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Annexin A1 interacting proteins in chronic myeloid leukemia KCL22 cells

ABSTRACT: In the present study, we used a functional proteomic approach to identify Annexin A1 (Anxa1) interacting proteins in the Philadelphia-positive KCL22 cell line. We focused on Anxa1 because it is one of the major proteins upregulated in imatinib-sensitive KCL22S cells versus imatinib-resistant KCL22R. Our proteomic strategy revealed 21 interactors. Bioinformatic analysis showed that most of these proteins are involved in cell death processes. Among the proteins identified, we studied the interaction of Anxa1 with two phosphatases, Shp1 and Shp2, which were recently identified as biomarkers of imatinib sensitivity in patients affected by chronic myeloid leukemia. Our data open new perspectives in the search for annexin-mediated signaling pathways and may shed light on mechanisms of resistance to imatinib that are unrelated to Bcr-Abl activity. For data analysis, we used the MASCOT software (version 2.4) Peptide Mass Fingerprinting search program (http://www.matrixscience.com) selecting NCBInr aug2010 database (11592891 sequences) (http://www.ncbi. nlm.nih.gov), and the following six parameters: specificity of the proteolytic enzyme used for hydrolysis (trypsin); up to 1 missed cleavage; cysteines such as S-carbamidomethylcysteines; unmodified N- and C-terminal ends; unmodified and oxidized methionines; putative Gln-induced pyroGlu formation; a precursor peptide maximum mass tolerance of 400 ppm, and a maximum fragment mass tolerance of 0.6 Da. Using the probability-based Mowse score, the ion score is 10 x Log(p), p being the probability that an eventual match is a random occurrence. All MS/MS spectra with a MASCOT score of or higher than 41 (p =< 0.05) had a good S/N, so the interpretation of the data was unambiguous. MS/MS spectra of peptides that had a MASCOT score below 41 were inspected manually and included in the statistics only in the presence of at least four continuous y or b ions.

INSTRUMENT(S): ultraflex TOF/TOF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Irene Colavita

LAB HEAD: Irene Colavita

PROVIDER: PXD000030 | Pride | 2013-08-13

REPOSITORIES: Pride

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Identification of Annexin A1 interacting proteins in chronic myeloid leukemia KCL22 cells.

Colavita Irene I Esposito Nicola N Quintarelli Concetta C Nigro Ersilia E Pane Fabrizio F Ruoppolo Margherita M Salvatore Francesco F

Proteomics 20130716 16

In the present study, we used a functional proteomic approach to identify Annexin A1 (Anxa1) interacting proteins in the Philadelphia-positive KCL22 cell line. We focused on Anxa1 because it is one of the major proteins upregulated in imatinib-sensitive KCL22S cells versus imatinib-resistant KCL22R. Our proteomic strategy revealed 21 interactors. Bioinformatic analysis showed that most of these proteins are involved in cell death processes. Among the proteins identified, we studied the interacti ...[more]

PMID: 23754495

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Project description:Inhibition of deregulated protein kinases by small molecule drugs has evolved into a major therapeutic strategy for the treatment of human malignancies. Imatinib mesylate has emerged as the leading compound to treat chronic myeloid leukemia (CML), through its inhibition of Bcr- Abl tyrosine kinases, and other cancers. However, resistance to imatinib develops frequently, particularly in late-stage disease and has necessitated the development of new BCR-ABL inhibitors. The synthesis of a new series of phenylaminopyrimidines, structurally related to imatinib showed large interest since the introduction of the nilotibin. To identify the cellular pathways affected by new synthesized compounds, we applied mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in K562 cells that were untreated or treated with imatinib and imatinib derivates. Further, the global proteome of the K562 cells treated with imatinib were quantitatively compared with the cells treated with the new compounds. This study enriched our knowledge about direct cellular targets of kinase selective drugs. Further the results offered important new knowledge for gaining insights into the structural effects of action of the new compounds. Samples were analyzed on a longer column (30cm) and a longer gradient (180min). Raw data files were processed with Mascot distiller 2.3. The mgf files were searched with Mascot daemon 2.3. The quantification was also done by Mascot Distiller. All data was stored in ms_lims. The manual validation of false peptide ratios was done with Rover (part of ms_lims). Fixed modifications: none. Variable modifications: acetylation of peptide N-terminus, pyroglutamate formation of N-terminal glutamine, methionine oxidation. Enzyme: trypsine with one missed cleavage allowed. Precursor mass tolerance: 10 ppm. Peptide fragment mass tolerance: 0.5 Da Quantitation method: SILAC arginine and lysine +6 Da. Overview of the 17 different analyses: B SK23 vs DMSO C Y22 vs DMSO D SK20 vs DMSO E Y18 vs DMSO I SK20 vs DMSO K Y18 vs DMSO O Y22 vs DMSO R Imatinib vs Water Z Imatinib vs Water J SK20 vs Imatinib M SK23 vs Imatinib N Y22 vs Imatinib P SK23 vs Imatinib Q Y18 vs Imatinib S Y22 vs Imatinib T SK20 vs Imatinib Y Y18 vs Imatinib

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