NPC Valorisation Voucher - Netherlands Proteomics Centre

esistance by targeting downstream pathways essential fortransformation. Therefore, it is important to fully understandthese pathways. Large-scale (phospho)proteomics experimentshave addressed the phosphoproteome of Bcr-Abl positivecells, leading to the identification of an impressive number ofserine/threonine-phosphorylated sites (e.g. [7]). Typically, asmentioned above tyrosine-phosphorylation events remainedunderrepresented in these efforts. Since Bcr-Abl is itself atyrosine kinase and many of the protein-protein interactions inits network are dependent on tyrosine phosphorylation as well[6], we started out to map these.Enrichments Therefore we enriched, identified and quantifiedphosphotyrosine peptides by mass spectrometry [4] in order toexamine the effect of Imatinib on the CML blast crisis cell lineK562. First, the phosphorylation status and stability of severalkey proteins involved in Bcr-Abl signalling were evaluated inresponse to Imatinib (see Figure 1A). We treated cells withImatinib at concentrations of 0, 1, and 10 µM for 4 hours. Celllysates were subsequently digested with the proteases Lys-Cand trypsin, followed by stable isotope dimethyl labellingof the resulting peptides. The tri-plexed labelling approachallowed us to distinguish three peptide pools (light, intermediateand heavy), which were mixed in equal concentrations.Subsequently, tyrosine phosphorylated peptides were enrichedby immunoprecipitation and analysed by LC-MS. Analysis of thequantitative changes in tyrosine phosphorylation yielded 201unique quantifiable phosphotyrosine peptide triplets belonging14 | NPC Highlights 16 | Arjen Scholtento 141 proteins, by far exceeding all previous reports [7]. Ofthese, 87 peptides showed at least a 2-fold down-regulationafter treatment with Imatinib (see Figure 1B).Down-regulated Imatinib significantly decreased the tyrosinephosphorylation of many peptides originating from Bcr-Abland its core interactors (c-Cbl, CrkL and SHIP-2, see Figure1B and 2A). Furthermore, several proteins that have beenshown to play pivotal roles in Bcr-Abl dependent signalling(Gab1, Gab2, Shc1, Crk, ERK-2, STAT5A/B and Yes) displayedreduced tyrosine phosphorylation, often on multiple sites.Also, Src family kinase substrates exhibited reduced tyrosinephosphorylation, e.g. Cortactin, Catenin delta-1, nPKC-deltaand Paxillin. Finally, Imatinib reduced the tyrosine phosphorylationof several proteins involved in cytoskeletal regulation,such as MEMO1, Intersectin-2, Catenin delta-1, HEPL, GRF-1,Centaurin delta 2, and Plakophilin, which have not previouslybeen linked to Bcr-Abl signalling.A motif analysis on the sequences of significantly downregulatedtyrosine phosphorylated sites revealed a distinctenriched motif, YxxP, which is also recognised by Bcr-Abl. Intotal, 80 percent (23 out of 29) of the peptides harbouring thisphosphorylation motif were significantly down-regulated uponImatinib treatment. The YxxP motif resembles a classic bindingsite for SH2-domains and a consensus target sequence forphosphorylation by Bcr-Abl [8]. Peptides found in our screencontaining this motif include Bcr-Abl (Tyr115 and 128), STAT5Aand B (Tyr682 and 699, respectively), several sites in the adaptorproteins Gab1 (Tyr259, 373, and 406) and Gab2 (Tyr266,and 409) and all four phosphotyrosine containing peptides ofthe docking protein HEPL (Tyr174, 195, 244, and 329), indicatingthat the latter may represent a new Bcr-Abl substrate (seeFigure 1B).Several members of the Bcr-Abl core proteome [6] are adaptor/scaffoldproteins required for the generation of SH2- orPTB-domain binding sites that lead to tightly regulated proteininteractions [9]. Several of these peptides also contain theconsensus sequence YxxP of the enriched phosphorylationmotif. As these proteins bind to Bcr-Abl, the known and novelFigure 1 | Exploring the effect on tyrosine phosphorylation upon Imatinibinhibition of K562 cells.A. Overview of the quantitative proteomics workflow. Cells were treatedwith different doses of Imatinib (0, 1 and 10 µM) for 4 hours, followed bycell lysis and protein digestion. The peptides from each Imatinib treatmentwere then differentially labelled using stable isotope dimethyl labelling. Thethree differentially labelled digests were combined, followed by simultaneousenrichment of tyrosine phosphorylated peptides using immobilisedphosphotyrosine specific antibodies. The enriched fraction was analysed byLC-MS and changes in tyrosine phosphorylation quantified.B. Quantitative profiles of site-specific tyrosine phosphorylation upon Imatinibinhibition of Bcr-Abl. The changes in tyrosine phosphorylation versus thecontrol are represented on a log scale, marked in red, phosphorylation sitescontaining the YxxP motif.