The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation

The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck–WIP–N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose.

T he tyrosine Src family kinases (SFKs) play a fundamental role in a wide variety of cellular processes including morphogenesis and proliferation 1 , phagocytosis 2 and hostpathogen interactions 3,4 . Furthermore, SFKs are overexpressed and/or aberrantly activated in a wide variety of cancers 5 . In humans, SFKs comprise eight members, with Src, Fyn and Yes being ubiquitously expressed 6 . SFKs consist of an N-terminal myristoylation/palmitoylation site, SH3 and SH2 proteininteraction domains and a C-terminal kinase domain (SH1). In their inactive state, SFKs assume an autoinhibited conformation that is mediated by intramolecular interactions 7 ( Supplementary  Fig. 1). Interaction between the SH2 domain and a C-terminal tyrosine Y527, when it is phosphorylated by Csk, promotes the autoinhibited conformation 8,9 , while removal of Y527 results in a constitutively activated kinase 10 . Dephosphorylation of Y527 as well as binding of ligands to the SH2 or SH3 domains alleviates the autoinhibitory state of Src, leading to autophosphorylation of Y416 and maximal kinase activity 10,11 . Upon binding of immunoglobulin (Ig)G-coated particles to FcgRs, active SFK phosphorylate the immunoglobulin tyrosine activation motif of FcgR 2 , which in turn initiates actin-driven opsono-phagocytosis downstream of Cdc42, Rac1, Nck and N-WASP 12,13 . We previously reported that opsono-phagocytosis via FcgRIIa could be inhibited by the enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) effector EspJ through an unknown mechanism 14 . Here we show that EspJ inhibits opsono-phagocytosis through inactivation of Src, disrupting phosphorylation of the FcgRIIa. EspJ inhibits Src activity by a unique post-translational modification mechanism involving amidation and adenosine diphosphate (ADP) ribosylation of a key kinase-domain residue, which is conserved across the protein kinase superfamily.

Results
EspJ resembles ADP ribosyltransferases. The Phyre protein fold recognition server 15 indicated that extensive structural homology exists between EspJ and the ADP-ribosyltransferase (ART) domain of the Pseudomonas syringae effector AvrPphF-ORF2 (ref. 16) (E-value ¼ 1.3 Â 10 À 8 ), including the b-sheet fold characteristic of ARTs (Fig. 1a,b; Supplementary Fig. 2). ARTs mediate transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD þ ) onto target proteins, modulating their interactions and subsequent signalling 17 . To explore whether EspJ can bind NAD þ , we recorded 1 H-15 N two-dimensional heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra of recombinant 15 N-labelled EspJ  (lacking the N-terminal secretion signal) in the presence and absence of NAD þ . The addition of NAD þ caused substantial chemical shift perturbations for several resonances, consistent with a significant interaction with EspJ (Fig. 1c).
To characterize this interaction further, we mutated R79 and D187 in EspJ, which would contribute directly to NAD þ binding and catalytic activity based on comparisons with AvrPphF-ORF2 and the canonical ART diphtheria toxin 16,18 (Fig. 1a,b; Supplementary Fig. 2). While the positions of some 1 H-15 N HSQC peaks for EspJ  R79A and EspJ 28-217 D187A were altered relative to the EspJ 28-217 spectra, the excellent spectra dispersion indicates that the mutations have not affected the overall protein structure. Upon titration with NAD þ no significant chemical shift perturbations were observed for EspJR79A. When NAD þ was present at a 10-fold molar equivalent some altered chemical shifts were observed for EspJ28-217 D187A, consistent with D187 contributing to NAD binding, but not being essential for recognition (Fig. 1c).
NAD binding is necessary for the biological activity of EspJ. To identify the cellular targets of EspJ, we first examined whether the EspJ-R (R79A), -D (D187A) and -R/D (R79A/D187A) mutants could inhibit phagocytosis of opsonized red blood cells (RBCs). J774A.1 macrophages were infected with EPEC and subsequently challenged with IgG-coated RBC (IgG-RBC). Infection with EPEC resulted in inhibition of phagocytosis of IgG-RBC to o2% compared with B40% in uninfected cells (Fig. 2a). In contrast, there was B23% RBC internalization in cells infected with EPECDespJ. The level of phagocytosis inhibition was restored by complementation with plasmid-encoded EspJ (pespJ), but not EspJ-R, -D or -R/D mutants (Fig. 2a). J774A.1 cells infected with EPEC and challenged with IgG-RBC also revealed that only EPEC or EPECDespJ-expressing EspJ, but not EspJ-R/D, significantly reduced the level of pTyr and actin accumulation at RBC attachment sites (Fig. 2b,c). In addition, co-expression of EspJ and FcgRIIa in Cos-7 cells, outside the context of EPEC infection, demonstrated that EspJ, but not EspJ-R/D, significantly reduced RBC internalization (Fig. 2d). Immunoblot analysis of FcgRIIa, crosslinked with anti-FcgRIIa antibodies, demonstrated that expression of EspJ, but not EspJ-R/D, reduced phosphorylation of FcgRIIa to that of its non-phosphorylatable Y282F/Y298F mutant ( Fig. 2e; Supplementary Fig. 3). This suggests that EspJ suppresses phagocytosis by inhibiting SFK-mediated phosphorylation of FcgRIIa.
EspJ blocks the kinase activity of Src. To determine whether other Src-dependent phosphorylation events were also inhibited, we examined the impact of EspJ on EPEC and Vaccinia virusinduced actin polymerization. The ability of EPEC and Vaccinia virus to stimulate actin polymerization is dependent on Src-and Abl-mediated phosphorylation of Tir and A36, respectively 3,4,19,20 . Phosphorylation of Tir and A36 results in recruitment of a signalling network consisting of Nck, WIP and N-WASP, which is required for Arp2/3-dependent actin polymerization 9,21 . We found that ectopically expressed EspJ, but not EspJ-R/D, inhibited actin polymerization induced by EPEC and Vaccinia virus (Fig. 3a,b). In contrast, EspJ had no impact on the phosphorylation-independent actin polymerization ability of EHEC O157:H7 (which naturally expresses the type-III secretion system effector TccP/EspF U ) or EPEC transformed with a plasmid encoding TccP/EspF U ( Supplementary Fig. 4a,b). This is not surprising as TccP/EspF U -driven actin polymerization circumvents the requirement of Tir tyrosine phosphorylation by directly activating N-WASP independently of Nck 9,22 .
While ectopic expression of EspJ did not interfere with Tir translocation, a marked reduction in Tir phosphotyrosine staining and Nck recruitment at bacterial attachment sites was observed in cells ectopically expressing EspJ, but not EspJ-R/D ( Supplementary Fig. 5a-c). Moreover, EspJ, but not EspJ-R/D, decreased the level of active SFKs (pY416) beneath adherent EPEC, although green fluorescent protein (GFP)-Src was recruited to bacterial attachment sites ( Fig. 3c; Supplementary Fig. 6a). Consistent with this, we found that EspJ inhibited EPEC-induced actin polymerization even in the presence of constitutively active SrcY527F ( Supplementary  Fig. 6b). Furthermore, immunoblot analysis demonstrated that EspJ inhibits the marked increase in levels of tyrosinephosphorylated cellular proteins seen following expression of Src or constitutively active SrcY527F, as well as autophosphorylation of the kinase on Y416 (Figs 3d and 4a; Supplementary Fig. 7). This inhibition was absent in cells expressing EspJ-R/D (Figs 3d and 4a).
To test whether the inhibition is due to direct inactivation of the kinase itself, we measured the kinase activity of SrcY527F immunoprecipitated from cells co-expressing EspJ or EspJ-R/D ( Fig. 4b; Supplementary Fig. 8). We found that SrcY527F immunoprecipitated from cells expressing EspJ-R/D, but not EspJ, readily phosphorylated the C terminus of Tir EPEC (TirC) forming a doublet on a-pTyr immunoblot, suggesting that Tir is phosphorylated on multiple tyrosines by Src (Fig. 4b). Furthermore, EspJ also inhibited autophosphorylation of the kinase domain in Src (SH1), as well as phosphorylation of Tir in vitro (Fig. 4a,b). Interestingly, one of the phosphorylated Tir bands was still observed in the presence of    SrcSH1, likely due to incomplete inhibition by EspJ. SrcSH1 immunoprecipitated from cells expressing EspJ-R/D was, as expected, fully active (Fig. 4b). Taken together, these results demonstrate that EspJ permanently inactivates the activity of Src, in a NAD þ -binding-dependent manner, by directly targeting its SH1 kinase domain.  EspJ amidates and ADP-ribosylates Src. We next investigated whether the inactivation of Src was through ADP ribosylation by performing an in vitro ADP-ribosylation assay on purified glutatione S-transferase (GST)-tagged Src, Src-K295M (kinase inactive) and SrcSH1-K295M in the absence and presence of EspJ or EspJ-R/D. Using radiolabelled NAD þ revealed that EspJ, but not EspJ-R/D, could ADP-ribosylate the three Src proteins ( Fig. 4c; Supplementary Fig. 9). The site of Src ADP-ribosylation was then determined by mass spectrometry (MS). In vitro ADP-ribosylated Src-K295M was separated by SDS-polyacrylamide gel electrophoresis (PAGE), and the Coomassie-stained protein band was excised and digested with either trypsin, thermolysin or elastase. All digests were analysed by nanoliquid chromatography (LC)-MS/MS using higher-energy C-trap dissociation (HCD), as well as electrontransfer dissociation (ETD) fragmentation. HCD spectra of ADPribosylated peptides were identified by filtering the spectra for the presence of ADP-ribosyl-specific marker fragments (m/z 250.09, 348.07 and 428.04) 23 . The spectrum shown in Fig. 4e shows typical ADP-ribosyl-specific marker fragments (for example, m 4 , m 6 and m 8 ) as well as a series of b ions (b 8 -b 12 ) that enabled the identification of the peptide, as well as the localization of the ADP ribosylation to E310. Unexpectedly, in addition to the ADP ribosylation, E310 was also amidated, so that E310 was converted to Q310. This finding was confirmed by searching all fragment ion spectra against a custom database containing the sequences of both Src-K295M and Src-K295M/E310Q. Whereas no ADPribosylated peptide was mapped to the sequence of Src-K295M, 27 ADP-ribosylated peptides (all containing Q310) were mapped to the sequence of Src-K295M/E310Q (Supplementary Table 4). ADP ribosylation of E310 was completely abolished when E310 was mutated to either A or Q (Fig. 4d). Additional evidence for the simultaneous modification of E310 of Src-K295M by amidation and ADP ribosylation came from a comparison of the tryptic digests of EspJ-treated and untreated Src-K295M (Fig. 4f). As expected, the tryptic peptide modified by amidation and ADP ribosylation was only detectable in the EspJ-treated sample. However, in addition we were able to identify the same tryptic peptide modified by amidation (E310Q) only, specifically in the EspJ-treated sample ( Supplementary Fig. 10). Accordingly, the MS data provide clear evidence for the simultaneous modification of E310 of Src-K295M by amidation and ADP ribosylation. The exact molecular mechanism of the concurrent modifications remains to be uncovered. However, since Src-K295M/E310Q is no longer modified by ADP ribosylation (Fig. 4d), we postulate a one-step mechanism where amidation and ADP ribosylation are directly coupled to each other rather than a two-step mechanism with successive amidation and ADP ribosylation.

Discussion
Here we show the modification and inactivation of the host cell kinase Src by a bacterial effector protein, resulting in the inhibition of a number of Src-mediated actin polymerization events including opsono-phagocytosis and pathogen-induced actin polymerization. EspJ targets the kinase domain of Src, disrupting Src autophosphorylation and the phosphorylation of Src substrates by post-translational modification of residue E310. Protein kinase domains are highly conserved sharing a common bilobal structure and 12 conserved motifs 24 . Phosphotransfer occurs in the cleft between the two lobes 25 , and E310 is part of the catalytic C helix, which projects into the catalytic cleft forming a salt bridge with K295, required for phosphotransfer 7,11 . Addition of ADP ribose on E310 would therefore disrupt salt-bridge formation and abrogate catalytic activity. By post-translationally modifying a highly conserved residue such as E310, EspJ may be able to inactivate multiple kinases ( Supplementary Fig. 11). This is further supported by the fact that EspJ can inhibit EPEC and Vaccinia actin polymerization, which rely on both Src and Abl family kinases 19,20 .
Host cell tyrosine kinases are hijacked by many bacterial and viral pathogens during infection to enhance their own adhesion or spread, trigger internalization or ensure they remain extracellular. For example, Shigella initiates host cell signalling to induce membrane ruffles facilitating their invasion requiring Src and Abl/Arg kinases 26,27 . The Helicobacter pylori effector protein CagA is phosphorylated by SFK and Abl/Arg with pleiotropic effects within the cell, including cytoskeletal rearrangements and cell elongation 28 . Interestingly, phosphorylated CagA then initiates a negative-feedback loop to inhibit Src activity by activating Csk, which then phosphorylates Src on Y527 inducing the inactive conformation 29 . In this study, we show that in addition to using host cell kinases for actin pedestal formation, EPEC translocates an effector protein to inhibit Src signalling. Inhibition of Src, Abl and possibly further tyrosine kinases by EspJ could contribute to EPEC and EHEC virulence by blocking phagocytosis and pedestal formation by secondary EPEC infection, or by antagonizing Tir signalling and promoting pedestal disassembly during late stages of infection. However, given Src is required for many signalling events, the full role during infection requires further analysis.
The post-translational modification ADP ribosylation is used by many pathogens to target host cell signalling. For example, Corynebacterium diptherium diphtheria toxin inhibits protein synthesis by ADP-ribosylating elongation factor 2 (refs 30,31). Several other bacterial toxins and effector proteins use ADP ribosylation to disrupt host cell cytoskeletal signalling. P. aeruginosa type-III secretion system effector ExoT ADPribosylates the Crk adaptor proteins 32 , which are involved in phagocytosis 33 , while the Clostridium botulinum ADPribosylating toxins C2 and C3 target monomeric actin and Rho GTPases, respectively 34,35 . The P. syringae effector HopF2, which shares homology with EspJ, ADP-ribosylates Mitogen-activated protein kinase (MAPK) disrupting plant responses to infection 36 . However, the activity of EspJ is novel as the combined amidation and ADP ribosylation of a target protein has not previously been reported. Furthermore, the inability to detect Src Q310 by MS and the lack of modification of Src E310Q suggest that amidation and ADP ribosylation are coupled rather than individual reactions. This represents a novel mechanism of action, the biochemical details of which require further investigation. EspJ therefore adds to the growing repertoire of bacterial effectors, which post-translationally modify host cell proteins, in this case using a novel amidase and ADP-ribosyltransferase activity to inactivate the tyrosine kinase Src.
Bacterial strains and growth conditions. The bacterial strains used in this study are listed in Supplementary  ARTICLE broth at 37°C, with ampicillin (100 mg ml À 1 ), chloramphenicol (34 mg ml À 1 ) or kanamycin (50 mg ml À 1 ) as appropriate. For EPEC infections, overnight cultures were primed in DMEM by diluting 1:100 and incubating statically at 37°C and 5% CO 2 for 3 h as described 37 . For EHEC cultures, bacteria were grown in LB shaking for 8 h, then diluted 1:100 in DMEM and incubated overnight statically at 37°C and 5% CO 2 . Bacterial cultures were induced with 0.05 mM isopropyl-beta-Dthiogalactopyranoside (IPTG) 30 min before infection if required. EPEC JPN15 espJ mutant was constructed using the lambda red method 38 . Briefly, a PCR product was generated by amplifying the kanamycin resistance cassette from the pKD4 template plasmid using primers (EPEC-espJ-pKD4-f and EPEC-espJ-pKD4-r shown in Supplementary Table 2), which add 50 nucleotides of flanking DNA regions homologous to the 5 0 and 3 0 ends of the espJ gene. The PCR product was transformed into EPEC JPN15 containing the pKD46 plasmid. Clones were grown on LB medium containing kanamycin, the pKD46 cured by growth at 42°C and the mutation verified by PCR using primers flanking espJ gene and primers into the antibiotic resistance gene.
Plasmid construction. Oligonucleotides used for gene amplification and sitedirected mutagenesis are shown in Supplementary Table 2, and plasmids used in this study are listed in Supplementary Table 3. For pcDNA-NTAP constructs, genes optimized for mammalian expression encoding EspJ EHEC , EspJ EPEC and EspJ EPEC R79A/D187A were synthesized by GeneArt and subcloned into pcDNA-NTAP. For all other constructs, bacterial sequences were amplified from EPEC O127:H6 E2348/69 genomic DNA. The QuikChange II Site-Directed Mutagenesis kit (Stratagene) was used as per the manufacturer's instructions to generate pcDNA-NTAP-espJ EHEC R79A/D187A and pSA10-espJ R79A, D187A and R79A/ D187A. Mutated espJ EPEC was then subcloned into pET28a and pRK5. pCB6-Src expression constructs were a kind gift from Professor Michael Way. Full-length Src (chicken c-Src) and Src SH1 (250-533) sequences were subcloned into pEGFP-N1 for expression with a myc tag or pGEX-KG for expression as a GST fusion. Sitedirected mutagenesis was used to insert E310A and E310Q mutations. All constructs were verified by DNA sequencing.
Protein expression and purification. BL21 (pET28a-espJ  ) strains were grown in LB overnight at 37°C and then diluted 1:100 in LB or in minimal media containing 0.07% 15 NH 4 Cl for 1 H 15 N HSQC analysis. Cultures were grown until an OD 600 of B0.6, induced with 1 mM IPTG and grown for a further 18 h at 37°C. For purification, the culture was centrifuged at 2,400 relative centrifugal force (RCF) for 20 min and the pellet resuspended in 25 ml denaturing protein buffer (8 M urea, 50 mM NaPO 4 pH 7.4, 200 mM NaCl, 10 mM imidazole and 5 mM bmercaptoethanol) with cOmplete EDTA-free protease inhibitors (Roche) and lysed with three passes through an Emulsiflex B-15. Samples were clarified at 17,000 RCF for 20 min and the supernatant loaded onto His-bind resin (Novagen) pre-equilibrated in denaturing protein buffer. Resin was washed with denaturing protein buffer, denaturing protein buffer containing 30 mM imidazole and the sample eluted with denaturing protein buffer containing 200 mM imidazole. Eluents were dialysed against 1 M urea, 50 mM NaPO 4 pH 7.4, 200 mM NaCl and 5 mM bmercaptoethanol followed by the same buffer containing no urea and finally gel filtered using a Superdex-75 gel filtration column (GE healthcare). His-tagged TirC EPEC was purified from BL21(pET28a-TirC EPEC ) grown in LB as above, except bacteria were grown at 30°C for 4 h following IPTG induction and purification was performed on ice with non-denaturing buffers. TirC EPEC was eluted in 5 ml protein buffer with 200 mM imidazole and dialysed against 50 mM Tris pH 7.5, 200 mM NaCl and 5 mM b-mercaptoethanol 10% glycerol. GST and GST-tagged Src derivatives were purified from BL21 carrying the appropriate pGEX-KG construct, following induction at 30°C for 4 h using Glutathione Sepharose resin (GE Healthcare) as per manufacturers' instructions.

NMR analysis. NMR 1 H 15 N HSQC experiments were performed on a Bruker
Avance II 800 MHz spectrometer equipped with a TXI cryoprobe at 295 K using 0.25 mM 15 N-labelled EspJ in 50 mM NaPO 4 pH 7.4, 200 mM NaCl, 5 mM bmercaptoethanol and 10% D 2 O in the presence and absence of NAD þ (Sigma) prepared in the same buffer. Data were processed with NMRpipe 39 and analysed with NMRview 40 .
Eukaryotic cell transfection. Cos-7 cells and Swiss 3T3 cells were seeded onto glass coverslips in 24-well plates at a density of 5 Â 10 4 or 7.5 Â 10 4 cells per well 24 h prior to transfection with FuGene 6 (Roche) or Lipofectamine 2000 (Invitrogen), respectively, according to the manufacturer's instructions. Cells were incubated at 37°C with 5% CO 2 and assayed 15 h post transfection.
EPEC/EHEC infection of eukaryotic cells. J774.A1 cells were seeded on glass coverslips in a 24-well plate at a density of 1.5 Â 10 5 cells per well and cultured overnight. Cells were starved in serum free (SF)-DMEM before infection with 200 ml JPN15 cultures, grown as described above. Plates were centrifuged at 500 RCF for 4 min and incubated for 1 h at 37°C in 5% CO 2 . Infected cells were washed three times with PBS and challenged with opsonized RBCs to assay phagocytosis, as described below. Transfected Swiss 3T3 cells were infected with 100 ml EPEC or EPEC (pSA10-TccP) culture, grown as described above, and incubated for 1 or 3 h, respectively. For EHEC 85-170 infection, 25 ml culture was added and plates centrifuged at 500 RCF for 4 min, incubated at 37°C in 5% CO 2 for 2.5 h washed three times and incubated for a further 2.5 h. For all infections, cells were washed three times in PBS and fixed with 4% paraformaldehyde for 20 min at room temperature (RT).
Vaccinia infection. Vaccinia expression vectors pEL-NTAP-espJ EHEC and pEL-NTAP-espJ EHEC R79A/D187A were generated by replacing GFP with the relevant insert in a previously described pEL vector 3 . For Vaccinia infection assays, HeLa cells were seeded on fibronectin-coated coverslips at B50% confluency and cultured overnight. Cells were infected with a WR strain of Vaccinia virus expressing an RFP-tagged version of the viral core protein A3 (ref. 41). Approximately 4 h post infection, cells were transfected with indicated constructs or pEL-CFP (control) using the Fugene transfection protocol (Promega) and then fixed in 4% paraformaldehyde B9 h post infection. Cells were permeabilized with PHEM buffer (60 mM PIPES, 25 mM Hepes, 10 mM EGTA and 1 mM Mg-acetate, pH 6.9), incubated with a-FLAG M2 (Sigma), washed with PBS, incubated with Alexa350 a-mouse and Alexa488 Phalloidin (Invitrogen) and mounted in mowiol.
RBC opsonization and phagocytosis assay. 0.3 or 0.1 ml of sheep RBC per well (J774.A1 or Cos-7, respectively) were opsonized with an equal volume of a-sRBC IgG (Sigma) previously diluted 1:50 in gelatin veronal buffer (Sigma) and rotated in a total volume of 500 ml gelatin veronal buffer for 30 min at RT. Opsonized RBCs were pelleted at 1,500 RCF for 2 min and resuspended in 500 ml DMEM per well. Prior to challenge, cells were serum-starved for at least 1 h. To assay for % internalization, infected cells were challenged with opsonized RBC for 30 min and transfected Cos-7 cells were challenged for 90 min. Where actin accumulation or phosphotyrosine staining were assessed, infected macrophages were incubated at 4°C for 15 min and then 37°C for 8 min after the addition of IgG-RBC. Where differential staining of RBC was required, cells were chilled on ice, washed with PBS and external RBC were stained with Alexa647 conjugated a-rabbit antibody (1:500, Invitrogen) before fixing with 4% paraformaldehyde for 20 min at RT.
Protein digest. For in-gel digestion, the excised gel bands were destained with 30% acetonitrile, shrunk with 100% acetonitrile and dried in a vacuum concentrator (Concentrator 5301, Eppendorf, Hamburg, Germany). Digests with trypsin, elastase and thermolysin were performed overnight at 37°C in 0.05 M NH 4 HCO 3 (pH 8). Approximately 0.1 mg of protease was used for one gel band. Peptides were extracted from the gel slices with 5% formic acid.
NanoLC-MS/MS analysis. NanoLC-MS/MS analyses were performed on an LTQ-Orbitrap Velos Pro (Thermo Scientific) equipped with an EASY-Spray Ion Source and coupled to an EASY-nLC 1000 (Thermo Scientific). Peptides were loaded on a trapping column (2 cm Â 75 mm inner diameter, PepMap C18 3 mm particles, 100 Å pore size) and separated on an EASY-Spray column (25 cm Â 75 mm inner diameter, PepMap C18 2 mm particles, 100 Å pore size) with a 45-min linear gradient from 3 to 30% acetonitrile and 0.1% formic acid. MS scans were acquired in the Orbitrap analyzer with a resolution of 30,000 at m/z 400; MS/MS scans were acquired in the Orbitrap analyzer with a resolution of 7,500 at m/z 400 using HCD fragmentation with 30% normalized collision energy. A TOP5 data-dependent MS/ MS method was used; dynamic exclusion was applied with a repeat count of 1 and an exclusion duration of 30 s; singly charged precursors were excluded from selection. Minimum signal threshold for precursor selection was set to 50,000. Predictive AGC was used with an AGC target value of 1e6 for MS scans and 5e4 for MS/MS scans. The same options were used for ETD fragmentation except for the following settings: a TOP3 method was applied, singly and doubly charged precursors were excluded, ETD activation time was set to 60 ms for triply and 45 ms for quadruply charged precursors and the AGC target was set to 300,000 for fluoranthene. Lock mass option was applied for internal calibration in all runs using background ions from protonated decamethylcyclopentasiloxane (m/z 371.10124).
Mascot Distiller 2.4 was used for raw data processing and for generating peak lists, essentially with standard settings for the Orbitrap Velos (high/high settings). Mascot Server 2.4 was used for database searching with the following parameters: peptide mass tolerance: 8 p.p.m., MS/MS mass tolerance: 0.02 Da, enzyme: 'trypsin' with three missed cleavage sites allowed for trypsin or 'none' for elastase and thermolysin; fixed modification: carbamidomethyl (C), variable modifications: Gln-4pyroGlu (N-term. Q), oxidation (M) and ADP ribosylation (RKCEDNQ). Database searching was performed against a small custom database containing Src sequence (K295M and K295M þ E310Q).