Letters to the Editor 743 REFERENCES 8 Pekarsky Y, Palamarchuk A, Maximov V, Efanov A, Nazaryan N, Santanam U et al. 1 Sgambati MLM, Devesa S. Chronic Lymphocytic Leukemia, Epidemiological, Famil- Tcl1 functions as a transcriptional regulator and is directly involved in the iar, and Genetic Aspects. Bruce Cheson, Ed Marcel Dekker, Inc: New York, 2001, pp pathogenesis of CLL. Proc Natl Acad Sci USA 2008; 105: 19643–19648. 33–62. 9 Harper JW, Elledge SJ, Keyomarsi K, Dynlacht B, Tsai LH, Zhang P et al. Inhibition 2 Fabbri G, Rasi S, Rossi D, Trifonov V, Khiabanian H, Ma J et al. Analysis of the of cyclin-dependent kinases by p21. Mol Biol Cell 1995; 6: 387–400. chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational 10 Mullican SE, Zhang S, Konopleva M, Ruvolo V, Andreeff M, Milbrandt J et al. activation. J Exp Med 2011; 208: 1389–1401. Abrogation of nuclear receptors Nr4a3 and Nr4a1 leads to development of acute 3 Puente XS, Pinyol M, Quesada V, Conde L, Ordonez GR, Villamor N et al. Whole- myeloid leukemia. Nat Med 2007; 13: 730–735. genome sequencing identifies recurrent mutations in chronic lymphocytic 11 Tanoue T, Moriguchi T, Nishida E. Molecular cloning and characterization of a leukaemia. Nature 2011; 475: 101–105. novel dual specificity phosphatase, MKP-5. J Biol Chem 1999; 274: 19949–19956. 4 Balatti V, Bottoni A, Palamarchuk A, Alder H, Rassenti LZ, Kipps TJ et al. NOTCH1 12 Stawowczyk M, Van Scoy S, Kumar KP, Reich NC. The interferon stimulated gene mutations in CLL associated with trisomy 12. Blood 2012; 119: 329–331. 54 promotes apoptosis. J Biol Chem 2011; 286: 7257–7266. 5 Aster JC, Blacklow SC, Pear WS. Notch signalling in T-cell lymphoblastic 13 Huang RP, Fan Y, de Belle I, Niemeyer C, Gottardis MM, Mercola D et al. Decreased leukaemia/lymphoma and other haematological malignancies. J Pathol 2011; 223: Egr-1 expression in human, mouse and rat mammary cells and tissues correlates 262–273. with tumor formation. Int J Cancer 1997; 72: 102–109. 6 Koch U, Radtke F. Notch in T-ALL: new players in a complex disease. Trends 14 Shields JM, Christy RJ, Yang VW. Identification and characterization of a gene Immunol 2011; 32: 434–442. encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol 7 Palomero T, Lim WK, Odom DT, Sulis ML, Real PJ, Margolin A et al. NOTCH1 Chem 1996; 271: 20009–20017. directly regulates c-MYC and activates a feed-forward-loop transcriptional 15 Xiao S, Li D, Zhu HQ, Song MG, Pan XR, Jia PM et al. RIG-G as a key mediator of the network promoting leukemic cell growth. Proc Natl Acad Sci USA 2006; 103: antiproliferative activity of interferon-related pathways through enhancing p21 18261–18266. and p27 proteins. Proc Natl Acad Sci USA 2006; 103: 16448–16453. Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu) Imatinib-dependent tyrosine phosphorylation profiling of Bcr-Abl- positive chronic myeloid leukemia cells Leukemia (2013) 27, 743–746; doi:10.1038/leu.2012.243 signaling were evaluated in response to Imatinib. Although Imatinib strongly reduced tyrosine phosphorylation, none of the five investigated proteins were degraded (Figure 1a). For qMS Bcr-Abl is the major cause and pathogenetic principle of chronic studies, we treated cells with 0, 1 and 10 mM Imatinib for 4 h as in myeloid leukemia (CML). Bcr-Abl results from a chromosomal previous studies (Figure 1b).5,7 Cell lysates were subsequently translocation that fuses the bcr and abl genes, thereby generating digested with the proteases Lys-C and trypsin, followed by stable a constitutively active tyrosine kinase, which stimulates several isotope dimethyl labeling of the resulting peptides. The labeling signaling networks required for proliferation and survival. regime allows to distinguish three peptide pools (light, Bcr-Abl’s oncogenic properties comprise both a kinase and a intermediate and heavy), which were mixed in equal scaffold protein.1 A number of Bcr-Abl interaction partners and concentrations. Subsequently, tyrosine-phosphorylated peptides downstream effectors have been described, improving our were enriched by immunoprecipitation and analyzed by liquid understanding of the signaling networks deranged in CML. The chromatography–mass spectrometry (LC-MS).6 Analysis of the ‘core interactome of Bcr-Abl’ entails seven major interaction quantitative changes in tyrosine phosphorylation yielded 201 partners: GRB2, Shc1, Crk-l, c-Cbl, p85, Sts-1 and SHIP2.2 unique quantifiable phosphotyrosine peptide triplets belonging to The introduction of the Bcr-Abl tyrosine kinase inhibitor (TKI) 141 proteins (Supplementary Table S1), by far exceeding all Imatinib (Gleevec) has been a landmark in the treatment of CML.3 previous reports.5,7 Of these, 87 peptides showed at least a However, the development of Imatinib resistance poses major twofold downregulation after treatment with Imatinib challenges to the clinical management of CML, and although (Supplementary Table S2 and Figure 1c). second-generation TKIs can block many Imatinib-resistant Imatinib significantly decreased the tyrosine phosphorylation of mutants they are ineffective against the common T315I many peptides originating from Bcr-Abl and its core interactors mutation. An alternative strategy is to circumvent Imatinib (c-Cbl, CrkL and SHIP-2, Figure 1c). Furthermore, several resistance by targeting downstream pathways essential for proteins that have been shown to have pivotal roles in transformation.4 Therefore, it is important to fully understand Bcr-Abl-dependent signaling (Gab1, Gab2, Shc1, Crk, ERK-2, these pathways. Large-scale (phospho)proteomics experiments STAT5A/B and Yes) displayed reduced tyrosine phosphorylation, have addressed the phosphoproteome of Bcr-Abl-positive cells, often on multiple sites. In addition, Src family kinase substrates leading to the identification of an impressive number of serine/ exhibited reduced tyrosine phosphorylation, for example, threonine-phosphorylated sites (for example, Pan et al.5), although Cortactin, Catenin delta-1, nPKC-delta and Paxillin. Finally, Imatinib tyrosine-phosphorylation events remained underrepresented. reduced the tyrosine phosphorylation of several proteins involved However, many of the protein–protein interactions in the in cytoskeletal regulation, such as MEMO1, Intersectin-2, Catenin oncogenic Bcr-Abl network are dependent on tyrosine delta-1, HEPL, GRF-1, Centaurin delta 2 and Plakophilin, which phosphorylation, either directly or indirectly through Bcr-Abl.2 have not been linked to Bcr-Abl signaling. Here, we have enriched and identified phosphotyrosine A motif analysis on the sequences of significantly down- peptides by quantitative mass spectrometry (qMS)6 in order to regulated tyrosine-phosphorylated sites revealed a distinct examine the effect of Imatinib in the CML blast crisis cell line enriched motif, YxxP (Figures 1d and e). In total, 80% (23 out of K562 (Supplementary Methods). First, the phosphorylation 29) of the peptides harboring this phosphorylation motif were status and stability of several key proteins involved in Bcr-Abl significantly downregulated upon Imatinib treatment. The YxxP Accepted article preview online 27 August 2012; advance online publication, 14 September 2012 & 2013 Macmillan Publishers Limited Leukemia (2013) 718 – 757 Letters to the Editor 744 Figure 1. Exploring the effect on tyrosine phosphorylation upon Imatinib inhibition of K562 cells. (a) K562 cells were treated with Imatinib or vehicle for 4 h. Western blotting was performed to assess the abundance of several key proteins of Bcr-Abl-dependent signaling, as well as the effect of Imatinib treatment (10 mM) on total phosphotyrosine levels. (b) Overview of the quantitative proteomics workflow. Cells were treated with different doses of Imatinib (0, 1 and 10 mM) for 4 h, followed by cell lysis and protein digestion. The peptides from each Imatinib treatment were then differentially labeled using stable isotope dimethyl labeling. The three differentially labeled digests were combined, followed by simultaneous enrichment of tyrosine-phosphorylated peptides using immobilized phosphotyrosine-specific antibodies. The enriched fraction was analyzed by LC-MS and changes in tyrosine phosphorylation quantified. (c) Quantitative profiles of site-specific tyrosine phosphorylation upon Imatinib inhibition of Bcr-Abl. The changes in tyrosine phosphorylation versus the control are represented on a log scale, marked in red, phosphorylation sites containing the YxxP motif. (d) Peptides showing a more than twofold decrease in tyrosine phosphorylation upon treatment with Imatinib were subjected to motif analysis using the Motif-X algorithm. The YxxP motif shown was significantly overrepresented in the data set. (e) Frequency of the motif YxxP is very prominent in the downregulated phosphotyrosine peptides; 87 of all 201 observed peptides are downregulated (43%), whereas 23 of 29 YxxP-containing peptides are downregulated (79%). Figure 2. (a) Imatinib treatment of K562 cells leads to destruction of the core Bcr-Abl complex (green; adapted from Brehme et al.2). A small selection of peripheral interactors are depicted in blue. GAB2 (yellow) is used as flag-tagged entry point for interaction analysis upon Imatinib treatment. LC-MS/MS revealed the release of GAB2 from the Bcr-Abl core proteome, except GRB2 (bold red). Dashed lines indicate yet-to-be- explored lost/intact interactions within the Bcr-Abl Core proteome upon Imatinib treatment. (b) Mutation of all regulated GAB2 tyrosine-