB (lanes 2+6), with a FLAG-RelB TS/A -mutant (T84A/S552A, lanes 3+7) or with a FLAG-RelB TS/E -mutant (T84/S552E, lanes 4+8). or K48/63R-ubiquitin (lanes 13-18). 90% of the whole cell extracts were used for an anti-FLAG IP under denaturing conditions (upper part) and 10% for control western blot analysis (input, lower panel). The anti-RelB immunoblot was included to monitor the expression levels of full length RelB.

Biochemical

  Journal Immediate Publication. Published on 11 Jul 2008 as manuscript BJ20080432 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080432 S t a g e 2 ( a ) P O S T -P R I N T

  Biochemical Journal Immediate Publication. Published on 11 Jul 2008 as manuscript BJ20080432 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080432 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 11 Jul 2008 as manuscript BJ20080432 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080432 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 6 :

 6 Figure 6: Characterization of the poly-ubiquitin conjugates attached to RelB. For an in vivo ubiquitination assay of Flag-RelB (A) or Flag-NEMO (C) 293 HEK cells were transiently transfected with 1 µg Flag-RelB or Flag-NEMO alone or in conjunction with 1 µg of expression vectors for wild type HA-ubiquitin or HA-ubiquitin mutants with an arginine substitution of either Lsy 48 (K48R) or Lys 63 (K63R) as indicated. The cells were lysed in RIPA-buffer and the resulting whole cell extracts were subjected to an anti-FLAG IP under denaturing conditions prior to immunoblot analyses with the indicated antibodies. B, NF-B reporter gene assay after transient transfection of 293 HEK cells with 100 ng of the FLAG-RelB vector alone or in combination with 400 ng of the different HA-ubiquitin variants as indicated. D, In vivo ubiquitination analysis of Flag-RelB in conjunction with a HA-K48/63Rubiquitin mutant. The cells were either left untreated (upper part) or were pretreated with the proteasome inhibitor MG132 (lower part).

Figure 7 :

 7 Figure 7: Mapping of the RelB-domain modified by ubiquitin-attachment.A, Schematic representation of the Flag-tagged RelB-deletion mutants used. B, In vivo ubiquitination assay of ectopically expressed Flag-tagged RelB deletion mutants in combination with either wild type HA-ubiquitin (lanes 7-12) or K48/63R-ubiquitin (lanes 13-18). 90% of the whole cell extracts were used for an anti-FLAG IP under denaturing conditions (upper part) and 10% for control western blot analysis (input, lower panel). The anti-RelB immunoblot was included to monitor the expression levels of full length RelB.

Figure 8 :Figure 9 :

 89 Figure 8: Identification of the lysine residues critical for the basal activity of RelB and the ubiquitin-induced augmentation of RelB-activity.A, Schematic representation of the RelBmutants with lysine to arginine substitutions. Note: Some of the mutants harbor a single mutation and several mutants contain the combined substitutions of closely located lysine residues. B, NF-B reporter gene assays. The relative basal activity of the various RelB mutants in comparison to wild type RelB is depicted. The increase in NF-B activity mediated by wild type RelB was arbitrarily set to 100%, the results from at least four independent experiments were included. C, Comparison of the ubiquitin-induced activity increase of the different RelB variants as monitored by NF-B reporter gene assays. Here, the fold increase in activity upon HA-ubiquitin co-expression in comparison to the activity of the respective RelB-variant alone is depicted in relation to wild type RelB (arbitrarily set to 100%). D, NF-B reporter gene analysis with the indicated RelB-variants alone or in combination with p50 or p52 expression vectors.

Figure 10 :

 10 Figure 10: Analysis of the poly-ubiquitination of the carboxy-terminus of RelB. A, In vivo ubiquitination analysis of the carboxy-terminal Flag-tagged RelB deletion mutants carrying either asparagine substitutions at the positions Lys 386 , Lys 387 , Lys 389 (K386N, lanes 4 and 8), at positions Lys 410 , Lys 413 , Lys 414 and Lys 415 (K410N, lanes 5 and 9) or at all positions (CT-DM), in comparison with the wild type counterpart (lanes 6 and 10). B, A similar in vivo ubiquitination analysis of wild type FLAG-RelB or the FLAG-RelB M15-mutant harboring lysine to arginine substitutions at the positions 386, 387, 389, 410, 413 and 415.

  Figure 5

Figure 7 Biochemical

 7 Figure 6

  Publication. Published on 11 Jul 2008 as manuscript BJ20080432 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080432 S t a g e 2 ( a ) P O S T -P R I N T

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ma 2367/3-1) and the German-Israeli-Foundation (2133-1599-11/2006). We thank Drs. Bernd Baumann and Thomas Wirth (Ulm University) for reagents and for the lively discussion of this project. The expression vectors for the HA-tagged K48R-and K63R-ubiquitin-mutants or the HAtagged K48/63R-ubiquitin-mutant were kindly provided by Dr. Ivan Dikic (Johann-Wolfgang-Goethe-University, Frankfurt) and Dr. Daniel Krappmann (GSF München), respectively.


Federica Gibellini

William N Hunter

Terry K Smith

BIOCHEMICAL CHARACTERISATION OF THE INITIAL STEPS OF THE KENNEDY PATHWAY IN TRYPANOSOMA BRUCEI -THE ETHANOLAMINE AND CHOLINE KINASES

Ethanolamine (EtN) and choline (Cho) are major components of the trypanosome membrane phospholipids, in the form of phosphatidylethanolamine (GPEtn) and phosphatidylcholine (GPCho). Ethanolamine is also found as an integral component of the glycosylphosphatidylinositol (GPI) anchor that is required for membrane attachment of cell surface proteins, most notably the variant surface glycoproteins.

The de novo synthesis of GPEth and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative choline/ethanolamine kinases (C/EKs) in the T. brucei genome, which were cloned, over-expressed, purified and characterised. TbEK1 was shown to be catalytically active as an ethanolaminespecific kinase, i.e. no choline kinase activity. The Km values for ethanolamine and ATP were found to be 18.4 ± 0.9 µM and 219 ± 29 µM, respectively. TbC/EK2, on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a Km 80 times lower than that of ethanolamine. The Km values for choline, ethanolamine and ATP were; 31.4 ± 2.6 µM, 2.56 ± 0.31 mM and 20.6 ± 1.96 µM respectively. Further substrate specificity analysis revealed both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C2 but substitutions on C1 and elongations of the carbon chain were not well tolerated.

INTRODUCTION

The zwitterionic glycerophospholipids phosphatidylcholine (GPCho) and phosphatidylethanolamine (GPEtn) are the two most abundant phospholipid species in eukaryotic cells and many bacteria [START_REF] Dowhan | Molecular basis for membrane phospholipid diversity: Why are there so many lipids?[END_REF]. In the parasitic protozoa Trypanosoma brucei, the aetiology of African sleeping sickness, GPCho accounts for approximately half of all phospholipids in both life cycle stages, whereas GPEtn forms between sixteen and twenty-one percent [START_REF] Patnaik | Molecular species analysis of phospholipids from Trypanosoma brucei bloodstream and procyclic forms[END_REF]. These phospholipids contribute an important structural role to the membrane and in addition determine membrane fluidity and cell surface charge.

Their biosynthesis and utilisation is, not surprisingly, implicated in a variety of cellular processes [START_REF] Dowhan | Molecular basis for membrane phospholipid diversity: Why are there so many lipids?[END_REF]. In mammals GPCho is an important source of signalling molecules and the biosynthetic intermediate phosphocholine (PCho) has been reported to be a mitogen required for DNA synthesis induced by growth factors [START_REF] Cuadrado | Phosphorylcholine: a novel second messenger essential for mitogenic activity of growth factors[END_REF].

Due to its physical-chemical properties GPEtn has a tendency to form non-bilayer structures and its incorporation into membranes affects the processes of membrane trafficking and the folding, stabilization and incorporation of certain proteins into membranes [START_REF] Bürgemaister | Contribution of different biosynthetic pathways to species selectivity of aminoglycerophospholipids assembled into mitochondrial membranes of the yeast Saccharomyces cerevisiae[END_REF]. Moreover, GPEtn is utilised in the biosynthesis of glycosylphosphatidylinositol (GPI), a complex glycolipid that anchors proteins to the exterior leaflet of eukaryotic plasma membranes [START_REF] Imhof | Phosphatidylethanolamine is the donor of the phosphorylethanolamine linked to the α-1,4-linked mannose of yeast GPI structure[END_REF][START_REF] Menon | Phosphatidylethanolamine is the donor of the phosphoethanolamine group in trypanosome glycosyl-phosphatidylinositol[END_REF][START_REF] Menon | Phosphatidylethanolamine is the donor of the ethanolamine residue linking a glycosylphosphatidylinositol anchor to protein[END_REF]. This is particularly important for the bloodstream form of T. brucei, which expresses a dense cell-surface coat (10 7 molecules per parasite) of a GPI-anchored Variant Surface Glycoprotein (VSG). This coat prevents attack by components of the innate immune system and by undergoing antigenic variation, allows the parasite to evade a specific immune response by the host.

The biosynthesis of the GPI-anchor has been genetically [START_REF] Nagamune | Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei[END_REF][START_REF] Chang | Cloning of the Trypanosoma brucei and Leishmania major genes encoding the GlcNAc-phosphatidylinositol de-N-acetylase of biosynthesis that is essential to the African sleeping sickness parasite[END_REF][10] and chemically validated [START_REF] Smith | Chemical validation of GPI biosynthesis as a drug target against African sleeping sickness[END_REF] as a drug target in bloodstream form T. brucei. Thus disruption of the biosynthesis of GPEtn could prevent the formation of mature GPI anchored VSG and lead to clearance of the parasite by the host's immune system.

Due to the major structural and functional roles of GPCho and GPEtn in T. brucei, disruption of their biosynthetic pathways is likely to interfere with the parasite biology in multiple ways and thus the enzymes involved become of interest as possible novel targets for chemotherapy. Indeed it has been proposed that the mode of action of lysophospholipid analogues (miltefosine, edelfosine, ilmofosine) currently used against other trypanosomatid parasites, i.e. Leishmania, are involved in the inhibition of GPCho biosynthesis [START_REF] Urbina | Mechanisms of action of lysophospholipid analogues against trypanosomatid parasites[END_REF].

GPEtn and GPCho are de novo synthesised via two branches of the same metabolic pathway; the Kennedy pathway [START_REF] Kennedy | function of cytidine coenzymes in the biosynthesis of phospholipids[END_REF] (Fig. 1). Ethanolamine (EtN) and choline (Cho) are readily phosphorylated to generate phosphoethanolamine (P-EtN) and phosphocholine (P-Cho), which are subsequently activated to high-energy donors; CDP-EtN and CDP-Cho respectively, before they are coupled to diacylglyceride (DAG) to form GPEtn and GPCho. For this reason the two branches of the Kennedy pathway are often referred to as the CDP-Etn or CDP-Cho pathways. A second major pathway for the formation of GPEtn is via phosphatidylserine (GPSer) decarboxylation: GPSer, which is formed through the action of a GPSer synthase (PSS), is decarboxylated by a GPSer decarboxylase (PSD) to form GPEtn. In most eukaryotic cells GPCho can also be generated from GPEtn, by three methylation steps. However, according to the tri-Tryp genomes (http://www.genedb.org) these methyl-transferase genes are neither present in T. brucei, nor T.cruzi, but interestingly are present in Leishmania, this has been confirmed experimentally (Smith, unpublished data).

Although the presence of the Kennedy pathway was demonstrated in T.brucei [START_REF] Rifkin | Specificity of ethanolamine transport and its further metabolism in Trypanosoma brucei[END_REF]

EXPERIMENTAL PROCEDURES

Organisms and reagents

Trypanosoma brucei strain 427 was used as a source of genomic DNA. Escherichia coli strains DH5α, XL-1 blue and TOP10 (Invitrogen) competent cells were used for routine manipulation, and strain BL21(DE3)Gold (Novagen) for protein overexpression. All chemicals were purchased from either Sigma-Aldrich or Fluka.

Restriction endonucleases and DNA modifying enzymes were from New England Biolabs or Promega. [2-3 H] Ethanolamine (50.0 Ci/mmol) and [methyl-3 H] Choline (82.0 Ci/mmol) were purchased from Amersham.

RNA isolation and cDNA synthesis

Total RNA was isolated from bloodstream form T. brucei using the RNeasy mini kit (Qiagen). A TbC/EK2 specific cDNA was generated and amplified using a mini-exon

Expression and Purification of TbC/EK1 and TbC/EK1

Overproduction and sample preparation

The pET15bTEV-TbC/EK1 and the pET15bTEV-TbC/EK2 constructs were transformed in BL21(DE3)Gold cells and clones selected on LB-agar plates containing carbenicillin (100 µg/ml). Single colonies were grown at 37°C in LB medium containing carbenicillin (50 µg/ml) until OD 600nm was between 0.5 and 0.

Purification and gel filtration

The cleared lysate was applied to a 1 ml HisTrap TM FF crude column or a 5 ml HisTrap TM column (GE-Healthcare) preloaded with Ni 2+ . Unbound proteins were removed by washing the column with 15 column volumes of buffer A, while

TbC/EK1 and TbC/EK2 were eluted with an imidazole gradient in the same buffer.

Assay of TbC/EK activity

Ethanolamine/choline kinase activity was measured by a spectrophotometric coupled assay as described previously [START_REF] Choubey | Molecular characterization and localization of Plasmodium falciparum choline kinase[END_REF]. The assay contained 50 mM MOPS pH 7.8, 150 The Brenner's motif (Figure 2) has been identified in many enzymes catalysing phosphotransfer reactions, such as protein kinases and atypical kinases (the superfamily that includes the choline/ethanolamine kinase family) [START_REF] Scheef | Structural evolution of the protein kinase-like superfamily[END_REF]. The second conserved cluster (Figure 2) is found specifically in members of the choline/ethanolamine kinase family and is referred to as the choline kinase motif.

Previous site-directed mutagenesis studies and structural studies in Caenorhabditis elegans CKα2 [START_REF] Yuan | Identification of critical residues of choline kinase A2 from Caenorhabditis elegans[END_REF][START_REF] Peisach | The crystal structure of choline kinase reveals a eukaryotic protein kinase fold[END_REF], and the analysis of the crystal structures of human CKα [START_REF] Malito | Elucidation of human choline kinase crystal structures in complex with the products ADP or phosphocholine[END_REF] highlighted the importance of several of the amino acids in substrate binding. Of particular importance are the two Asp residues marked by an asterisk in the alignment (Figure 2). These residues are involved in the binding of ATP and the stabilization of the transition state of the reaction and their mutation leads to complete enzyme inactivation [START_REF] Yuan | Identification of critical residues of choline kinase A2 from Caenorhabditis elegans[END_REF][START_REF] Malito | Elucidation of human choline kinase crystal structures in complex with the products ADP or phosphocholine[END_REF].

Recombinant expression and purification of TbC/EK1 and TbC/EK2

In order to confirm that the putative TbE/CK1 and TbE/CK2 are catalytically active, the two ORFs were cloned into the vector pET-15bTEV. This vector encodes an Nterminal hexa-histidine epitope-tag followed by a TEV protease site, which allowed purification of the recombinant proteins via nickel-ion affinity chromatography (Fig. 3A andB). MALDI mass spectrometry of the recombinant TbC/EK1 and TbC/EK2

were very close to their theoretical masses of 51655 and 72614 Da respectively. The proteins TbC/EK1 and TbC/EK2 were further purified by gel-filtration (Fig. 3C).

Elution from the gel filtration column showed that both TbC/EK1 (filled arrow) and

TbC/EK2 (empty arrow) exhibit a dimeric state in solution (Fig. 3D). This is similar to most of the choline/ethanolamine kinases from other sources [START_REF] Gee | Multiple isoforms of choline kinase from Caenorhabditis elegans: cloning, expression, purification, and characterization[END_REF][START_REF] Ayoama | Structure and function of choline kinase isoforms in mammalian cells[END_REF][START_REF] Kim | Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae[END_REF] with the notable exception of the monomeric P. falciparum choline kinase [START_REF] Choubey | Molecular characterization and localization of Plasmodium falciparum choline kinase[END_REF]. Typical yields were 10 mg and 15 mg per litre of bacterial culture for TbC/EK1 and TbC/EK2, respectively. TbC/EK1 was found to lose activity over time, being completely lost after a couple of days. It is also prone to precipitation upon freeze thawing, and therefore assays were preformed on freshly prepared batches. TbC/EK2 on the other hand was stable and freeze thawing did not lead to loss of activity or precipitation.

Kinetic analysis of TbC/EK1 and TbC/EK2

Previously characterised choline/ethanolamine kinases from various sources [START_REF] Choubey | Molecular characterization and localization of Plasmodium falciparum choline kinase[END_REF][START_REF] Gee | Multiple isoforms of choline kinase from Caenorhabditis elegans: cloning, expression, purification, and characterization[END_REF][START_REF] Ayoama | Structure and function of choline kinase isoforms in mammalian cells[END_REF][START_REF] Kim | Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae[END_REF][START_REF] Clary | Substrate specificity of choline kinase[END_REF][START_REF] Wharfe | Lipid metabolism in germinating seeds. Purification of ethanolamine kinase from bean[END_REF] have exhibited different specificity towards the two substrates; choline and ethanolamine. It was therefore necessary to test the activity of the purified proteins in order to understand if they were able to catalyse the phosphorylation of both choline and ethanolamine or if they showed selectivity to each branch of the Kennedy pathway. TbC/EK1 was found to be able to catalyse the formation of phosphoethanolamine from ethanolamine, in an ATP and magnesium dependant manner (Fig. 4A andB).

However the enzyme was unable to catalyse the formation of phosphocholine from choline, in the presence of ATP and magnesium (Tables 1 and2). This restricts the specificity of this kinase to the CDP-ethanolamine branch of the Kennedy pathway and for this reason TbC/EK1 was renamed TbEK1.To our knowledge this is the first ethanolamine-specific kinase to be recombinantly expressed, purified to homogeneity and extensively characterised.

Kinetic analysis showed that TbEK1 displayed a pH optimum at pH 8.0 (Fig. 5A), with a Km of 18.4 ± 0.9 µM for ethanolamine (Fig. 5B) and a Km of 219 ± 29 µM for ATP (Fig. 5C), which is in reasonable agreement with a previously reported Km value of 2.75 µM for ethanolamine kinase activity from crude trypanosome homogenates [START_REF] Rifkin | Specificity of ethanolamine transport and its further metabolism in Trypanosoma brucei[END_REF]. The Vmax of TbEK1 is 7.62 ± 0.07 µmol/min/mg, corresponding to a kcat of 6.56 s -1 (Table 1), this turnover rate / specific activity compares favourably with the previously reported activities of 346 nmol/min/mg for the yeast EK [START_REF] Kim | Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase[END_REF] and 10.73 µmol/min/mg for a soybean EK [START_REF] Wharfe | Lipid metabolism in germinating seeds. Purification of ethanolamine kinase from bean[END_REF]. Two catalytically inactive mutants of TbEK1

were generated by substitution of Asp267 and Asp286 (Fig. 4B andC) with Ala, thus confirming the crucial importance of these two residues in the catalytic mechanism of this kinase.

TbC/EK2, on the other hand, was found to be able to phosphorylate both ethanolamine (Figure 6B) and choline (Figure 6A and 6C), even though choline was the preferred substrate (Table 1 and2), with a Km 80 times lower than that of ethanolamine. TbC/EK2 was also dependent upon ATP and magnesium for activity (Figure 6B and 6C) with a pH optimum of 7.8 (Fig. 7A). Kinetic analysis showed that TbC/EK2 displayed Km values for choline, ethanolamine and ATP of 31.4 ± 2.6 µM, 2.56 ± 0.31 mM and 20.6 ± 1.96 µM respectively with a Vmax of 10.67 ± 0.18 µmol/min/mg and a kcat of 12.91 s -1 (Figures 7B-D, Table1). This value is in the range of activities reported for other eukaryotic C/EKs; and more than ten times higher than the choline kinase from P. falciparum [START_REF] Choubey | Molecular characterization and localization of Plasmodium falciparum choline kinase[END_REF]. Since the TbC/EK2 Km value for ethanolamine is several orders of magnitude higher than TbEK1 and the fact that levels of ethanolamine in human plasma are in the micromolar range [START_REF] Baba | High-performance liquid chromatographic determination of serum aliphatic amines in chronic renal failure[END_REF], one can reasonably assume that TbEK1 accounts for the vast majority of the ethanolamine-phosphate formation in the trypanosome. There were no differences in the kinetics between the His-tagged and untagged (TEV cleaved) versions of either TbEK1 or TbC/EK2, thus the tagged versions were used as this decreases preparation time and increases stability, especially important for TbEK1.

Acceptor specificity of TbEK1 and TbC/EK2

The ability of the two enzymes to utilise chemically modified substrates for the phosphotransfer reaction was investigated using a variety of ethanolamine and choline analogues, with modifications either at the amino-or the hydroxyl-moieties, or with additional substituents on the two-carbon backbone. Each analogue was compared with ethanolamine (for TbEK1) or choline (for TbC/EK2) at a constant substrate concentration (1 mM) and a fixed, saturating concentration of ATP to give a relative rate (ethanolamine, choline = 100) (Tables 234).

TbEK1 was able to tolerate various modifications at the amino group, including secondary and tertiary amino modifications despite having been shown

earlier not to process choline, (Table 2). As shown in Table 3 single methyl and ethyl substituents on the carbon backbone at C2 (R(-)2-amino-1-butanol, S(+)2-amino-1butanol, R(-)2-amino-1-propanol, S(+)2-amino-1-propanol) are well tolerated, but the introduction of a second methyl group at C2 (2-amino-2-methyl-1-propanol) or a negatively charged carboxyl group (L-serine) resulted in greatly reduced or non detectable activity, respectively. Methyl substitutions on C1 (R(-)1-amino-2-propanol,

S(+)1-amino-2-propanol)

diminished the ability of the compound to act as a substrate, but to varying degrees in a stereospecific fashion. The increase of the carbon backbone length by one methylene unit (3-amino-1-propanol) led to a pronounced decrease in activity, whereas the increase by an ethylene unit (4-amino-1-butanol), completely eliminated substrate activity.

TbC/EK2 was able to accept analogues modified at the amino group as substrates of the reaction, in fact secondary and tertiary amino modifications were better than choline. Notably, of the compounds tested, ethanolamine was the poorest acceptor substrate (Table 2), these findings are in contrast to those with CK from brewer's yeast [START_REF] Clary | Substrate specificity of choline kinase[END_REF], where N-methylethanolamine and N,N-dimethylethanolamine were as poor substrates as ethanolamine. Compounds with methyl and ethyl alterations on C2 are good substrates only in the (S) configuration; R(-)2-amino-1butanol and R(-)2-amino-1-propanol are much poorer substrates, as well as 2-amino-2-methyl-1-propanol, which bears two methyl groups at C2 (Table 3). A reduction of activity is seen with the elongation of the carbon chain, but it is not abolished even when increased by an ethylene unit (4-amino-1-butanol). The introduction of a negative charge (L-serine) as well as the introduction of substituents at C1 (R(-)1amino-2-propanol, S(+)1-amino-2-propanol) are not tolerated (Table 3). That the enzyme is relatively more tolerant of substituents on C2 is in agreement with the previous studies on the yeast orthologue [START_REF] Clary | Substrate specificity of choline kinase[END_REF]. As expected, modifications at the hydroxyl group (Table 4) resulted in no detectable reactivity for both TbEK1 and TbC/EK2.

Analogues deemed as non-substrates were tested as potential inhibitors by preincubation at 1 mM for 5 min prior to the addition of ethanolamine or choline (0.1 mM) to TbEK1 and TbC/EK2 respectively. However, none of the potential inhibitory analogues showed any inhibitory effect (<5 %) on either TbEK1 or TbC/EK2 activity.

Previous studies in T. brucei [START_REF] Rifkin | Specificity of ethanolamine transport and its further metabolism in Trypanosoma brucei[END_REF] demonstrated that ethanolamine analogues, such as N-methylethanolamine, N-ethylethanolamine, N,N-dimethylethanolamine, S-(+)-2-amino-1-butanol, R-(-)-2-amino-1-butanol, can be recognized by the ethanolamine transport system, whereas choline is not. There was ambiguity as to whether these compounds were acting as inhibitors of the transporter or merely competing with ethanolamine to be transported into the cell. If the latter case were true, our results suggest these analogues could be phosphorylated by either TbEK1

FOOTNOTES

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession numbers AM939568 and AM939569.

ABBREVIATIONS

The abbreviations used are: GPEtn, phosphatidylethanolamine;

Biochemical

  Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T

  the constituent enzymes have never been characterized. Here, we report investigations into the first steps of both branches of the Kennedy pathway, involving molecular cloning, recombinant expression, purification and detailed kinetic characterisation of both ethanolamine and choline kinases (EK and CK) from T. brucei. Biochemical Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T

3 '

 3 specific for the sequence of TbC/EK2 using the SuperScript III One step RT-PCR kit with Platinum Taq (Invitrogen). The amplified cDNAs were purified (QIAquick PCR purification kit, Qiagen), ligated into pCR®-Blunt II TOPO® (Invitrogen) and sequenced.Cloning of T. brucei EK1 gene and construction of the pET15b-TbEK1 expression vectorsUsing the Saccharomyces cerevisiae ethanolamine kinase gene sequence as a query, two putative C/EK genes were identified in the T. brucei genome database (Sanger Centre, http://www.genedb.org) using tBlastN. The two putative open reading frames (Tb11.18.0017 TbC/EK1, and Tb927.5.1140 TbC/EK2) together with ~ 350bp of their 5'-and 3'-untranslated regions, were amplified from T. brucei strain 427 genomic DNA using Pfu DNA polymerase and the forward and reverse primers 5; 5'-ATAAGTAAGCGGCCGCAAGTGCGTTGTGCAGGTCGGCGACGGT-3' and 5'-ATAAGTAAGCGGCCGCCGTTGGAAGGAGGAAAACGGCCGAGG-Biochemical Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

3 '

 3 for C/EK2. The resulting 2.1 Kb (C/EK1 and UTRs) and 3.7 Kb (C/EK2and UTRs) fragments were cloned into pCR®-Blunt-II TOPO® vector (Invitrogen). Clones were sequenced and compared with the annotated Gene Data Bank sequences. A BamHI restriction site, internal to the TbC/EK1 open reading frame was silenced by site Directed Mutagenesis Kit (Stratagene). The TbC/EK1 open reading frame was PCR amplified from the TOPO® construct with Pfu polymerase using the forward and reverse primers 5amplicon was purified (QIAquick PCR purification kit, Qiagen) and ligated into pCR®-Blunt II TOPO® (Invitrogen) and sequenced. Using the NdeI and BamHI restriction site (underlined in primer sequences) the putative TbC/EK1 was ligated into the expression vector pET-15b (Novagen) modified with a Tobacco Etch Virus (TEV) protease cleavage site (in place of a thrombin cleavage site) via the same restriction sites generating the pET15bTEV-TbC/EK1 construct. The same procedure was employed to ligate the putative TbC/EK2 into the same vector using the forward and reverse primers 5'-GGAATTCCATATGATGGCATTACGACCGTTCCCG-3' and 5'-CGCGGATCCGCGTCAGGAAAGAAGCCCCTTCTC-3'. The mutants of TbEK1 D287A and D267A were generated by using the QuickChange Site Directed Mutagenesis Kit with the forward and reverse primers 5lower case letters in the primers highlight the mutated bases). All DNA sequencing was performed by The Sequencing Service (College of Life Sciences, University of Dundee, Scotland, www.dnaseq.co.uk) using Applied Biosystems Big-Biochemical Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.

Fractions

  containing TbC/EK1 or TbC/EK2 were pooled, dialysed against 50 mM Tris-HCl pH 8, 300 mM NaCl and further purified through a Superdex200 16/60 gel filtration column (GE-Healthcare), run in the same buffer at a rate of 1 ml/min. Low and High Molecular Weight Calibration Kits (GE-Healthcare) were used to determine the molecular weight and the oligomeric state of the proteins. Purity of protein samples was assessed by SDS-PAGE and matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (Proteomics Facility, College of Life Sciences, University of Dundee).

Biochemical

  Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N TmM KCl, 6 mM MgCl 2 , 0.5 mg/ml BSA, 1 mM phosphoenolpyruvate, 0.5 mM NADH, 7 units/ml pyruvate kinase, 32 units/ml lactate dehydrogenase, 2 µg/ml TbC/EK1 or 1 µg/ml TbC/EK2. Concentrations of ATP and ethanolamine (or choline) were varied and the rate of the reaction monitored by the decrease in absorbance at 340 nm (consequence of NADH oxidation) in a Shimadzu UV-1601 Spectrophotometer. Enzyme Kinetics, SigmaPlot (SPSS Inc.), was used to analyse and fit the kinetic data.The pH optimum was assessed by measuring TbC/EK1 and TbC/EK2 activities at various pHs: sodium acetate (pH 5.0), Bis-Tris propane or MOPS (pH 6.5), Tris-HCl (pH 7.0, 8.0, 8.5), MOPS (pH 6.0, 7.0, 7.4, 7.8) and glycine (pH 9.0). Various ethanolamine and choline analogues were tested as substrates at least in triplicate on 96 wells plates using a SpectraMAX 340PC plate reader (Molecular Devices) and those deemed as non-substrates were tested as inhibitors by preincubation at 1mM for 5 min prior to the addition of ethanolamine or choline (0.1mM).Direct ethanolamine and choline kinase activity assays were performed by assessing the production of phosphoethanolamine or phosphocholine, using a modified method of Kim et. al.,[START_REF] Kim | Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase[END_REF]. Briefly, 2 µg of purified protein is incubated with a reaction mixture (total volume 50 µl) of 100 mM MOPS pH 7.8, 6 mM MgCl 2 , 5 mM ATP, with either 2 mM ethanolamine and 0.2 µCi [ 3 H]ethanolamine or 2 mM choline and 0.2 µCi [ 3 H]choline at 37°C for 20 minutes and quenched by boiling at 100°C for 5 min. Substrates and products were separated by HPTLC using silica 60 plates with methanol: 0.6% sodium chloride: ammonium hydroxide (10:10:1, v/v) as solvent.Radiolabelled species were detected by fluorography at -70 o C after spraying with En 3 hance and using Kodak XAR-5 film with an intensifying screen. Unlabelled ethanolamine and phosphoethanolamine standards were run in parallel and visualized by spraying with ninhydrin (0.2% in water-saturated butan-1-ol). Unlabelled phosphocholine was run in parallel and visualised by iodine staining. Biochemical Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T RESULTS AND DISCUSSION Genomic identification and cloning of Tb C/EK1 and 2 Two putative Cho/EtN kinases were identified in the T. brucei genome database (Sanger Centre); these putative ORFs were PCR-amplified from genomic DNA, cloned and sequenced. Two potential initiating methionines were detected in the TbC/EK2 ORF, this ambiguity was resolved by amplification and sequencing of the first ~350 bp of the RT-PCR product using total RNA, a forward primer against the mini-exon and a reverse primer specific for the TbC/EK2 ORF. From the cDNA sequence (supplementary Fig 1) the second methionine is identified as the initiating residue. Interestingly there seems to be extra 'stuffer' sequence (65 bp) between this and the mini-exon (supplementary Fig 1), this may represent some form of regulatory element, a concept we are currently investigating. The predicted molecular masses of the deduced amino acid sequences are 49.1 kDa (431 amino acids) for TbC/EK1 and 70.1 kDa (628 amino acids) for TbC/EK2. An alignment of the translated sequences of TbC/EK1 and 2, with previously characterised or putative C/EKs from other organisms (Figure 2) shows significant similarities. Even though the amino-terminal region is not generally well conserved amongst C/EKs, the carboxy-terminal portion contains two highly conserved regions.

  Biochemical Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T Not surprisingly, TbC/EK1 and 2 are most closely related to C/EKs from other kinetoplastids. In the region of the alignment shown in Figure 2 TbC/EK1 is 63% identical to a putative C/EK from T. cruzi and TbC/EK2 is 40% identical to a putative C/EK from L. major. The similarity towards previously characterised C/EKs from human, yeast, C. elegans and Plasmodium falciparum is much lower (data not shown), with the exception of the Brenner's and the choline kinase motives. The TbC/EK1 and TbC/EK2 sequences have been submitted to GenBank Nucleotide Sequence Database with Accession numbers AM939568 and AM939569 respectively.

Biochemical

  Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  and/or TbC/EK2, and possibly proceed further in the Kennedy pathway leading to the formation of unusual phosphatidylethanolamine analogues, a concept we are currently pursuing. Biochemical Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T CONCLUSIONS African sleeping sickness is a neglected disease, which urgently requires new drug therapies. Characterising fundamental differences in the biochemical pathways and enzymes therein, of the parasite and its mammalian host is an important first step towards this goal. Thus, we present here for the first time, the identification and detailed molecular characterization of the two putative choline/ethanolamine kinases in T. brucei. TbEK1, is committed to only the CDP-ethanolamine branch of the Kennedy pathway since it is unable to phosphorylate choline; TbC/EK2 on the other hand, was able to phosphorylate choline much more efficiently than ethanolamine. The substrate specificities for each kinase were determined, providing crucial information on the structural requirements for ligand recognition. Analysis of the roles of TbEK1 and TbC/EK2 in parasite survival will be crucial to investigate their potential as drug targets for the development of new chemotherapeutic agents against African sleeping sickness and perhaps other protozoan parasites. The similarities in ligand recognition displayed by TbEK1 and TbC/EK2 may guide the design of inhibitors able to target both enzymes at once. Further work will characterise and assess the importance of all steps of the two branches of the Kennedy pathway, as well as their functional role in the biology of the trypanosome. Biochemical Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. A Tcoffee alignment of the predicted amino acid sequences of Trypanosoma brucei C/EK 1 and 2 with choline and ethanolamine kinases of other eukariotic organisms: Trypanosoma cruzi (Q4E3A9), Leishmania major (Q4FYH6, Q4FWX2) and Homo sapiens (Q9HBU6). Residues are shaded according to percentage similarity using the software Jalview 2.08.1. The boxes highlight the choline kinase motif and the Brenner's phosphotransferase motif. The two Asp residues marked by an asterisk were mutated in TbEK1 to generate the catalytically inactive mutants D267A and D286A.

Figure 3 .Fig. 4 .

 34 Figure 3. Expression and purification of recombinant TbC/EK1 and TbC/EK2 in E. coli. (A) TbC/EK1 protein samples from each purification step were separated on a 4-12 % SDS-PAGE gel and stained with Coomassie brilliant blue. Lane1, Total E. coli cellular protein after induction; Lane 2, Cleared cell lysate; Lane 3, nickel column wash at 20 mM imidazole; Lane 4, nickel column wash at 50 mM imidazole; Lanes 5-8 subsequent elutions from the nickel column at 250 mM imidazole.

Fig. 5 Fig. 6 .

 56 Fig. 5 Kinetic analysis of TbC/EK1 (A) Ethanolamine kinase activity was measured spectrophotometrically as a function of pH as described in Experimental. Data are presented as mean ± SD from three measurements. (B,C) Determination of TbEK1 Michaelis-Menten constants for ethanolamine and ATP (inserts Lineweaver-Burk plots). (B) ATP concentration was held constant (5 mM) while ethanolamine concentration was varied. (C) Ethanolamine concentration was held constant (2 mM) while ATP concentration was varied.

Fig. 7

 7 Fig. 7 Kinetic analysis of TbC/EK2 (A) Choline kinase activity was measured spectrophotometrically as a function of pH as described in Experimental. Data are presented as mean ± SD from three measurements. (B,C,D) Determination of TbC/EK2 Michaelis-Menten constants for choline, ethanolamine and ATP (inserts Lineweaver-Burk plots). ATP concentration was held constant (5 mM) while choline (B) or ethanolamine (C) concentrations were varied. (D) Choline concentration was held constant (2 mM) while ATP concentration was varied.

( a )

 a Analogues are compared with ethanolamine at the constant concentration of 1 mM and a saturating concentration of ATP to give a relative rate (ethanolamine = 100) (b) Analogues are compared with choline at the constant concentration of 1 mM and a saturating concentration of ATP to give a relative rate (choline = 100) Biochemical Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T

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  (a) (b) See Table2

Biochemical

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Figure 1 CholineFigure 2 BiochemicalFigure 3 BiochemicalFigure 4 Biochemical

 1234 Figure 1

Figure 5 Biochemical

 5 Figure 5

Figure 6 Biochemical

 6 Figure 6

Figure 7 Biochemical

 7 Figure 7

Table 1 : Kinetic values for recombinantly expressed and purified TbEK1 and TbC/EK2

 1

	Enzyme	Vmax (µmol/ min/mg protein)	Km Cho (µM) Km Etn (µM)	Km (µM)	ATP	kcat (s -1 )	kcat/Km Cho (s -1 mM -1 )	kcat/Km Etn (s -1 mM -1 )	kcat/Km ATP (s -1 mM -1 )
	Tb EK1	7.62 ± 0.07	0.0 ± 0.0	18.4 ± 0.9	219.2 ± 29	6.56	-	356.53	29.93
	Tb CK2	10.67 ±0.18	31.4 ± 2.6	2600 ± 310	20.6 ± 1.96	12.91	411.25	5.04	626.86

Biochemical Journal Immediate Publication. Published on 20 May 2008 as manuscript BJ20080435 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080435 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 2 : Substrate specificity of TbEK1 and TbC/EK2 to utilise analogues modified at the amino group of the acceptor substrate.

 2

	TbEK1	TbC/EK2

Table 3 : Substrate specificity of TbEK1 and TbC/EK2 to utilise analogues modified by extra substitutents on the carbon backbone of the acceptor substrate.

 3

	TbEK1	TbC/EK2

Table 4 : Substrate specificity of TbEK1 and TbC/EK2 to utilise analogues modified at the hydroxyl group of the acceptor substrate.

 4

	TbEK1	TbC/EK2

© 2008 The Authors Journal compilation © 2008 Biochemical Society

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ACKNOWLEDGEMENTS

This work was supported by a Wellcome Trust Senior Research Fellowship 067441(TKS), Wellcome Trust grants 082596 and 083481 (WNH) and a Wellcome Trust PhD studentship (FG).