EUKARYOTIC CELL, Apr. 2009, p. 617–626 Vol. 8, No. 41535-9778/09/$08.00�0 doi:10.1128/EC.00366-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification and Specific Localization of Tyrosine-PhosphorylatedProteins in Trypanosoma brucei�†

Isabelle R. E. Nett,1 Lindsay Davidson,2 Douglas Lamont,1 and Michael A. J. Ferguson1*Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland,

United Kingdom,1 and Division of Molecular Physiology, College of Life Sciences, University of Dundee,Dundee DD1 5EH, Scotland, United Kingdom2

Received 12 November 2008/Accepted 16 January 2009

Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellularand intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinasesin the genome of the single cell organism Trypanosoma brucei, phosphorylation on trypanosomal proteintyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphor-ylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phospho-tyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cellextracts of procyclic form T. brucei. A significant proportion of the phosphotyrosine-containing proteinsidentified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknownfunction and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly,immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentrationof tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and inthe nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that thefunction of signaling molecules is controlled by their precise location in T. brucei, a principle well known fromhigher eukaryotes.

Reversible protein phosphorylation by protein kinases andphosphatases is a major regulatory mechanism of most cellularprocesses in eukaryotic organisms (7). Dysregulation of pro-tein phosphorylation networks is responsible for a myriad ofdiseases from cancers to immune disorders and neurodegen-erative diseases (8, 9). These findings have prompted the char-acterization of the protein kinase complements (“kinomes”) ofa number of organisms (2, 6, 14, 16, 18, 21, 24, 27, 32, 41), andan important turning point came with the realization that par-asite kinomes are substantially different from those of the hoststhey infect. One major difference is the absence of genes cod-ing for any recognizable tyrosine-specific kinases in the ge-nome of Trypanosoma brucei (29; I. R. E. Nett, D. M. A.Martin, D. Miranda-Saavedra, D. Lamont, J. D. Barber, A.Mehlert, and M. A. J. Ferguson, submitted for publication). T.brucei causes human African trypanosomiasis (also known asAfrican sleeping sickness) and is responsible for �30,000deaths per annum (35). However, despite the lack of conven-tional tyrosine kinases in this parasite and related trypano-somes, there is evidence that several proteins are phosphory-lated on tyrosine residues in these organisms (10, 30). Tyrosinephosphatase activity has also been observed in cell extracts ofbloodstream form and procyclic form T. brucei (1). More re-cently, the identification and biochemical characterization of a

T. brucei protein tyrosine phosphatase 1 suggest a role fortyrosine phosphorylation in the regulation of the trypanosomelife cycle (36). In addition, the characterization of the T. bruceiprotein phosphatase complement (“phosphatome”) revealedthe presence of 19 dual-specificity protein phosphatases. How-ever, orthologues of the human mitogen-activated protein ki-nase (MAPK) phosphatases are missing in the T. brucei ge-nome (3), although plant-like MAPK phosphatase homologuesare present. This is an interesting observation, since our large-scale phosphoproteomics analysis of bloodstream form T. bru-cei cells (Nett et al., submitted for publication) revealed phos-phorylation on tyrosine residues of 13 protein kinases, of whichtwo belong to the MAPK family (GeneDB accession no.Tb927.6.4220 and Tb10.61.0250).

Phosphorylation of protein tyrosine residues regulates im-portant cell functions in higher eukaryotes, but the role of thisposttranslational modification is largely unknown for T. brucei.Here, we used a phosphotyrosine-specific mass spectrometry(MS)-based approach to identify proteins carrying this modi-fication in the procyclic form of the parasite and reveal thatphosphorylation of tyrosine residues within canonical se-quence motifs is conserved in T. brucei. However, by usinganti-phosphotyrosine-specific antibodies, we show a localiza-tion pattern for phosphotyrosine-containing proteins in T. bru-cei that is substantially different from that in mammalian cells.

MATERIALS AND METHODS

Cell preparation and lysis. The procyclic form T. brucei cell line 29-13-6 (44)was cultured in SDM-79 medium (5), containing G418 (Gibco) at 15 �g/ml andhygromycin B (Roche) at 50 �g/ml. Human HeLa cells were grown in Dulbeccomodified Eagle medium (Gibco) supplemented with 10% fetal calf serum(Gibco) and 2 mM L-glutamine (Gibco) at 37°C.

For the isolation of phosphotyrosine-containing peptides from trypanosomes,

* Corresponding author. Mailing address: Division of BiologicalChemistry and Drug Discovery, Wellcome Trust Biocentre, College ofLife Sciences, University of Dundee, Dow St., Dundee DD1 5EH,Scotland, United Kingdom. Phone: 011 44 1382 345645. Fax: 011 441382 345764. E-mail: m.a.j.ferguson@dundee.ac.uk.

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 30 January 2009.

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1 liter of cells was grown to a density of 1 � 107 cells/ml at 28°C in a water-jacketed incubator. Cells were treated with 800 �M hydrogen peroxide for 30min before lysis. Cell lysis and phosphopeptide preparation were performedusing the PhosphoScan P-Tyr-100 kit (Cell Signaling) according to the manufac-turer’s instructions.

Cell lysis for Western blotting. Before lysis, cells were washed three times inice-cold phosphate-buffered saline (PBS) buffer and then lysed in ice-cold RIPAlysis buffer (10 mM Tris-HCl, pH 7.5; 1 mM sodium-�-glycerophosphate; 1 mMsodium-pyrophosphate; 1 mM sodium fluoride; 5 mM EDTA; 0.5% NP-40; 0.2%sodium-deoxycholate; 0.2% sodium dodecyl sulfate [SDS]; EDTA-free proteaseinhibitor tablet [Roche]; 100 �M activated sodium-orthovanadate) at a ratio of1 � 109 cells per 1 ml of lysis buffer. The lysate was sonicated two times for 30 sat 80% power of an ultrasonic processor machine (Jencons) at 4°C and centri-fuged at 14,000 rpm for 20 min at 4°C. The supernatant was transferred into afresh tube, and the protein concentration determined using the Micro BCAprotein assay kit (Pierce) according to the manufacturer’s instructions. Approx-imately 10 �g of proteins were mixed with the appropriate volume of 4�Laemmli sample buffer and 10� sample-reducing agent (Invitrogen), heated,and separated on a precast Novex 4 to 12% Bis-Tris SDS-polyacrylamide elec-trophoresis gel. After electrophoresis, proteins were transferred onto a polyvi-nylidene difluoride (PVDF) membrane, and the membrane was blocked with 4%bovine serum albumin in Tris-buffered saline (TBS) (150 mM NaCl, 50 mMTris-HCl, pH 7.2) containing Triton X-100 (0.2%) for 1 h at room temperature.The membrane was incubated with the anti-phosphotyrosine monoclonal anti-body 4G10 (1 mg/ml; Upstate) at a dilution of 1:10,000 for 1 h at room temper-ature, then washed, and incubated with secondary antibody (anti-mouse horse-radish peroxidase; Roche) at 1:5,000. Western blots were developed using theECL plus Western blotting detection reagent (GE Healthcare) according to themanufacturer’s instructions and were exposed to Hyperfilm (GE Healthcare).

To examine phosphotyrosine-containing proteins of cytosol and cytoskeletalfractions, aliquots of 3 � 107 procyclic form T. brucei cells were washed in PBSbuffer and lysed in either 100 �l ice-cold MME buffer (10 mM MOPS, pH 6.9;1 mM EGTA; 1 mM MgSO4) containing EDTA-free protease inhibitor tablet(Roche), 0.2% Triton X-100, and a phosphatase inhibitor cocktail of 1 mMsodium-�-glycerophosphate, 1 mM sodium-pyrophosphate, 1 mM sodium fluo-ride, and 100 �M activated sodium-orthovanadate or in 100 �l RIPA lysis buffer(see above). The MME lysate was incubated on ice for 30 min and centrifugedat 13,000 � g for 2 min at 4°C. The supernatant was transferred into a fresh tube,and the pellet was washed three times with 1 ml MME buffer containing 0.2%Triton X-100 with protease and phosphatase inhibitors, as described previously.The insoluble cytoskeletons were resolubilized in 100 �l RIPA lysis buffer, and20-�l aliquots (6 � 106 cell equivalents) of the MME soluble fraction and theRIPA-solubilized cytoskeletal fraction were resolved on a 4 to 12% NuPAGE gel(Invitrogen), alongside 20 �l of the whole-cell RIPA-lysate, and transferred toPVDF. Replicate blots were incubated with either alkaline phosphatase buffer(50 mM Tris-HCl, pH 8.5; 5 mM MgCl2) containing 15 units of shrimp alkalinephosphatase at 30°C overnight or with 10 �g of recombinant glutathione S-transferase-SHP-1 in 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.03% Brij 35,0.1% �-mercaptoethanol. The control blot was incubated in glutathione S-trans-ferase-SHP-1 buffer. Blots were rinsed three times with TBS containing 0.2%Triton X-100 (TBS-T), and Western blotting for phosphotyrosine was performedas described previously. Western blots were stripped in 50 mM Tris-HCl (pH 7),2% SDS, 0.1% �-mercaptoethanol for 30 min at 65°C and then rinsed three timeswith TBS containing 0.2% Triton X-100. For a loading control, blots wereincubated with the anti-tubulin clone DM1A (1 mg/ml; Abcam) at a dilution of1:10,000 and then with goat anti-mouse horseradish peroxidase (1:10,000) anddeveloped by enhanced chemiluminescence.

Immunolocalization. Mid-log procyclic trypanosomes (�1 � 107 cells/ml)were settled onto coverslips for 15 min, and in the case of cytoskeleton prepa-rations, settled cells were incubated with MME buffer (see above) on ice for 10min with three changes of buffer. Isolated flagellar complexes were obtained byincubating the cells with MME buffer containing 1 M NaCl, 0.2% Triton X-100,and protease and phosphatase inhibitors, as described previously, on ice for 15min with three changes of buffer. Cells were washed in PBS and fixed in either4% paraformaldehyde (PFA) in PBS or 100% ice-cold methanol. Coverslipswere blocked with 2% fish gelatin in TBS/0.2% Triton X-100 for 1 h at roomtemperature before antibody labeling with approximately 0.25 �g per coverslip of4G10 antibody (Upstate), fluorescein isothiocyanate-conjugated 4G10 antibody(Upstate), PY-20 antibody (Transduction Laboratories), or PY-100 antibody(Cell Signaling Technologies) or with a 1:30 dilution in 2% fish gelatin inTBS/0.2% Triton X-100 of BBA4 or Rib72 monoclonal antibody tissue culturesupernatant (kind gifts of Keith Gull). Rib72 labeling was visualized with anti-mouse immunoglobulin G (IgG) Alexa Fluor 633 (2 �g/ml; Invitrogen) second-

ary antibody and 4G10 labeling with either anti-mouse IgG Alexa Fluor 633 (2�g/ml; Invitrogen) or anti-mouse IgG Alexa Fluor 488 (2 �g/ml; Invitrogen).BBA4 labeling was visualized with anti-mouse IgM Alexa Fluor 488 (2 �g/ml;Invitrogen). Cells were counterstained with anti-mouse fluorescein isothiocya-nate-conjugated anti-� tubulin (1.5 �g/ml; Sigma) and/or 4�-6-diamidino-2-phe-nylindole (2 �g/ml; Sigma) before mounting with Vectashield (Vector Labora-tories). For staining of the nucleolus, procyclic cells were incubated with acarboxy-terminal tetramethyl rhodamine isothiocyanate (TRITC)-labeled deca-arginine peptide, TRITC-D(R10) (Peptide Specialty Laboratories GmbH), at a 7�M final concentration for 1 h at 28°C. Cells were washed once in PBS, resus-pended in SDM-79 medium and incubated for another 2 h at 28°C. Cells werethen harvested, fixed in 4% PFA/PBS, and mounted as described above.

For the dephosphorylation assay, methanol-fixed trypanosomes were incu-bated with 2 ml alkaline phosphatase buffer (see above) containing 10 units ofalkaline shrimp phosphatase in a six-well plate overnight at 37°C. Positive controlcells were incubated in 2 ml alkaline phosphatase buffer only, without the addi-tion of alkaline shrimp phosphatase.

For immunofluorescence studies of human HeLa cells, 2 � 105 cells wereseeded onto a square coverslip (22 mm by 22 mm) and incubated at 37°C for 24 h.The cells were washed twice with 1 ml of PBS and fixed in 4% PFA/PBS.Antibody incubations and mounting of cells were performed as described fortrypanosomes.

Images were collected using a DeltaVision Spectris restoration wide-fielddeconvolution microscope (Applied Precision LLC) equipped with a CoolSnapHQ cooled charge-coupled-device camera. Optical sections were processed usingSoftWoRx software (Applied Precision LLC) and Adobe Photoshop (Adobe).

LC-MS. Liquid chromatography (LC) was performed on a fully automatedUltiMate 3000 Nano LC system (Dionex) fitted with a 1- by 5-mm precolumnPepMap C18 (LC Packings, Dionex) and a 75-�m by 15-cm reverse-phase C18

nano-column (LC Packings, Dionex). Samples were loaded in 0.1% formic acidcontaining 2% acetonitrile (buffer A) and separated using a binary gradientconsisting of buffer A and buffer B (90% acetonitrile, 0.08% formic acid).Peptides were eluted with a linear gradient from 5 to 40% buffer B over 130 min.The high-pressure liquid chromatography system was coupled to an LTQ-Orbi-trap mass spectrometer (Thermo Electron) equipped with a proxeon nanosprayionization source.

With the LTQ-Orbitrap mass spectrometer, a survey scan was performed overa mass range of m/z 335 to 1,800 in the Orbitrap analyzer (R � 60,000), eachtriggering five tandem MS (MS/MS) LTQ acquisitions of the five most intenseions. The Orbitrap mass analyzer was internally calibrated on the fly using thelock mass of polydimethylcyclosiloxane at m/z 445.120025.

Raw peak list files obtained from the LTQ-Orbitrap were converted to Mascotgeneric files using Raw2msm software (gift from Matthias Mann) and weresearched against concatenated forward and reverse sequence databases consist-ing of amino acid sequences from T. brucei strain 927 and translated openreading frames from T. brucei strain 427 using the Mascot search engine (MascotV2.1; Matrix Science). The search criteria were as follows: up to two missedcleavages were allowed; carbamidomethylation, oxidation, and phosphorylationwere set as variable modifications. The precursor ion mass tolerance was set to10 ppm and 0.8 Da for all MS/MS spectra acquired in the LTQ mass analyzer.

RESULTS

Detection of tyrosine-phosphorylated proteins by immuno-blotting using 4G10 anti-phosphotyrosine antibody. To initiatean analysis of tyrosine-phosphorylated proteins in T. brucei, weinvestigated the usefulness of commercially available anti-phosphotyrosine antibodies to bind to trypanosomal phospho-tyrosine-containing proteins by Western blotting. The 4G10anti-phosphotyrosine antibody (Upstate) revealed several pro-tein bands, especially high-molecular-weight proteins, in totalcell lysates of both procyclic (Fig. 1, lane 1) and bloodstreamform T. brucei (see Fig. S1 in the supplemental material). Theassociation of phosphotyrosine-containing proteins with micro-tubular structures, indicated by immunofluorescence micros-copy (see below), was also analyzed by Western blotting usingwhole-cell, TX-100-soluble and cytoskeleton extracts of procy-clic form T. brucei cells (Fig. 1). Anti-phosphotyrosine reactiveproteins were detected in both fractions to different degrees

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(Fig. 1, compare lanes 2 and 3). A higher proportion of ty-rosine-phosphorylated proteins was found in the cytoskeletonextract (Fig. 1, lane 3) than for TX-100-soluble proteins ob-tained from an equal amount of cells (Fig. 1, lane 2). To assessthe possibility of nonspecific antibody binding, parallel incuba-tions of PVDF membranes containing the same amount ofprocyclic cell lysate were performed in the absence (Fig. 1,control) and presence of alkaline phosphatase (Fig. 1) or thephosphotyrosine-specific phosphatase SHP-1 (Fig. 1). A reduc-tion of signal intensity was clearly visible for the majority of thebands after treating the PVDF membrane with alkaline phos-phatase; however, the signal was completely abolished aftertreating PVDF membranes with the protein tyrosine phos-phatase SHP-1. These results confirm the specificity of the4G10 antibody for phosphotyrosine-containing proteins.

Tyrosine phosphorylation is hydrogen peroxide sensitive.We next examined the level of tyrosine phosphorylation aftertreating the cells with different concentrations of hydrogenperoxide (Fig. 2). Hydrogen peroxide has been shown to beinvolved in the regulation of redox signaling pathways by in-activating protein tyrosine phosphatases and, as a result, in-creasing tyrosine phosphorylation of target proteins (33). Inprocyclic trypanosomes, first responses to the hydrogen perox-ide treatment were detected at concentrations of 200 �M, anddetection levels reached their maximum in cell lysates obtainedfrom cells that had been incubated with 800 �M hydrogenperoxide for 20 min or 30 min (Fig. 2). In bloodstream form

cells (see Fig. S2 in the supplemental material), clear responseto the hydrogen peroxide treatment was detected at 400 �M ofthe oxidizing agent at all time points measured (17, 23, and 30min), which could be increased with higher hydrogen peroxideconcentrations up to 800 �M where the signal reached satu-ration. These results suggest the possible expression of hydro-gen peroxide-sensitive phosphatases in the trypanosome and/orthe presence of redox-regulated protein tyrosine kinases. Thelatter, if they exist, would have to be dual-specificity kinases,since conventional tyrosine kinase genes are absent in T. bru-cei. It is worth noting that peroxovanadium [bpV(phen)] hadno effect on phosphotyrosine levels under conditions in whichit had clear effects on mammalian cell control experiments runalongside (data not shown).

Immuno-affinity purification of phosphotyrosine-containingpeptides from procyclic form T. brucei. In order to identify T.brucei tyrosine-phosphorylated proteins, we immunoprecipi-tated tyrosine-phosphorylated peptides from tryptic digests ofwhole-cell lysates of the procyclic form and analyzed the im-munoprecipitate by LC-MS/MS. Procyclic trypanosomes weretreated with 800 �M hydrogen peroxide for 30 min before celllysis to increase the levels of phosphorylation on tyrosine res-idues and thus improve the detection of phosphorylated pro-teins by MS. In total, 45 phosphotyrosine-containing peptideisoforms from 34 proteins were identified in two separate ex-periments using approximately 40 mg starting material (seeTable S1 in the supplemental material). An example of a frag-mentation mass spectrum of a tyrosine-phosphorylated peptideis shown in Fig. 3. A mass increment of 243 Da, which corre-sponds to a phosphorylated tyrosine residue (pY), was ob-

FIG. 1. Detection of T. brucei tyrosine-phosphorylated proteins byimmunoblotting. Aliquots of total cell lysates (T) and TX-100-soluble(S) and insoluble (P) fractions from 6 � 106 procyclic form trypano-some equivalents were subjected to SDS-polyacrylamide gel electro-phoresis and Western blotting with 4G10 antibody before (Control,lanes 1 to 3) and after alkaline phosphatase (alk. phosphatase, lanes 4to 6) or SHP-1 phosphotyrosine-specific phosphatase (SHP-1, lanes 7to 9) treatment of the blot. Following the 4G10 Western blotting, themembranes were stripped and probed with anti-tubulin antibodies as aloading control (lower panels). �-pTyr, anti-phosphotyrosine.

FIG. 2. T. brucei cells are sensitive to treatment with hydrogenperoxide. Procyclic trypanosomes were treated with 200 �M, 400 �M,and 800 �M hydrogen peroxide for 10 min, 20 min, and 30 min beforecell lysis. An aliquot of 15 �g protein was loaded in each lane. Lane 1,protein lysate of untreated cells. Bands that show a clear response tothe hydrogen peroxide treatment are indicated with an asterisk.�-pTyr, anti-phosphotyrosine.

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served for residue Y188 in the phosphopeptide sequence GVGVNVTSpYVVTR of GeneDB accession no. Tb927.6.1780.The loss of a phosphate group in the form of phosphoric acid(H3PO4) was observed for T186, suggesting this residue as asecond site of phosphorylation in the same peptide.

The majority of proteins found to be phosphorylated ontyrosine residues were protein kinases belonging to the CMGCkinase group, and one kinase was designated a NEK groupmember (Table 1). T. brucei protein kinases were annotatedaccording to the classification described in the work of Nett etal. (submitted for publication). Of the CMGC kinases, weidentified 10 putative T. brucei mitogen-activated protein ki-nases (TbMAPK) homologous to the 15 putative MAPKs en-coded in the Leishmania genome (42), including the experi-mentally characterized TbMAPK2 (28) (Table 1). We alsodetected the T. brucei homologue of LmjMPK15, which fallsinto the T. brucei CDK kinase family (Tb10.329.0030).

The signature TXY motif in the activation loop of MAPKs(25) was found fully phosphorylated in 8 of the 10 TbMAPKs(Table 1), and the remaining 3 kinases (including the identifiedTbCDK) were phosphorylated only on the tyrosine residue.The TXY motif present in the putative TbMAPKs showed arather variable central amino acid; five TbMAPKs were foundto contain a TEY motif, three TbMAPKs displayed a TDYmotif, and the remaining two TbMAPKs had either THY or

TSY. Another variation of the TXY motif was found in the T.brucei homologue of LmjMPK15, which showed a TFY signa-ture motif in the activation segment.

Other tyrosine-phosphorylated proteins identified in ourstudy are involved in protein synthesis (GeneDB accessionno. Tb927.5.3120 translation initiation factor, putative; ri-bosomal protein S27, putative), energy metabolism (glyco-somal phosphoenolpyruvate carboxykinase; ATP-dependentphosphofructokinase), and RNA metabolism (ATP-depen-dent DEAD/H RNA helicase, putative). Tyrosine-contain-ing peptides were also found for nonclassified proteins (10“hypothetical proteins”) (see Table 2).

Localization studies of tyrosine-phosphorylated proteins inT. brucei. We next investigated the localization pattern of ty-rosine-phosphorylated proteins using the anti-phosphotyrosinemonoclonal antibodies 4G10, PY-20, and PY-100 on PFA-fixed procyclic trypanosomes (Fig. 4). Phosphotyrosine-con-taining proteins appeared to localize specifically to punctatestructures in the posterior of the cell (Fig. 4A) and to thenucleus (Fig. 4A to C). A phosphotyrosine signal was alsoobserved along the flagellum (Fig. 4A to C), which faded outtoward its distal tip, particularly in the case of 4G10 and PY-100 staining. It is worth noting that the fixation method (4%PFA versus 100% methanol) had no effect on the overall stain-ing pattern; however, the recognition of phosphotyrosine-con-

FIG. 3. Mass spectrometric analysis of T. brucei phosphotyrosine-containing peptides. Shown is the fragmentation spectrum of the diphos-phorylated peptide GVGVNVpTSpYVVTR (p indicates phosphorylated residue) of a putative TbMAPK (GeneDB accession no. Tb927.6.1780)measured on an LTQ-Orbitrap mass spectrometer. Phosphorylation at the threonine and tyrosine residues of the TSY motif could be deduced dueto the neutral loss of phosphoric acid starting from the y7 and b7 ions (P) and the observed mass increment of 243 Da (�P) between the y4 andy5 ions, respectively.

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taining structures using methanol-fixed procyclic form cellsseemed to be weaker compared to that using PFA fixation(data not shown). A no-primary antibody control was alsoincluded to demonstrate that the staining was a consequence ofthe primary antibody and not a nonspecific background (Fig.4D). The specificity of the anti-phosphotyrosine antibody 4G10to recognize phosphotyrosine-containing residues on fixed try-panosomes was tested by treating coverslips with alkaline phos-phatase overnight and then with 4G10 staining and immuno-fluorescence microscopy (see Fig. S3 in the supplemental

material). The treatment with alkaline phosphatase abolishedthe immunofluorescence signal, demonstrating the selectivityof the 4G10 antibody after cell fixation and ruling out thepossibility of cross-reactivity.

In order to investigate whether the proteins visualized by theanti-phosphotyrosine antibodies were associated with microtu-bular structures, procyclic form T. brucei total cytoskeletonswere extracted with detergent, fixed in 4% PFA, and immuno-stained with the anti-phosphotyrosine antibodies 4G10, PY-20,and PY-100 (Fig. 5). Immunofluorescence images of the cy-

TABLE 1. Tyrosine-phosphorylated protein kinases of whole-cell procyclic trypanosomesa

T. bruceiGeneDB

accession no.cGeneDB annotation Kinase group Kinase family MW

(in thousands)d Phosphopeptideb Reference

Tb10.70.7040 CRK1 CMGC CDK 34.3 11 IGEGSpYGVVFR 21 37, 38, 39Tb10.389.1730* MAPK11, putative CMGC MAPK 47.3 173 EESDQGEHMTDpYVTMR 188Tb10.6k15.2790 MAPK1, putative CMGC MAPK 39.3 160 CFNTQGGDNDLTEpYIATR 177Tb927.7.2420 GSK3�, putative CMGC GSK3 55.3 274 NVPpYIFSR 281Tb927.3.690* PK, putative CMGC MAPK 63.8 170 ARPPYTDpYVSTR 181Tb927.7.3580 PK, putative NEK 66.5 429 FNGGpYNSSSVSGNGVVK 445Tb10.70.2070* MAPK2, putative CMGC MAPK 49.7 166 EQVARPVLTDpYIMTR 180Tb11.01.4230 PK, putative CMGC CLK 53.1 99 KVTpYALPNQSR 109Tb10.329.0030 PK, putative CMGC CDK 64.4 234 DAQASDTFpYVCTR 246Tb927.8.3770* PK, putative CMGC MAPK 46.9 182 EDTQDPNKTHpYVTHR 196Tb09.211.0960* PK, putative CMGC MAPK 41.7 152 GLHVSQPLTEpYVSTR 166Tb10.70.2210 CRK3 CMGC CDK 35.0 26 MDILGEGTpYGVVYR 39 39Tb11.02.0640 PK, putative CMGC DYRK/class II 51.5 266 LFTpYIQSR 273Tb11.01.8550* ECK1 CMGC MAPK 72.1 155 GNYTEpYVATR 164 15Tb10.61.1850* MAPK9, putative CMGC MAPK 42.7 158 SRPPFTEpYVSTR 169Tb10.61.0250 MAPK2 CMGC MAPK 41.8 180 DDQCTQTSALTEpYVVTR 196 28Tb927.6.1780* PK, putative CMGC MAPK 46.1 180 GVGVNVTSpYVVTR 192Tb927.7.7360 CRK2 CMGC CDK 39.2 52 VGEGSpYGIVYK 62 38, 39Tb10.61.3140 PK, putative CMGC GSK3 40.3 178 LAADEPNVApYICSR 191

a Shown are proteins identified in our phosphotyrosine-specific proteomics study with at least one tyrosine-phosphorylated residue.b pY indicates a tyrosine-phosphorylated residue. Numbers preceding and following the sequences specify the start and the end of the phosphopeptide sequence.c TbMAPKs with a fully phosphorylated TXY motif are indicated with an asterisk.d MW, molecular weight.

TABLE 2. Non-protein kinase tyrosine-phosphorylated proteins of whole-cell procyclic trypanosomesa

T. bruceiGeneDB

accession no.Protein description MW

(in thousands)c Phosphopeptideb Reference

Tb927.5.3120 Translation initiation factor, putative 34.9 287 SVQAVGSATpYSAQVGKR 303Tb11.01.1475 Ribosomal protein S27, putative 9.6 25 LVQGPNSpYFMDVK 37Tb927.2.4210 Glycosomal phosphoenolpyruvate carboxykinase;

glycosomal protein P6058.5 172 EQVILGTEpYAGEMK 185

Tb927.3.3270 TbPFK ATP-dependent phosphofructokinase;6-phospho-1-fructokinase

53.5 54 DKTDYIMpYNPRPR 66 23

Tb09.211.3510 ATP-dependent DEAD/H RNA helicase, putative 82.7 138 FDVDFpYDRPR 147Tb09.211.0170 Hypothetical protein, conserved 28.8 96 FTGGpYSSTPYVTSDAR 111Tb927.4.2040 Hypothetical protein, conserved 20.8 2 PSpYPPRDEYR 11Tb10.6k15.2400 Hypothetical protein, conserved 97.7 536 GAEEAQAALpYAVGK 549Tb11.02.0420 Hypothetical protein, conserved 21.4 7 CIICAGNSAHpYR 18Tb10.70.3380 Hypothetical protein, conserved 52.9 34 SILGKpYPIR 42Tb09.160.1160 Hypothetical protein, conserved 85.9 19 IGGDpYEWSNTLAR 31Tb11.01.2800 Hypothetical protein, conserved 41.6 287 THDpYDEPQELIR 298Tb927.4.3130 Hypothetical protein, conserved 30.6 178 VGVTSGpYANTQGR 190Tb927.3.1400 Hypothetical protein, conserved 37.2 279 YLQSQNCRPTGNAGpYGGGN

GPASSAEVR 306Tb10.6k15.3460 Hypothetical protein, conserved 284.7 385 HVMEQHpYGTIAK 396

a Shown are proteins identified in our phosphotyrosine-specific proteomics study with at least one tyrosine-phosphorylated residue.b pY indicates a tyrosine-phosphorylated residue. Numbers preceding and following the sequences specify the start and the end of the phosphopeptide sequence.c MW, molecular weight.

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toskeleton preparations gave the same staining pattern thatwas previously observed with whole cells, except for the nu-cleus (compare with Fig. 4). Identical results were obtainedwith bloodstream form T. brucei cytoskeletons using the anti-phosphotyrosine antibody 4G10 (see Fig. S4 in the supplemen-tal material). The strong signal for the punctate structures inthe posterior of the cell was present in both life cycle stages,and the position and shape of this organelle indicated thelabeling of the basal body (mature and immature probasalbody). The lack of any gap between the signal for the basalbody and the labeling of the flagellum (Fig. 5A), as well as thefact that the signal for the flagellum connects to only one (themature basal body), strongly pointed to the flagellum signalbeing axonemal rather than paraflagellar rod or flagellum at-tachment zone filament (Keith Gull, personal communica-tion). Moreover, the signal did not seem to vary much throughthe cell cycle, as it could be detected on the old and newflagella (Fig. 5B).

In order to define the individual components within thecytoskeleton that the phosphotyrosine signal was associatedwith, we performed immunolocalization studies using 4G10 in

association with the monoclonal antibodies BBA4 and Rib72.BBA4 is a specific marker for the proximal poles of both thebasal and probasal bodies (45) and labels the basal body struc-ture as a two-dot pattern in the posterior of a trypanosome cell(12). Colabeling experiments indicate that phosphotyrosine-containing proteins are localized toward the distal end of boththe basal and probasal bodies, as the staining patterns for 4G10and BBA4 show very close proximity but do not overlap (Fig.6C). This specific labeling has previously been observed inprocyclic trypanosomes when using the anti-phosphopeptidemonoclonal antibody MPM2 (12). In addition, the phosphoty-rosine signal seems to be on the distal end of the basal bodyrather than around this organelle, as determined by takingdifferential interference contrast images of anti-phosphoty-rosine 4G10-labeled flagellum extractions (Fig. 6E).

We next investigated the localization of phosphotyrosine-containing proteins observed along the length of the flagellumby using the monoclonal anti-axonemal Rib72 antibody. Rib72extends from the basal body to the tip of the flagellum but isnot present in the basal body itself (Keith Gull, personal com-munication). Colabeling experiments with 4G10 (Fig. 7B) and

FIG. 4. Indirect immunofluorescence using anti-phosphotyrosine antibodies. Whole-cell procyclic trypanosomes were fixed in 4% PFA andstained with 4G10 (A), PY-20 (B), and PY-100 (C). (D) A no-primary-antibody control was included and demonstrated that the staining was aconsequence of the primary anti-phosphotyrosine antibodies. Tyrosine-phosphorylated proteins (in green) localized to punctate structures in theposterior of the cells (white arrows) and were also found along the length of the flagellum (yellow arrows) and in the nucleus (white arrowheads).(E and F) Control HeLa cells labeled with 4G10 showed concentrated anti-phosphotyrosine staining (in red) at focal adhesion regions. DNA (blue)is visualized by 4�-6-diamidino-2-phenylindole (DAPI) staining. Anti-�-tubulin is shown in green. p-Tyr, tyrosine-phosphorylated proteins. Whitebar corresponds to a length of 5 �m.

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Rib72 (Fig. 7C) revealed that the staining pattern of bothmonoclonal antibodies overlapped in most of the areas (Fig.7D). Thus, the phosphotyrosine signal occurs as a continuouspattern between the basal body and the tip of the flagellum,indicating that T. brucei tyrosine-phosphorylated proteins areassociated with, or are immediately adjacent to, the flagellumaxoneme. However, part of the phosphotyrosine signal doesnot colocalize with the Rib72 stain but separates from theaxoneme and occurs along the cell body (Fig. 7D). This obser-vation suggested that phosphotyrosine-containing proteinscould also be associated with membranous structures close tothe flagellum in T. brucei. An axonemal location of phospho-tyrosine proteins was also supported by flagellum extraction ofprocyclic form cells and staining with the anti-phosphotyrosine4G10 antibody (Fig. 6E). The phosphotyrosine signal on thebasal body and up the flagellum survived the high-salt treat-ment applied to obtain these isolated flagellum preparations,indicating that phosphotyrosine-containing proteins are tightlyassociated with structures forming the flagellum.

To determine the subnuclear localization of phosphoty-rosine-containing proteins observed by immunofluorescencemicroscopy (Fig. 4B), we used a fluorescent marker synthetic

peptide [TRITC-D(R10)] that targets the nucleolus (22). Thissubcompartment of the nucleus was chosen for the colocaliza-tion studies because the nucleolar T. brucei proteinNOPP44/46 (GeneDB accession no. Tb927.8.760) has beenidentified as having developmentally regulated tyrosine phos-phorylation (11, 31). The nucleolus-specific TRITC-D(R10)label colocalized with the anti-phosphotyrosine signal in a dis-tinct region of the nucleus (see Fig. S5 in the supplementalmaterial), thus confirming previous evidence by the Parsonsgroup of tyrosine-phosphorylated proteins in the nucleolus.

DISCUSSION

In this study, we used advanced MS-based phosphotyrosine-specific proteomics to shed light on the extent of protein ty-rosine phosphorylation in T. brucei. We show that proteintyrosine phosphorylation is a relatively abundant posttransla-tional modification in an organism that lacks conventional ty-rosine kinases (Nett et al., submitted for publication). Of the34 phosphotyrosine-containing proteins we detected in thisstudy, 19 protein kinases and 2 metabolic kinases (phos-phoenolpyruvate carboxykinase and 6-phospho-1-fructokinase)

FIG. 5. Indirect immunofluorescence analysis of cytoskeletal preparations using anti-phosphotyrosine antibodies. Procyclic form cytoskeletonswere fixed in 4% PFA and stained with 4G10 (A), PY-20 (B), and PY-100 (C). (D) A no-primary-antibody control was included and demonstratedthat the staining was a consequence of the primary anti-phosphotyrosine antibodies. The staining pattern suggested labeling of the basal bodiesin the posterior of the cell and was observed along the length of the flagellum (in green). DNA (blue) is visualized by 4�-6-diamidino-2-phenylindole(DAPI) staining. White bar corresponds to a length of 5 �m.

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could be identified, which suggests that in T. brucei tyrosinephosphorylation preferentially occurs on proteins that possessputative kinase activity. Of the 19 protein kinases, phosphory-lation sites on 11 T. brucei homologues of Leishmania MAPKscould be identified. MAPK family members require phosphor-ylation of both a threonine residue and a tyrosine residue inthe activation loop for full enzymatic activity of the kinase. Wefound the highly conserved TXY motif in the activation seg-ment of MAPKs fully phosphorylated on eight of the identifiedputative TbMAPKs. Phosphorylation of the tyrosine residueallows the activation loop to adopt the active conformation,and subsequent phosphorylation of the threonine residue ac-tivates the kinase fully by promoting the correct alignmentof the catalytic residues (17). Thus, the remaining putativeTbMAPKs found to be phosphorylated on only the tyrosineresidue, including the previously characterized TbMAPK2(28), are likely to represent the kinase in an inactive state.These results suggest that the activation of MAPKs in thishighly divergent parasite follows the same mechanistic princi-ples as those in higher eukaryotes. The expression of phosphor-ylated STE kinases, which are thought to represent TbMAPKupstream activator proteins (29) in bloodstream form trypano-somes (Nett et al., submitted for publication), could be evi-dence for the existence of canonical signal transduction viaMAPK in T. brucei.

Other protein kinases identified in this study include thecell division cycle 2 (Cdc2)-related protein kinases CRK1(GeneDB accession no. Tb10.70.7040), CRK2 (GeneDB ac-cession no. Tb927.7.7360), and CRK3 (GeneDB accession no.Tb10.70.2210), all of which were found to be tyrosine phos-phorylated in their N terminus (Y16, Y57, and Y34, respec-tively). All three CRK isoforms have previously been detectedphosphorylated on the same residues in our global phospho-proteome study of bloodstream form T. brucei (Nett et al.,submitted for publication), suggesting that life cycle stage-specific tyrosine phosphorylation of these residues can be ex-cluded. Interestingly, the phosphorylated tyrosine sites areflanked by an S15 in CRK1, S56 in CRK2, and T33 in CRK3,which could correspond to the conserved human CDK1 T14

and Y15 residues. Phosphorylation of the conserved Y15 ofCDK1 by the Wee1 dual-specificity tyrosine kinase inactivatesthe CDK complex (20), and as a Wee1 kinase is encoded by theT. brucei genome, it is possible that CRKs in T. brucei areregulated by a similar mechanism.

Our detection of previously identified trypanosomal phos-phorylation sites within the activation segments of both iso-forms of the glycogen synthase kinase-3 (GeneDB accessionno. Tb927.7.2420 and Tb10.61.3140) and a DYRK homologue(GeneDB accession no. Tb11.02.0640) (Nett et al., submittedfor publication) strongly supports the view that known kinase-

FIG. 6. Colocalization studies of phosphotyrosine staining using the anti-basal body marker BBA4. Indirect immunofluorescence of procyclicform T. brucei cytoskeletons colabeled with the anti-phosphotyrosine antibody 4G10 (A) and BBA4 (B), a specific marker for the proximal poleof both basal and probasal structures. Images shown in panels A and B are shown merged in panel C. Differential interference contrast (DIC)images of T. brucei flagella were taken (D) and merged with immunofluorescence images labeled with the anti-phosphotyrosine 4G10 antibody (E).White arrows, labeling of the basal body. Colabeling structures that were observed are enlarged and surrounded by a white box. p-Tyr,tyrosine-phosphorylated proteins. White bar corresponds to a length of 5 �m.

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activating sites are conserved in T. brucei and indicates thatseveral segments of known signal transduction pathways areconserved in this organism.

This study also provided an insight into the localization oftyrosine-phosphorylated proteins in both life cycle stages of T.brucei. Proteins carrying this posttranslational modificationwere found to be concentrated at the cytoskeleton, specificallywith the axoneme and the basal bodies of the flagellum, as wellas the nucleolar compartment of the parasite, as determined byimmunofluorescence microscopy. This is in stark contrast tothe localization pattern of tyrosine-phosphorylated proteins inmammalian cells, in which the signal for this posttranslationalmodification is concentrated at the plasma membrane in focaladhesion points that are known as locations for high tyrosinekinase activity (40). In these studies, it is difficult to correlatephosphotyrosine proteome data with localization data, sincethe former are based on the purification of phosphotyrosine-containing tryptic peptides, whereas the latter are based onantibody recognition in the context of folded and fixed pro-teins. Therefore, we cannot say what proportion of the phos-phoproteome is associated with the cyctoskeletal and nucleolarcompartments nor what subset of phosphoproteins might bespecific for those locations. Nevertheless, the lack of conven-tional receptor tyrosine kinases and nonreceptor tyrosine ki-nases in T. brucei might be one aspect of the different stainingpatterns observed between trypanosomes and mammaliancells. The different locations of phosphotyrosine-containingproteins in the human system and the parasite might alsodirectly reflect different functions of phosphotyrosine-based

signaling in the organisms due to their substantially differentrequirements for survival.

Work by Das et al. (11) has identified a tyrosine-phosphor-ylated NOPP44/46 protein with a nucleolar localization in pro-cyclic trypanosomes. This observation is consistent with ourdetection of a phosphotyrosine signal in the nucleolus of pro-cyclic form cells by immunofluorescence. It remains unclearwhether our microscopy studies also detected the NOPP44/46protein, as it was not detected in our focused tyrosine phos-phoproteomics screen of whole procyclic form cells. However,analysis of the NOPP44/46 amino acid sequence (GeneDBaccession no. Tb927.8.760) reveals that, of the five tyrosineresidues it contains, at least four of them are undetectable byMS. One is the C-terminal residue in the sequence RY (thetryptic product would be too small to detect), and three residein tryptic peptides with masses of 6,947 Da (two sites) and7,440 Da (one site) that are too large to provide meaningfulMS-MS spectra. Only the N-terminal tyrosine residue (Y) inthe sequence MEGFYGVEVSAGQKVK would be potentiallydetectable by our method. However, there is no guarantee thatthis is a phosphorylation site in NOPP44/46, and in any phos-phoproteome analysis there is no guarantee that every occu-pied site will be detected. NOPP44/46 has been shown to bindnucleic acid (11), and RNA interference of the T. brucei-spe-cific protein in the procyclic form affects large-subunit rRNAprocessing (19). This raises the interesting question of whethergrowth control and development of T. brucei are mediated bynucleolus-localized tyrosine-phosphorylated proteins, with ri-bosome synthesis as the obvious target of regulation. A moretargeted phosphoprotomics study of T. brucei nucleoli couldprovide answers to the questions of how these nucleolus-local-ized tyrosine-phosphorylated proteins operate and if there is aconnection to known signaling pathways.

The observation of tyrosine-phosphorylated proteins locatedon the basal body and along the axoneme of the flagellumextending to its distal tip is intriguing, as it suggests that sig-naling molecules could be associated with microtubules, whichhas been shown with mammalian cells (26). In fact, in mam-malian cells, many kinases, such as MAPK, MEK1, MEK2,Raf1 (MEKK), CDK5, and CDC2, as well as many phosphata-ses, such as protein phosphatase 2A (PP2A), protein phos-phatase 2B (PP2B), and protein phosphatase 1 (PP1), weredetected in microtubule pellets, indicating that microtubuleassembly and stability might be regulated by an interplay ofthese signaling molecules. The same study also revealed thatthe pool of microtubule-associated MAPK was constitutivelyactive, which would be consistent with our observation that thephosphotyrosine signal along the flagellum and on the basalbody was cell cycle independent.

Interestingly, a recent study of the green alga Chlamydomo-nas reinhardtii has shown that the tyrosine-phosphorylated ac-tive form of GSK3 was enriched in the flagellum (43). RNAinterference of Chlamydomonas reinhardtii GSK3 resulted incells that had no flagella, suggesting a role for GSK3 in assem-bly and maintenance of flagella, presumably through the reg-ulation of intraflagellar transport. Our study showed thattyrosine-phosphorylated proteins are associated with the fla-gellum and the basal body, which is an organelle that providesa platform for recruiting proteins involved in intraflagellartransport (34). In addition, we identified TbGSK3 phosphory-

FIG. 7. Phosphotyrosine-containing proteins are associated withthe flagellum axoneme. Cytoskeleton preparations of procyclic form T.brucei (A) were colabeled with the anti-phosphotyrosine 4G10 anti-body (B) and the anti-axonemal Rib72 antibody (C). The overlap ofboth staining patterns in most of the areas (D) strongly suggested thatthe anti-phosphotyrosine signal is axonemal. The area of the phospho-tyrosine signal that did not colocalize with the Rib72 staining is indi-cated with arrows (D). White bar corresponds to a length of 5 �m.

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lated in its activation loop, suggesting that the kinase is activein the parasite. Both flagellum formation and basal body seg-regation are vital for T. brucei (4, 13), and our findings indicatethat tyrosine-phosphorylated proteins might play a role inthese essential processes.

ACKNOWLEDGMENTS

We thank Keith Gull at the University of Oxford for providing theBBA4 and Rib72 monoclonal antibodies, Silvana van Koningsbruggenat the University of Dundee for the TRITC-D(R10) synthetic peptide,and Lucia Guther for procyclic cells. We also thank Sam Swift of theCentre for High Resolution Imaging and Processing at the Universityof Dundee for help with light microscopy.

I.R.E.N. was supported by a Wellcome Trust Prize Ph.D. student-ship. M.A.J.F. is supported by a Wellcome Trust program grant(085622). L.D. is supported by an MRC program grant and theDundee DSTT consortium (Astra Zeneca, Boehringer Ingelheim,GlaxoSmithKline, Merck and Co., Merck KGaA, and Pfizer).

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