Báo cáo khoa học: Investigation of the kinetics and order of tyrosine phosphorylation in the T-cell receptor f chain by the protein tyrosine kinase Lck potx

Investigation of the kinetics and order of tyrosine phosphorylation in the T-cell receptor f chain by the protein tyrosine kinase Lck Hazel R. Housden 1 , Paul J. S. Skipp 1 , Matthew P. Crump 1 , Robert J. Broadbridge 1 , Tom Crabbe 2 , Martin J. Perry 2 and Michael G. Gore 1 1 Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, UK; 2 Celltech Group plc, Slough, UK We report experiments to investigate the role of the physio- logically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor f chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the f chain. Preliminary 1 H-NMR studies of recombinant f chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphoryla- tion kinetics were determined for each tyrosine by assaying the transfer of 32 P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the pro- posal that Lck phosphorylates the six f chain ITAM tyro- sines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor f chain. After in vitro phosphorylation by Lck, the partially phosphorylated f chain was digested with trypsin. Separation and identifica- tion of the f chain fragments using LC–MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six f chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contri- butes to the distinct patterns of phosphorylation observed in vivo. Keywords: immunoreceptor tyrosine-based activation motif (ITAM); mass spectrometry; NMR; protein tyrosine kinase Lck; T-cell receptor f chain. The T-cell receptor (TCR) complex is essential for T-cell function in the adaptive immune response. On binding of the TCR to appropriate antigens, Src-family protein tyrosine kinases (PTKs) such as Lck phosphorylate tyro- sines located within immunoreceptor tyrosine-based activa- tion motifs (ITAMs) [1,2]. ITAMs have the consensus sequence Y-X-X-(L or I)-X (6)8) -Y-X-X-(L or I) and are found in the intracellular portions of the TCR complex c, d, e and f chains, as well as in other immunoreceptors including the B-cell receptor [3] and several Fc receptors [4,5]. Doubly phosphorylated ITAMs form binding sites for pairs of Src homology domain 2 (SH2) domains, such as those found on ZAP-70 (f-associated protein of 70 kDa). Within 15 s of stimulation of the TCR, ZAP-70 binds to phosphorylated f chain and becomes activated [6]. A phosphorylation cascade ensues which culminates in T-cell activation. Therefore, phosphorylation of ITAM tyrosines is an absolute requirement for the TCR-mediated trigger of T-cell activation [2,7]. Probing the TCR with different stimuli leads to various patterns of f ITAM tyrosine phosphorylation which in turn alters the T-cell response [8]. Only full agonist ligands can enable full phosphorylation of all six ITAM tyrosines to make the phosphorylated f form Ôp23Õ [9] and bring about the full array of T-cell effector functions [including ZAP-70 recruitment, interleukin 2 production, calcium fluxing, and Ins(1,4,5)P generation]. Partial agonist ligands effect partial phosphorylation, sometimes generating the partially phosphorylated f form Ôp21Õ, and a partial or antagonist response. The observed, crucial, ordered phosphorylation of TCRf chain ITAMs may potentially be influenced by several different factors in vivo. We report data from experiments designed to investigate the role of PTK Lck in this process and ascertain whether the kinase has a preference for phosphorylation of certain ITAM tyrosines over others. The six TCRf ITAM tyrosines investigated will be described according to their location in the TCR, with the ITAM closest to the N-terminus/membrane referred to as 1, the next closest as 2, and the farthest as 3. The two tyrosines within each ITAM are then further classified as N or C, reflecting their locations relative to the N-terminus and C-terminus (Fig. 1). Six synthetic peptides were made based on the individual sequences of TCRf, and modified to con- tain only a single tyrosine. These served as model substrates for assessment of the phosphorylation kinetics of these individual tyrosines. The absence of secondary structure in Correspondence to M. G. Gore, Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK. Fax:+442380594459,Tel.:+442380594313, E-mail: mgg@soton.ac.uk Abbreviations: ITAM, immunoreceptor tyrosine-based activation motif; His-cTCRf, histidine-tagged cytosolic TCRf;PTK,protein tyrosine kinase; SH2, src homology domain 2; TCR, T-cell receptor. (Received 18 February 2003, revised 29 March 2003, accepted 2 April 2003) Eur. J. Biochem. 270, 2369–2376 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03604.x whole, cytosolic TCRf is shown by CD [10] and our 1 H-NMR spectroscopy, suggesting that it is unlikely that ITAM tyrosines are buried by the rest of the f chain. Consequently, tyrosines located on the peptides may be expected to exhibit the same kinetics as those located on the intact protein. Therefore, determination of the kinetics of phosphorylation of these peptides allows the order of phosphorylation of the six TCRf ITAM tyrosines, by the PTK Lck, to be deduced. Experimental procedures Peptide synthesis Peptides were made by manual solid-phase peptide synthesis using the tBoc method [11], purified by RP-HPLC, and analysed by MS. The following peptides were synthesized for direct determination of phosphorylation kinetics: 1N, QNQLYNELNLGRREEFDVLDNle; 1C, QNQLFNEL NLGRREEYDVLDNle; 2N, QEGLYNELQKDKMAE AFSEIG; 2C, QEGLFNELQKDKMAEAYSEIG; 3N, HDGLYQGLSTATKDTFDALH, 3C, HDGLFQGLST ATKDTYDALH. The nonstandard amino acid norleucine (Nle) was used to replace a methionine in the ITAM1-based peptides, which exhibited a tendency for oxidation during synthesis. A phosphotyrosine (pY)-containing peptide, based on f ITAM1, (pp1, QNQLpYNELNLGRREEpY DVLD) was synthesized using Fmoc chemistry [12], for use as an experimental control. An unrelated Ôcontrol peptideÕ (CP, GAHNITEEEDTWQKLC) was also used. The con- centrations of most of the peptides were determined from their A 280 , using absorption coefficients of 5690 M )1 Æcm )1 for tryptophan, 1280 M )1 Æcm )1 for tyrosine, and 0 M )1 Æcm )1 for phenylalanine [13]. The concentration of phosphotyro- sine-containing peptides was determined at 267 nm using molar absorption coefficients of 652 M )1 Æcm )1 for phospho- tyrosine [14] and 1427 M )1 Æcm )1 for tyrosine [15]. Gene cloning, protein expression, and purification of glutathione S -transferase (GST)–Lck The coding region of human p56 Lck (amino acids 1–509) was placed in-frame with the coding sequence of GST from Schistosoma japonicum [16] in a pEE12 vector [17], which was then stably transfected into the mouse myeloma NS0 cell line. Cells were grown in mass culture before harvesting and lysis in 50 m M Tris/HCl, pH 7.3, containing 1% Nonidet P40 and protease inhibitors. GST–Lck was purified using glutathione-linked resin, washing in buffer A (25 m M Pipes/NaOH, 500 M NaCl, pH 6.8), and then eluting the bound GST–Lck in 20 m M glutathione (reduced) in buffer A, before storage in aliquots at )70 °CinbufferAwith 10% (v/v) glycerol. Gene cloning, protein expression, and purification of His-cTCRf The cytosolic portion of human TCRf was cloned into the pQE-30 vector (Qiagen) giving it an N¢-terminal His 6 tag. Sequencing of this cTCRf gene revealed it to have a slightly different nucleotide, and consequently amino-acid, sequence from the published sequence for the cytosolic portion of TCRf. Instead of the nucleotide sequence gcagag at positions 249–254 published by Weissman et al. [18], corresponding to residues Glu60 and Phe61 in the whole protein (SwissProt accession number P20963), we found the nucleotide sequence gacgcc in our His-tagged cystosolic TCRf chain (His-cTCRf) gene, corresponding to residues Asp21 and Ala22 in His-cTCRf. The variation in sequences is probably due to natural polymorphism of this gene and is thought unlikely to influence the phosphorylation kinetics, as the residues involved do not lie in any of the ITAMs or the ITAM peptide mimic sequences used in our studies. To produce His-cTCRf, a single colony of freshly transformed Eschericia coli JM103 cells was grown over- night in Luria–Bertani broth containing 100 lgÆmL )1 ampicillin. Six flasks containing 750 mL Luria–Bertani broth and ampicillin were inoculated and grown at 37 °Cin an orbital shaker. When the A 600 reached 0.6, expression of His-cTCRf wasinducedwith1m M isopropyl thio-b- D - galactoside (final concentration), and the cells grown for a further 3 h before harvesting by centrifugation. Cell pellets were resuspended in buffer A [50 m M Tris/HCl, 500 m M NaCl, 0.01% (w/v) sodium azide, pH 7.5] also containing lysozyme (1 mgÆmL )1 ; Sigma), a Complete TM EDTA-free protease inhibitor cocktail tablet, and DNase I (5 l M ; both Fig. 1. Nomenclature used to describe positions of the six ITAM tyrosines in TCRf and simulated trypsin digest of His-cTCRf. The ITAM tyrosines have been named according to the ITAM they are in and their position within each ITAM (towards the N¢ or C¢ terminus). Thus the individual tyrosines are named 1N, 1C, 2N and so on, as shown in bold above the corresponding tyrosine. The location of trypsin cleavage sites (fl)inHis- cTCRf is such that complete digestion will separate all of the ITAM tyrosines on to different peptide fragments (numbered in italics), which contain between8and22residues. 2370 H. R. Housden et al.(Eur. J. Biochem. 270) Ó FEBS 2003 from Roche Diagnostics GmbH, Mannheim, Germany) and sonicated. The soluble His-cTCRf was separated from insoluble debris by centrifugation at 40 000 g and purified using a 5-mL Hi-trap chelating column (Amersham Phar- macia Biotech UK Ltd) charged with Ni 2+ ions. Buffer A containing 50 m M imidazole was used to remove weakly binding contaminants from the coordinated Ni 2+ . The pure His-cTCRf wasthenelutedin200m M imidazole [in 50 m M Tris/HCl, 500 m M NaCl, 0.01% (w/v) sodium azide, pH 7.5] and exhaustively dialysed against buffer A to remove imidazole. Assay of radioactivity incorporation Peptides were prepared at a range of concentrations between 0 and 140 l M and incubated with recombinant PTK GST-Lck (0.03–0.1 mgÆmL )1 )and[c- 32 P]ATP (1–5 lCi per reaction; Amersham Pharmacia Biotech UK Ltd) in 50 m M Tris/HCl, pH 7.5, containing 150 m M NaCl, 10 m M MgCl 2 ,10m M MnCl 2 and 50 l M nonradioactive ATP. Each 40 lL reaction was incubated for 30 min at 30 °C, and then stopped by adding 8 lL acetic acid. Aliquots (12 lL) of stopped reaction mixture were applied to strips of P81 phosphocellulose paper (Whatman), in triplicate. Once completely dry, the strips were washed in 1% (v/v) phosphoric acid for 3 · 10 min, then rinsed for 5 min in acetone, and air-dried. The strips were immersed in scintillation fluid (Optiphase HiSafe 3; Wallac), and a Beckman LS 6500 scintillation counter was used to detect 32 P. As a negative control, 50 l M assayed peptide was incubated without the GST-Lck. To determine the total radioactivity present for each experiment, 12 lLlotsof pooled, stopped reaction mixture were pipetted on to three phosphocellulose strips which, after drying, were transferred directly into the scintillation fluid, without washing. NMR of His-cTCRf The protein was resuspended in 500 lLH 2 Oand50lL D 2 O, and the pH adjusted to 6 with dilute HCl. Standard 1D and 2D NOESY and DQF-COSY experiments were recorded at 600 MHz on a Varian INOVA spectrometer at the University of Southampton. Spectra were processed and analysed using VNMR. Analysis of the phosphorylation of His-cTCRf using on-line LC-MS A 1.6-mL reaction was prepared, containing 84 l M His- cTCRf,24lgÆmL )1 GST-Lck, 300 l M ATP, 10 m M MgCl 2 ,10m M MnCl 2 ,20m M Tris/HCl, pH 7.5. It was incubated at 37 °C, and 200 lL samples were removed after 0, 5, 15, 30, 60, 90, 120 and 180 min and mixed with 40 lL acetic acid to stop the reaction. The His-cTCRf was then purified from the other reaction components using RP- HPLC. For each timepoint sample, 200 lL stopped reac- tion mix was loaded on to a 50 · 4 mm Genesis C 4 column with 4-lm diameter beads with 300-A ˚ pores (Jones Chro- matography USA Inc., Lakewood, CO, USA) using a Hewlett-Packard series 1050 HPLC at 0.7 mLÆmin )1 in solvent A [0.1% (v/v) trifluoroacetic acid in H 2 O]. A linear gradient was run from 5 to 40% solvent B [0.05% (v/v) trifluoroacetic acid in acetonitrile) over 24 min following the A 216 . All of the His-cTCRf phosphospecies were eluted at about 20 min and were collected. Samples of these were analysed using MS to identify the phosphospecies present. Mass spectra were collected using a Fisons VG Quattro II electrospray mass spectrometer in positive ion mode with a scan range of 500–2500 m/z, a source temperature of 100 °C, and capillary and cone voltages of 4230 V and 29 V, respectively. Mass profiles were deconvoluted using the maximum entropy software MAX ENT (Micromass UK Ltd., Manchester, UK) initially over a 5–25 kDa, and then finally to 1 Da resolution between 14 and 15.5 kDa (the only region containing significant peaks). The HPLC-purified, partially phosphorylated, His- cTCRf samples were concentrated to dryness under vacuum and then redissolved in solution containing 50 m M ammo- nium bicarbonate, 5 m M CaCl 2 , 10% (v/v) acetonitrile and HPLC-grade trypsin (Roche Diagnostics GmbH; 4% of the mass of His-cTCRf) and incubated at 37 °Cfor24hto separate each ITAM tyrosine on to a different fragment (Fig. 1). The 200-lL samples were loaded on to a Synergi 4 l RP-polar HPLC column (250 · 4.6 mm; Phenomenex, Torrance, CA, USA) using a Hewlett–Packard series 1050 HPLC at 8% solvent B [where solvent A is 0.1% (v/v) trifluoroacetic acid in water and solvent B is 0.05% (v/v) trifluoroacetic acid in acetonitrile] at 0.7 mLÆmin )1 .The peptide fragments were separated on a linear gradient of 8–40% solvent B, over 99 min, and, on exiting the column, passed through a UV detector set to 216 nm; 10% of the flow was directed into the Fisons VG Quattro II electro- spray mass spectrometer, set in positive ion mode, for continuous recording. Scans were set up to detect ions with m/z values in the range 500–2500, with a source temperature of 100 °C and capillary and cone voltages of 4230 V and 29 V, respectively. Results The phosphorylation was investigated by incubating a range of concentrations of each ITAM peptide with recombinant GST–Lck, [c- 32 P]ATP and unlabelled ATP over 30 min. The excess of ATP ensured that none of the reactions were rate-limited by ATP concentration. The levels of incorpor- ation of 32 P into the peptide were used to calculate the kinetics of phosphorylation by Lck at each tyrosine, and from these an order of phosphorylation was determined. The results of these peptide studies were then followed up using whole (cytosolic) f chain. The recombinant f chain was phosphorylated under limited ATP conditions, so that, if phosphorylation were ordered, then only the first tyrosine in the series would become significantly phosphorylated. Samples of the partially phosphorylated f chain were then digested with trypsin so that each ITAM tyrosine was on a different fragment. On-line LC-MS was then used to separate and identify the fragments, allowing clear identi- fication of the first tyrosine phosphorylated by Lck, as tyrosine 1N, in agreement with the peptide-based studies. Assay of 32 PO 4 incorporation For each peptide representing a tyrosine from His-cTCRf, triplicate scintillation count data were obtained at a range Ó FEBS 2003 Phosphorylation of the T-cell receptor f chain (Eur. J. Biochem. 270) 2371 of peptide (substrate) concentrations. The triplicate values were averaged, and the negative control subtracted. The short half-life of 32 P necessitated determination of the spe- cific radioactivity of the phosphate at the time of the assay from the Ôtotal radioactivityÕ samples, to allow conversion from c.p.m. to mol phosphate. The phosphorylation was shown to occur at a constant rate over the 30 min duration of the assay (data not shown) and was not limited by concentrations of peptide or ATP, the latter giving rise to near saturation (95%) of the enzyme [19]. It was therefore assumed that the extent of phosphorylation at 30 min is directly proportional to the phosphorylation rate. For each set of peptide concentrations and corresponding rate values, kinetic parameters were calculated using the Hanes–Woolf derivative of the Michaelis–Menten equation (S/v) ¼ (K m /V max )+(S/V max ), where v ¼ therateofreaction, V max ¼ the maximum rate at infinite substrate concentra- tion, S ¼ the substrate concentration, and K m(app) ¼ the Michaelis constant under these conditions. From the calculated value of V max , the turnover number [k cat(app) ] was also determined under these conditions. The values obtained for phosphorylation of each of the six f ITAM tyrosines are presented in Table 1. Here it is seen that the K m(app) of Lck for each of the tyrosines ranges from 2.3 · 10 )5 to 21.7 · 10 )5 M ,andinallcasesishigherthan the published K m of PTK Lck for an ÔartificialÕ,single tyrosine-containing peptide AEEEIYGVLFAKKKK (1.7 · 10 )5 M ) [19] and for a peptide based on whole, cytosolic, TCRf, containing all three ITAMs (0.65 · 10 )5 M ) [20]. The k cat(app) ranges from 2.3 · 10 )4 to 98 · 10 )4 s )1 for phosphorylation of the different f ITAM tyrosines. Our values are lower than the k cat value calculated from published data for the PTK c-Src for phosphorylation of enolate (250 · 10 )4 s )1 ) [21], and also those for PTK Csk with a-casein as substrate (ranging from 330 · 10 )4 s )1 [22] to 2400 · 10 )4 s )1 [23]). The specificity constant [k cat(app) / K m(app) ], a measure of the enzyme’s efficiency with different tyrosine substrates, allows a direct comparison of its effectiveness at phosphorylating the different tyrosines, with a high specificity constant indicating high efficiency. It can be seen that Lck shows marked differences in specificity towards the six tyrosines investigated, suggesting that it will phosphorylate TCRf in the order 1N first [k cat(app) / K m(app) ¼ 122 M )1 Æs )1 ], then 3N, 3C, 2N, 1C and lastly 2C [k cat(app) /K m(app) ¼ 1.76 M )1 Æs )1 ], provided that all sites are equally accessible. Supplementary experiments were performed to investi- gate the effect of the phosphotyrosine product on the ability of Lck to phosphorylate tyrosine substrates. The radio- activity-incorporation assay was performed for peptide 1N in the presence of either a doubly phosphorylated ITAM1- based peptide (pp1) or an unrelated control peptide (CP). The K m(app) and k cat(app) values obtained for phospho- rylation of peptide substrate 1N in the presence (22.8 ± 5.5 · 10 )5 M and 28.7 ± 15.9 · 10 )4 s )1 , respect- ively) and absence (25.1 ± 5.1 · 10 )5 M and 23.1 ± 12.3 · 10 )4 s )1 , respectively) of pp1 are not significantly different, showing that there is no product inhibition. NMR of His-cTCRf The 1D NMR spectrum of His-cTCRf is presented in Fig. 2. The downfield region between 6.6 and 8.6 p.p.m. shows several sets of peaks that can be grouped by type. The two sharp peaks at 7.50 and 8.62 p.p.m. correspond to the aromatic protons of the His 6 tag. At 7.8–8.6 p.p.m. the amide resonances of the protein backbone show a poorly dispersed envelope considering the protein size of 14.3 kDa. In contrast, amide resonances show a well-dispersed envel- ope of peaks in a folded globular protein. This alone indicates that His-cTCRf has no defined secondary or tertiary structure. The two groups of peaks at 6.85 and 7.15 p.p.m. correspond to the aromatic resonances of the seven tyrosines in the protein, and the inset 2D expansion of the 1 H- 1 H COSY shows that the seven are indistinguishable from each other by chemical shift, showing just one overlapped cross-peak between the d and e protons. The remaining 2D spectrum shows few NOEs, again indicating that the protein has no overall structure. In conclusion, it appears that all of the seven tyrosines in His-cTCRf are in similar chemical environments and may therefore be equally exposed to solvent and potentially kinases. Following phosphorylation of His-cTCRf using MS Samples of His-cTCRf were taken after different lengths of incubation with GST-Lck, and their masses determined to assess the level of phosphorylation. For the unphosphoryl- ated sample shown in Fig. 3, the predominant mass was 14 274 Da (24 Da less than the mono-isotopic mass of 14 298 Da predicted from the amino-acid sequence using Biolynx software from Micromass). No single chemical process has been found to account for this, but it may well result from a combination of processes, perhaps including loss of the N¢-terminal methionine ()131 Da) and oxidation of some or all of the remaining five methionines (as observed by van Oers et al. [24]). Less abundant mass species are mostly plus multiples of 14 Da, which could be due to formylation. Mass determination of the samples of His-cTCRf that were incubated with Lck revealed the presence of several phosphospecies, which had masses that were multiples of 80 Da larger than the unphosphorylated protein. The deconvoluted calculated mass profile from a sample incubated for 30 min, presented in Fig. 3, shows that the sample contains His-cTCRf molecules phospho- rylated at zero, one, two, three or four different locations. Table 1. Kinetics of phosphorylation for each tyrosine of TCRf. 32 PO 4 -incorporation assays were used to determine the apparent Michaelis constant [K m(app) ], the apparent turnover number [k cat(app) ] and the specificity constant [k cat(app) /K m(app) ] for phosphorylation of peptides representing the six ITAM tyrosines in TCRf, by the kinase Lck. Values are mean ± SD. f tyrosine investigated K m(app) (· 10 )5 M ) k cat(app) (· 10 )4 s )1 ) k cat(app) /K m(app) ( M )1 Æs )1 ) No. 1N 2.3 ± 0.1 28.0 ± 3.0 121.8 2 1C 3.1 ± 0.2 2.3 ± 0.7 7.4 2 2N 3.9 ± 1.6 7.8 ± 3.0 20.0 3 2C 21.7 ± 2.0 3.8 ± 0.8 1.8 2 3N 9.2 ± 0.4 95.0 ± 6.8 103.8 2 3C 11.9 ± 1.8 98.4 ± 16.8 82.7 3 2372 H. R. Housden et al.(Eur. J. Biochem. 270) Ó FEBS 2003 As expected, the extent of phosphorylation of His-cTCRf increases with incubation time, and, in the 60 minute sample, evidence of complete ITAM phosphorylation (six phosphates) was seen. However, this analysis does not give any information about the location of the phosphate groups within the protein. To determine the phosphorylation status of each ITAM tyrosine in His-cTCRf, the protein was digested with trypsin, which cleaves His-cTCRf so that each of the ITAM tyrosine residues is on a different fragment, shown in Fig. 1. On-line LC-MS analysis of the samples revealed the presence of more products than would be expected after complete trypsin digestion in both the UV and total ion count recordings. Trypsin does not cut efficiently at pairs or groups of neighbouring Arg and Lys residues, of which there are several in the His-cTCRf sequence. Therefore, in practice, a range of fragments are generated for each predicted peptide, which differ by inclusion of additional Lys or Arg residues. The individual mass spectra of samples comprising each peak of the total ion count profile were examined for evidence of multiple ion species of the predicted peptide fragments. For each of the six f ITAM tyrosines, collections of mass ions were found generated from peptides containing the individual tyrosines, eluted at 32–58 min. Mass ions corresponding to the phosphorylated species of each of these fragments (with molecular mass of 80 Da more) were also Fig. 3. MS profiles of unphosphorylated and 30 minute phosphorylated His-cTCRf. Mass spectra of His-cTCRf were determined for HPLC-purified samples in  30% acetonitrile and 0.08% trifluoroacetic acid, using positive ion electrospray ionization MS. The mass spectra were combined and deconvoluted to 1 Da. The regions containing significant peaks are expanded above to show the different mass species present in sample phosphorylated for 0 min (left panel) and 30 min (right panel) of phosphorylation. The modal mass of the nonphosphorylated His-cTCRf is 14 274 Da, and the other mass species are mostly increased by multiples of 14 Da, which could be due to formylation. After phosphorylation, species that have gained between 0 and 4 · 80 Da in mass are detected, corresponding to the addition of 0–4 phosphate moieties. Fig. 2. 1 H-NMR spectroscopy of His-cTCRf. (A) Expansion of the 1D 1 HspectrumofHis-cTCRf. The aromatic resonances of the tyrosines are marked with arrows. (B) Expansion from the 1 H- 1 H DQF-COSY of His-cTCRf. The major cross-peak is the correlation between the d and e protons and is clearly overlapped for all seven tyrosines. Ó FEBS 2003 Phosphorylation of the T-cell receptor f chain (Eur. J. Biochem. 270) 2373 identified, eluting  5–12 min earlier in the HPLC gradient than their unphosphorylated partners, at 22–52 min). The majority of the peptide fragments were present as +2 and +3 ion species, with the +1 and +4 forms making a negligible contribution to the overall mass ion count. An attempt was made to use the second quadropole (MS-MS) to look for parent ions that lost 80 Da (phosphate). However, it was not possible to use parent ion scanning because, unlike phosphoserine and phosphothreonine, phosphotyrosine is unable to undergo b-elimination. To determine the order of tyrosine phosphorylation in His-cTCRf, the presence of each of the relevant fragment mass ions in the samples phosphorylated for different durations was determined. Figure 4 shows a reconstituted mass chromatogram for m/z values of 1225.3 and 1265.8, corresponding to the +2 ion species of peptide fragment 5, containing tyrosine 1N in its unphosphorylated and phos- phorylated forms, respectively. As expected, the level of phosphorylated peptide ÔproductÕ increases with time relat- ive to the level of unphosphorylated ÔsubstrateÕ. Integrating the area under each peak gives the total number of ions with aparticularm/z value in each sample. To compare the levels of phosphorylated and unphosphorylated tyrosine 1N from one sample to the next, the levels of +2 and +3 ion species of phosphorylated fragment 5 were expressed as a percent- age of the combined ion count from both the phosphoryl- ated and unphosphorylated forms of fragment 5 present in that sample. The analysis was repeated for fragment 5–6, the other high-yielding product of the (incomplete) trypsin digest in which the only ITAM Tyr was 1N. This procedure was performed for all of the other high-yielding products of trypsin digestion that contained single ITAM tyrosines at each time point. The level of phosphorylation was seen to increase mainly over the first 30 min, with the levels of each phosphotyrosine staying similar over the following 60 min. The average levels of phosphorylation of each ITAM tyrosine after 30 min of phosphorylation are presented in Table 2. Tyrosine 1N is clearly the most highly phosphory- lated tyrosine, being over 65% phosphorylated. The remaining five ITAM tyrosines are phosphorylated to a much lesser extent and at similar levels to one another (13–21%). These data, obtained in the presence of a limited supply of ATP, confirm that tyrosine 1N is indeed the first tyrosine to be phosphorylated. Discussion We have used peptide ITAM mimics to show that the PTK Lck phosphorylates the six ITAM tyrosines of TCRf with different efficiencies. An investigation into the specificity of the catalytic domain of PTK Lck using a peptide library Table 2. Presence of phosphate on trypsin-generated peptide fragments containing individual ITAM tyrosines from His-cTCRf phosphorylated for 30–90 min. His-cTCRf was phosphorylated for between 0 and 90 min, then digested with trypsin to separate each ITAM tyrosine on to a different fragment. The fragments, and their phosphorylation statuses, were identified using HPLC-MS. The fragment numbers refer to the fragments generated in a complete hypothetical digest of His- cTCRf, starting with the N¢-terminal fragment, as shown in Fig. 1. The ion count corresponding to m/z values of the M+2 and M+3 species of a specific phosphorylated peptide fragment were converted into a percentage of the combined ion count for m/z values from both the unphosphorylated and phosphorylated versions of the relevant fragment. (The levels of +1 and +4 ion species were found to be negligible.) The average percentage values obtained for samples that have been incubated for between 30 and 90 min are given. Phosphorylated tyrosine Fragment(s) of His-cTCRf Percentage of these fragments found to be phosphorylated after 30 min (± SD) n ¼ 3 1N 5 & 5–6 65.9 (± 7.6) 1C 6–8, 7–9 & 7–9 12.7 (± 2.8) 2N 12–14 & 12–15 19.9 (± 9.3) 2C 16–17 19.0 (± 9.3) 3N 21 & 20–21 21.0 (± 3.9) 3C 22 19.6 (± 8.3) Fig. 4. LC-MS profiles showing increased phosphorylation of His-cTCRf tyrosine 1N with time of incubation with Lck. The levels of fragment 5 (containing tyrosine 1N) detected with and without phosphate, after 0, 5 and 30 min of incubation with Lck are displayed above. Both the unmodified (U) and phos- phorylated (P) versions of the fragment were most commonly found as the M+2 ions (with m/z values of 1225.3 and 1265.8, respectively). These spectra are normalized to 100% of the highest peak at each time point, and show that the relative level of the phosphorylated species increases over the 30 min of incubation. 2374 H. R. Housden et al.(Eur. J. Biochem. 270) Ó FEBS 2003 based on the sequence Met-Ala-x-x-x-x-Tyr-x-x-x-x-Ala- Lys-Lys-Lys (where x is any of the 20 standard amino acids except Ser, Thr, Tyr, Cys or Trp) found the kinase to exhibit a preference for certain residues in the sites )3, )1and+1 to +4, relative to the tyrosine [25]. Particularly favoured were bulky hydrophobic residues (Phe, Ile, Leu or Val) at the Tyr )1 and Tyr +3 sites, and small residues (Gly or Ala) in the Tyr +1 position. All six of the ITAM-located tyrosines that we studied contain an optimal leucine residue at position Tyr +3. Comparison of the sequence prefer- ences with the other residues surrounding the six f ITAM tyrosines reveals that those found to be most efficiently phosphorylated, 1N and 3N, both contain an additional match, having a leucine at the Tyr )1 position. Meanwhile, the least efficiently phosphorylated tyrosine, 3N, has no additional favoured residue matches. However, 2N, the only f ITAM tyrosine to contain two additional matches, at the )1and)3 positions, has an intermediate specificity constant, which perhaps indicates that the effect of a favourable residue at one position can be reduced when certain other amino acids are nearby. We believe that the 20-residue and 21-residue peptides studied, each containing a single tyrosine, are good representatives of the individual tyrosines on the larger, multityrosine-containing TCRf chain because CD studies [10] and our NMR data show that the cytosolic portion of TCRf lacks classical secondary structure. Therefore it can be concluded from these studies that Lck phosphorylates TCRf in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last). Subsequent studies of recombinant TCRf, using trypsin digestion followed by LC-MS to identify the sites of in vitro phosphorylation by Lck, confirmed tyrosine 1N to be the first phosphorylated. Differences arising in the order of phosphorylation of the subsequent tyrosines in the whole cTCRf chain in vitro, relative to the single-tyrosine-containing peptides, may well be due to the presence of an SH2 domain in Lck which binds to phosphotyrosines in an ITAM context with high affinity [26]. On binding of the SH2 domain of Lck to a phosphorylated tyrosine in TCRf, an additional steric factor may well be introduced to the observed phosphorylation kinetics, as its site of binding on TCRf may influence the ability of its catalytic domain to reach the remaining unphosphorylated tyrosines of TCRf. However, our studies using peptides containing single tyrosines show that Lck is capable of phosphorylating all six of the tyrosines without being tethered to the substrate via such phosphotyrosine–SH2 domain interactions. It is also possible that, although TCRf lacks classical secon- dary structure, there may still be some steric restriction to Lck accessing certain tyrosine residues of the protein chain. Our studies have determined the efficiency of phosphory- lation of each of the six f ITAM tyrosines by PTK Lck, from which we can deduce the order in which Lck should perform the phosphorylations, in the absence of any other influencing factors. To determine whether the order observed in vivo is solely due to the differential phosphory- lation kinetics of Lck for the individual tyrosines, our results must be compared with previous reports of ordered phosphorylation. Kersh et al. [9] used phosphotyrosine antibodies, specific for the individual phosphotyrosines in TCRf, to detect which tyrosines were phosphorylated in response to partial and full agonist ligands in T-cell lines. Their results suggested a different phosphorylation order from that for Lck acting alone, starting with 2N (being phosphorylated even in resting cells) then 3C, 1C, 1N, 2C and lastly 3N (only phosphorylated in the presence of strong agonist ligands). Both findings contrast with the results of van Oers et al.[24],whousedHPLCandMStoidentify phosphorylated tyrosines in the partially phosphorylated p21 and more/fully phosphorylated p23 forms of f chain purified from human thymus samples. 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