Leishmania Parasites Reveals a Unique Stage-Specific P

Article pubs.acs.org/jpr

Phosphoproteomic Analysis of Differentiating Leishmania Parasites Reveals a Unique Stage-Specific Phosphorylation Motif Polina Tsigankov,† Pier Federico Gherardini,‡ Manuela Helmer-Citterich,‡ Gerald F. Spaẗ h,§ and Dan Zilberstein*,† †

Technion-Israel Institute of Technology, Haifa 32000, Israel Center for Molecular Bioinformatics, Department of Biology, University of Rome Tor Vergata, Rome, Italy § Institut Pasteur, CNRS URA2581, Unité de Parasitology moléculaire et Signalisation, 75015 Paris, France ‡

S Supporting Information *

ABSTRACT: Protists of the genus Leishmania are obligatory intracellular parasites that cause a wide range of cutaneous, mucocutaneous, and visceral diseases in humans. They cycle between phagolysosomes of mammalian macrophages and the sand fly midgut, proliferating as intracellular amastigotes and extracellular promastigotes, respectively. Exposure to a lysosomal environment, i.e. acidic pH and body temperature, signals promastigotes to differentiate into amastigotes. Time course analyses indicated that Leishmania differentiation is a highly regulated and coordinated process. However, the role of posttranslational events such as protein phosphorylation in this process is still unknown. Herein, we analyzed and compared the phosphoproteomes of L. donovani amastigotes and promastigotes using an axenic host-free system that simulates parasite differentiation. Shotgun phosphopeptide analysis revealed 1614 phosphorylation residues (p-sites) corresponding to 627 proteins. The analysis indicated that the majority of the p-sites are stage-specific. Serine phosphorylation in a previously identified trypanosomatid-specific “SF” motif was significantly enriched in amastigotes. We identified a few phosophotyrosines (pY), mostly in proteins known to participate in signal transduction pathways. The analysis indicated that Leishmania contains proteins with multiple p-sites that are phosphorylated at distinct stages of the life cycle. For over half of the phosphorylation events, changes in phosphoprotein abundance did not positively correlate with changes in protein abundance, suggesting functional regulation. This study compares, for the first time, the phosphoproteins of L. donovani axenic promastigotes and amastigotes and provides the largest data set of the Leishmania phosphoproteome to date. KEYWORDS: Phosphoroteomic analysis, phosphorylation motif, Leishmania differentiation

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INTRODUCTION Parasitic protozoa of the genus Leishmania are the causative agents of a wide range of cutaneous, mucocutaneous, and visceral diseases.1 These organisms exhibit a digenetic life cycle that includes the extracellular promastigote and intracellular amastigote forms. Extracellular promastigotes develop in the alimentary tract of sand flies. Following infection of humans, the promastigotes differentiate into obligatory intracellular amastigotes within macrophage phagolysosomes.2,3 This differentiation process can be simulated in a host-free axenic culture by shifting promastigotes from an insect-like (26 °C, pH 7) to a lysosome-like (37 °C, pH 5.5 and 5% CO2) environment.4−8 Genome-wide time course analysis of L. donovani differentiation established that promastigote to amastigote differentiation is a programmed and highly regulated process.9−12 These analyses revealed expression of stage-specific as well as constitutive proteins that correlated well with data from intracellular amastigotes.13,14 Changes in protein abundance indicated strong metabolic and functional adaptation to the two distinct environments of the midgut and the phagolysosome. Rosenzweig et al.15 found that during differentiation, Leishmania proteins undergo posttranslational modifications. © XXXX American Chemical Society

These included methylation, acetylation, glycosylation, fucosylation, and phosphorylation. Phosphorylation in axenic L. donovani amastigotes occurs on proteins whose biological functions are linked to protein degradation, stress response, and signal transduction.16 A trypanosomatid-specific phosphorylation prediction tool was developed and was employed on a training set that included experimentally identified Leishmania phosphorylation sites.17 The analysis indicated that in addition to consensus phosphorylation motifs, Leishmania contains novel motifs, characterized by a consensus of “SF” and “NxS”. We hypothesized that these motifs are involved in processes that are unique to Leishmania development inside its host. Furthermore, we found that the Leishmania phosphoproteome (as well as those of all other members of the trypanosomatid family) contains distinct p-sites in proteins that are highly conserved in eukaryotes. For example, the alpha subunit of the eukaryote initiation factor 2 (eIF2α) attenuates translation when Ser51 that is surrounded by a conserved motif is phosphorylated.18 Leishmania and Trypanosoma brucei eIF2α Received: March 20, 2013

A

dx.doi.org/10.1021/pr4002492 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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Figure 1. Phosphopeptide distribution in axenic L. donovani amastigotes and promastigotes. Shotgun phosphoproteomic analysis was done as described in Methods. (A) MS/MS spectrum of ERpSPTSLSIK peptide from a hypothetical protein (LinJ.25.0290). The peptide contains three potential p-sites. Manual inspection of the spectrum allowed the identification of the specific phosphorylated residue. (B) Venn diagram of the number of phosphorylated sites identified in each life-stage. The distribution of phosphorylated sites between promastigotes and amastigotes is shown as a pie chart.

To date, phosphorylation profiling has been carried out in axenic or intracellular amastigotes, but limited information is available on developmental regulation of protein phosphorylation. We therefore examined and compared protein phosphorylation in axenic L. donovani promastigotes and amastigotes using LC−MS/MS. Our analysis indicated that

also contain this motif, but instead of serine they are phosphorylated on a threonine (Thr166).10,19,20 The fact that Leishmania is phylogenetically close to prokaryotes and contains unique p-sites and motifs that differ from those of higher eukaryotes is intriguing, justifying further in-depth analysis of their phosphoproteome. B



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Figure 2. Phosphorylation motif enrichment in L. donovani promastigotes and amastigotes. Training sets containing promastigote- and amastigotespecific as well as constitutive phosphopeptides were analyzed using motif-x webserver. (A) Amastigote enriched motifs, (B) promastigote enriched motifs, and (C) motifs equally expressed in both life stages.

(Promega). Samples were then acidified and desalted using reversed phase solid phase extraction (SPE). Ten milligrams of TiO2 beads (GL Science, 10 μm diameter) was added to each sample for phosphopeptide enrichment. Washed TiO2 beads were transferred to StageTips with C8 frits for phosphopeptide elution. Samples were acidified with formic acid prior to LC− MS/MS analysis. 25−50% of each sample was injected per LC−MS/MS analysis using a Waters nano Acquity autosampler and nanoflow (300 nL/min) HPLC system coupled to a QSTAR Pulsar I operated in positive ion mode. MS/MS spectra were acquired in a data-dependent manner over the course of four injections. Following selection for MS/MS analysis, precursor ions were excluded from selection for MS/ MS analysis for 180 s. Following the first 2 h of LC−MS/MS analysis, a mass exclusion list was generated from the peptides identified using Mascot. A second 2-h LC−MS/MS analysis was performed using the mass exclusion list in order to detect peptides of lower abundance by restricting selection of previously identified ions. Gas phase fractionation was used to increase the total number of identified peptides in this experiment. Peptide annotation was done using L. infantum proteome as downloaded from TriTrypDB (http://tritrypdb. org/tritrypdb/). The above-described assays were repeated three times, using independent cultures of each life stage.

there is more stage-specific than constitutive phosphorylation; serine phosphorylation in the Leishmania-specific “SF” motif is highly enriched in amastigotes; and p-sites within multiply phosphorylated proteins are often not coordinated. While some p-sites are phosphorylated in promastigotes, others are phosphorylated in amastigotes or both (i.e., constitutive). This work constitutes an in-depth comparison of developmentrelated protein phosphorylation that significantly enriches the database of phosphorylation sites in Leishmania.

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METHODS

Phosphoproteomic Analysis

A cloned line of L. donovani 1SR was grown and subjected to differentiation as described previously.5 Briefly, promastigotes were grown in medium M199 supplemented with 10% heat inactivated fetal calf serum (FCS) and amastigotes in medium M199 supplemented with 25% FCS and titrated to pH 5.5. Mid-logarithmic phase promastigotes (1−1.5 × 107 cells/mL) were washed three times in ice-cold phosphate buffered saline (PBS) supplemented with phosphatase inhibitors. Amastigote cell cultures (1−1.5 × 107 cells/mL) were washed three times in ice-cold Earle’s buffer at pH 5.5 supplemented with phosphatase inhibitors. Frozen cell pellets were lysed using a buffer containing deoxycholate and phosphatase inhibitors. Lysis was completed by boiling the samples. One milligram of protein from each sample was reduced with dithiothreitol and cysteine sulfhydryls were alkylated with iodoacetamide. Samples were subjected to overnight digestion with trypsin

Stage-Specific Motif Enrichment

We used the motif-x21 webserver with default parameters (minimum number of motif occurrences: 20, significance C


D

LinJ.29.2140

LinJ.34.2680 LinJ.36.2170 LinJ.02.0350 LinJ.23.0860 LinJ.23.1190 LinJ.29.0640

LinJ.04.0030 LinJ.12.0610 LinJ.13.1350 LinJ.22.0930 LinJ.25.2450

LinJ.34.2680 LinJ.35.1030 LinJ.35.1070 LinJ.36.0640 LinJ.36.3580

LinJ.29.2140

LinJ.05.0390 LinJ.06.0860 LinJ.11.1110, LinJ.11.113 LinJ.15.0370 LinJ.17.009, LinJ.17.0100, LinJ.17.0110, LinJ.17.0170, LinJ.17.0180, LinJ.17.0190, LinJ.17.0200 LinJ.22.1260 LinJ.23.0600 LinJ.25.0940 LinJ.27.2350 LinJ.29.1530

accession

GKIpSFANLK AApSFFSMGR FADEpSFAGK DAVEMQGGGVDPSDIFApSFFGGGSRPR SEAQLEGSpSFVDQMTNAIK

centrin, putative,Ca2 -binding EF-hand protein 5−3 exonuclease XRNC, putative cyclophilin a heat shock protein DNAJ, putative clathrin coat assembly protein ap19, putative,sigma adaptin, putative serine/threonine protein kinase, putative,protein kinase, putative regulatory subunit of protein kinase a-like protein casein kinase, putative protein kinase, putative sec14, cytosolic factor vacuolar protein sorting-associated protein-like protein cAMP-specific phosphodiesterase, putative serine/threonine protein phosphatase-like protein MCAK-like kinesin, putative dynein heavy chain, cytosolic, putative serine/threonine-protein kinase, putative, protein kinase, putative regulatory subunit of protein kinase A-like protein serine/threonine protein phosphatase, putative FtsJ-like methyltransferase, putative 3-ketoacyl-CoA thiolase, putative hypothetical protein, unknown function ABC protein subfamily A, member 10, putative, ABC transporter, putative serine/threonine protein kinase, putative, protein kinase, putative pSFGSAPGGGFNTSSSQVAR

IApSFPTTPK SIGNApSFAEPFK LLNAPTPTGVALTDDEQQQPANSpSFSSPAPSPALGASPR KLpSFEQCNGEDPSNVK DVHEGEKNYQEEGEKpSFNDANVEATSKHQQCDKDDAAKDVEAADDAAK AIDDDEPHGApSFASCSLKK

MEEAPSTAEVIASRPPGGpSFSNR ISpSFTATIGSVGFGVNK pSFGSRPVTSSSPQK ASpSFSPSSLALLR ANpSFVGSPFYVAPDVLR

SpSFDKYVVQLVR IGSGpSFGEIFR GSSNSTGpSFLSSIK FIAPDAVSLpSFTPTSSSK GQQpSFSLLSR

VSDTSLSDGSGNGGPGGSIAVGpSFR

ANWNNPpSFAAALR ISASLLLEPQTVGAATpSFIK AVAAVRPDLADVpSFR HNAALTFNNpSFDDTLEALQR EAAEIGKApSFK

phosphorylated tryptic peptide

protein kinase, putative lipin, putative 60S ribosomal protein L28, putative ecotin, putative elongation factor 1-α

description

Table 1. Functionally Annotated Proteins That Contain the Trypanosomatid-Specific “SF” Motif

679

342 671 145 229 30 615

639 631 601 1088 212

3 21 408 389 14

462

99 781 97 89 153

109 539 107 221 53

Pposition

pro

const const pro pro pro pro

const const const const const

ama ama ama ama ama

ama

ama ama ama ama ama

ama ama ama ama ama

stage

Journal of Proteome Research Article



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threshold: 1 × 10−6). The complete L. infantum proteome was used as background to calculate the fold-enrichment of the motifs in the data set.

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had four distinct phosphorylation sites (Figure 3). We have also identified a few proteins with over six distinct phosphorylated

RESULTS

Leishmania Shows Stage-Specific Protein Phosphorylation

Three independent cultures of axenic promastigotes and amastigotes were harvested at midlog phase, their proteins extracted, treated with trypsin, subjected to a TiO2 column for phosphopeptide enrichment, and finally analyzed by QStar pulsar I Q-TOF.10,22−24 These experiments revealed 1614 phosphorylated amino acid residues (p-sites) representing 627 distinct proteins (Supplementary Table S1, Supporting Information). Manual analysis of all MS spectra was carried out to validate all phosphorylation sites (see example in Figure 1A). 1046 sites were identified in amastigotes and 998 in promastigotes. The analysis revealed an equal distribution of stage-specific phosphorylated sites between promastigotes and amastigotes, 35 and 38% respectively, but only 27% of the sites showed constitutive phosphorylation at both stages (Figure 1B). Phospho amino-acid distribution resembled that of higher eukaryotes as well as previous analyses in Leishmania:25−28 80% pS, 19.6% pT, and 0.4% pY. We used the motif-x webserver21 to identify phosphorylation motifs in each set of peptides (promastigote, amastigote, and constitutive). Of the two Trypanosomatid-specific motifs we recently discovered,17 only one, characterized by the “SF” consensus sequence, is significantly enriched in amastigotes (Figure 2A). Functionally annotated proteins that contain this motif are listed in Table 1. Among these are heat shock protein DNAJ (LinJ.30.1790), eEF1α (encoded by seven adjacent genes on chromosome 17), casein kinase 1 (LinJ.35.1030), and cyclophilin A (LinJ.25.0940), all with “SF” motifs that are phosphorylated exclusively in amastigotes. Interestingly, proteins with more than a single “SF” motif had at least one that is phosphorylated in amastigotes and the rest either in promastigotes or both (i.e., constitutive). For example, the regulatory subunit of protein kinase A-like protein (LinJ.34.2680; R′-PKA), a gene that exists only in Leishmania and Trypanosoma cruzi, has two “SF” sites: Ser3 is phosphorylated only in amastigotes and Ser342 is constitutively phosphorylated. A Ser/Thr kinase (LinJ.29.2140) is an additional example of a protein with two “SF” motifs; Ser462 is phosphorylated in amastigotes, and Ser679 is phosphorylated only in promastigotes. It is likely that distinct protein kinases phosphorylate the different sites. Phosphorylation sites that are promastigote-specific or constitutive (Figure 2, panels B and C, respectively) contained pS followed by acidic residues (aspartate or glutamate). According to Netphorest,29 a collection of sequence-based classifiers that predict which kinase group is more likely to phosphorylate a given substrate, casein kinase 2 (CK2) substrates are enriched with these types of motifs. Interestingly, we found that peptides predicted to be phosphorylated by CK2 are significantly under-represented in the amastigote-specific data set (p-value 2-fold increase or decrease relative to promastigotes. Three E



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Figure 4. Correlation between phosphorylation pattern and protein abundance. (A) Three types of relationships between protein phosphorylation and protein abundance changes during L. donovani differentiation. Protein abundance values in amastigotes are presented as log2 fold from promastigotes. (B) The frequency of correlation between phosphorylation pattern and protein abundance.

amastigotes emphasizes the uniqueness of signaling pathways involved in Leishmania development. Furthermore, having proteins that contain multiple “SF”s with distinct phosphorylation profiles suggests that parasite cells express several protein kinases that phosphorylate this motif. This, as well as previous studies, indicates that the Leishmania proteome contains pY residues in several proteins, most of which are protein kinases.26 All three tyrosine phosphorylated MAPKs found in our analysis contained pY in a single motif, namely, “TxY”. It is interesting that MAPKs in Leishmania are conserved in the way they are activated, notably by phosphorylation of the “TxY” motif through MAP Kinase Kinases (MKKs). Indeed, Leishmania genome contains 17 genes encoding for MKK.31 In addition, several studies suggest that in trypanosomatids, tyrosines in motifs other than “TxY” are substrates of dual specificity protein kinases such as CLK and DYRK.31 Comparative analysis of phosphorylation with protein abundance between L. donovani promastigotes and amastigotes indicates that about two-fifths are positively correlated, about half are not correlated, and the rest are negatively correlated. Positive correlation means that the level of phosphorylation per protein (net phosphorylation) did not change between amastigote and promastigote. Negative correlation likely indicates that phosphorylation repressed protein activity. In cases where no correlation is observed there are two possible explanations: (a) phosphorylation is stage-specific and there is no change in protein abundance, or (b) phosphorylation is constitutive and found in both promastigotes and amastigotes, yet protein abundance is up- or down-regulated. In both cases, net phosphorylation changed and phosphorylation affects protein function. An example that validates the above hypothesis is the phosphorylation of eIF2α during Leishmania differentiation; eIF2α phosphorylation increased by almost 4fold in amastigotes relative to promastigotes, whereas its abundance hardly changed during, but decreased toward the end of differentiation.10 Phosphorylation of LdeIF2α attenuates protein synthesis during differentiation as it does in all eukaryotes.18

types of correlation were defined as illustrated in Figure 4A: (I) positive correlation, (II) no correlation, and (III) negative correlation. As indicated in Table S2 and Figure 4B, 43.3% of psite patterns are positively correlated with changes in protein abundance, 51.3% are not correlated, while only 5.4% are negatively correlated. For example, pyruvate phosphate dikinase (LinJ.11.1000) shows positive correlation between phosphorylation and protein abundance changes; it is phosphorylated on Ser103 in amastigotes, while protein abundance increased >4 fold in amastigotes. In contrast, beta tubulin (LinJ.33.0860) demonstrates no correlation between phosphorylation and protein abundance; it is phosphorylated on Ser285 in promastigotes, but its protein abundance remains unchanged. Negative correlation was observed for peroxidoxin (LinJ.23.0050); this protein is phosphorylated on Ser171 in promastigotes, while protein abundance increased >2 fold in amastigotes. Thus, according to our hypothesis, this analysis predicted that more than half of the phosphorylations identified in this study affect protein function, either via protein activity, localization, or interaction with other proteins.

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DISCUSSION

Development of parasites inside their hosts is a process associated with signals that the two exchange.32−34 Posttranslational modifications such as protein phosphorylation likely transmit such signals and therefore play major role in these processes. The presence of Leishmania-specific phosphorylation strongly supports this idea.17 Our long-term goal is to characterize stage-regulated protein phosphorylation and to assess its role in Leishmania development. Here we have analyzed and compared phosphoproteomes of Leishmania donovani promastigotes and amastigotes. More than half of Leishmania putative protein kinase genes encode for yet unknown functions.12,31 The discovery of trypanosomatid-specific phosphorylation motifs, “SF” and “NxS”,17 supports the notion that organisms of this family either express novel protein kinases or possess unique substrates for known protein kinases. Our observation that proteins containing pS in “SF” motifs are enriched in F



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developmentally-regulated activities in axenic amastigotes of Leishmania donovani. Mol. Biochem. Parasitol. 1998, 95 (1), 9−20. (5) Barak, E.; Amin-Spector, S.; Gerliak, E.; Goyard, S.; Holland, N.; Zilberstein, D. Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response. Mol. Biochem. Parasitol. 2005, 141 (1), 99−108. (6) Debrabant, A.; Joshi, M. B.; Pimenta, P. F.; dwyer, D. M. Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics. Int. J. Parasitol. 2004, 34 (2), 205−217. (7) Goyard, S.; Segawa, H.; Gordon, J.; Showalter, M.; Duncan, R.; Turco, S. J.; Beverley, S. M. An in vitro system for developmental and genetic studies of Leishmania donovani phosphoglycans. Mol. Biochem. Parasitol. 2003, 130 (1), 31−42. (8) Bates, P. A.; Robertson, C. D.; Tetley, L.; Coombs, G. H. Axenic cultivation and characterization of Leishmania mexicana amastigotelike forms. Parasitology 1992, 105 (Pt 2), 193−202. (9) Rosenzweig, D.; Smith, D.; Opperdoes, F. R.; Stern, S.; Olafson, R. W.; Zilberstein, D. Retooling Leishmania metabolism: from sandfly gut to human macrophage. FASEB J. 2008, 22 (2), 590−602. (10) Lahav, T.; Sivam, D.; Volpin, H.; Ronen, M.; Tsigankov, P.; Green, A.; Holland, N.; Kuzyk, M.; Borchers, C.; Zilberstein, D.; Myler, P. J. Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania. FASEB J. 2011, 25 (2), 515− 525. (11) Paape, D.; Aebischer, T. Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development. J. Proteomics 2011, 74 (9), 1614−24. (12) Tsigankov, P.; Gherardini, P. F.; Helmer-Citterich, M.; Zilberstein, D. What has proteomics taught us about Leishmania development? Parasitology 2012, 139 (9), 1146−1157. (13) Pescher, P.; Blisnick, T.; Bastin, P.; Spath, G. F. Quantitative proteome profiling informs on phenotypic traits that adapt Leishmania donovani for axenic and intracellular proliferation. Cell Microbiol. 2011, 13 (7), 978−991. (14) Paape, D.; Barrios-Llerena, M. E.; Le Bihan, T.; Mackay, L.; Aebischer, T. Gel free analysis of the proteome of intracellular Leishmania mexicana. Mol. Biochem. Parasitol. 2010, 169 (2), 108−114. (15) Rosenzweig, D.; Smith, D.; Myler, P. J.; Olafson, R. W.; Zilberstein, D. Post-translational modification of cellular proteins during Leishmania donovani differentiation. Proteomics 2008, 8, 1843− 1850. (16) Morales, M. A.; Watanabe, R.; Dacher, M.; Chafey, P.; Osorio y Fortea, J.; Scott, D. A.; Beverley, S. M.; Ommen, G.; Clos, J.; Hem, S.; Lenormand, P.; Rousselle, J. C.; Namane, A.; Spath, G. F. Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (18), 8381−8386. (17) Palmeri, A.; Gherardini, P. F.; Tsigankov, P.; Ausiello, G.; Spath, G. F.; Zilberstein, D.; Helmer-Citterich, M. PhosTryp: a phosphorylation site predictor specific for parasitic protozoa of the family trypanosomatidae. BMC Genomics 2011, 12 (1), 614. (18) Dever, T. E. Gene-specific regulation by general translation factors. Cell 2002, 108 (4), 545−56. (19) Gosline, S. J.; Nascimento, M.; McCall, L. I.; Zilberstein, D.; Thomas, D. Y.; Matlashewski, G.; Hallett, M. Intracellular eukaryotic parasites have a distinct unfolded protein response. PLoS ONE 2011, 6 (4), e19118. (20) Moraes, M. C.; Jesus, T. C.; Hashimoto, N. N.; Dey, M.; Schwartz, K. J.; Alves, V. S.; Avila, C. C.; Bangs, J. D.; Dever, T. E.; Schenkman, S.; Castilho, B. A. Novel membrane-bound eIF2alpha kinase in the flagellar pocket of Trypanosoma brucei. Eukaryot. Cell 2007, 6 (11), 1979−1991. (21) Schwartz, D.; Gygi, S. P. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat. Biotechnol. 2005, 23 (11), 1391−1398. (22) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. Highly selective enrichment of phosphorylated

Our results show that a significant portion of the identified proteins have multiple phosphorylation sites that are independently regulated in a stage-specific manner. This indicates that phosphorylation is regulated differently on different sites of the same protein and that phosphorylation effect on protein function should be considered on a sitespecific basis. As was suggested in previous studies on human cell cultures, proteins with multiple phosphorylation sites might serve as platforms and/or junctions between different cellular pathways propagated by various kinases.27,28 However, in bacteria, both Gram-positive and Gram-negative, multiply phosphorylated proteins are associated with stress conditions. It has been suggested that multiple phosphorylations serve as degradation tags.35 We suggest that Leishmania, being phylogenetically closer to prokaryotes, adopted the use of multiply phosphorylated proteins as stress recognition tags that activate corresponding cellular responses. In fact, the Leishmania differentiation signal is composed of two stress factors: acidic pH and heat, which together function to stimulate differentiation. Leishmania are able to translate multiple stress conditions into a signal, enabling differentiation to a form adapted to the different environments in host and vector.36,37 This is likely a general phenomenon in all parasitic protozoa.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1: Phosporylated residues identified in promastigotes (pro), amastigotes (ama), or both (const.). Table S2: Correlation between phosphorylation patterns and protein abundance. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +972-4-2893647. Fax: +972-4-8225153. E-mail: danz@bi. technion.ac.il. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Moshe Ephros and Dr. Roni Nitzan-Koren for their help, and Dr. Christoph Borchers and Derek Smith from University of Victoria Proteomics Center, Canada, for the MS assays. This work was supported by the seventh Framework Programme of the European Commission through a grant to the LEISHDRUG programme and by Grant Number 33928 from the Chief Scientist of the Israel Ministry of Health Foundation.

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REFERENCES

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