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Alterations of Gab2 signalling complexes in imatinib and dasatinib treated chronic myeloid leukaemia cells

Sebastian Halbach1234, Kristoffer TG Rigbolt26, Franziska U Wöhrle12345, Britta Diedrich26, Christine Gretzmeier26, Tilman Brummer12357* and Jörn Dengjel256*

Author Affiliations

1 Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University Freiburg, Stefan-Meier-Str. 17, Freiburg 79104, Germany

2 Centre for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University Freiburg, Habsburgerstraße 49, Freiburg 79104, Germany

3 Institute for Biology III, Faculty of Biology, Albert-Ludwigs-University Freiburg, Schänzlestraße 1, Freiburg 79104, Germany

4 Spemann Graduate School of Biology and Medicine, Albertstraße 19A, Freiburg 79104, Germany

5 Centre for Biological Signalling Studies BIOSS, Schänzlestraße 18, Freiburg 79104, Germany

6 Freiburg Institute for Advanced Studies (FRIAS), Albert-Ludwigs-University Freiburg, Albertstraße 19, Freiburg 79104, Germany

7 Comprehensive Cancer Center Freiburg (CCCF), Universitätsklinikum Freiburg, Hugstetter Straße 55, Freiburg 79106, Germany

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Cell Communication and Signaling 2013, 11:30  doi:10.1186/1478-811X-11-30

Published: 22 April 2013

Abstract

Background

The Gab2 docking protein acts as an important signal amplifier downstream of various growth factor receptors and Bcr-Abl, the driver of chronic myeloid leukaemia (CML). Despite the success of Bcr-Abl tyrosine kinase inhibitors (TKI) in the therapy of CML, TKI-resistance remains an unsolved problem in the clinic. We have recently shown that Gab2 signalling counteracts the efficacy of four distinct Bcr-Abl inhibitors. In the course of that project, we noticed that two clinically relevant drugs, imatinib and dasatinib, provoke distinct alterations in the electrophoretic mobility of Gab2, its signalling output and protein interactions. As the signalling potential of the docking protein is highly modulated by its phosphorylation status, we set out to obtain more insights into the impact of TKIs on Gab2 phosphorylation.

Findings

Using stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS), we show now that imatinib and dasatinib provoke distinct effects on the phosphorylation status and interactome of Gab2. This study identifies several new phosphorylation sites on Gab2 and confirms many sites previously known from other experimental systems. At equimolar concentrations, dasatinib is more effective in preventing Gab2 tyrosine and serine/threonine phosphorylation than imatinib. It also affects the phosphorylation status of more residues than imatinib. In addition, we also identify novel components of the Gab2 signalling complex, such as casein kinases, stathmins and PIP1 as well as known interaction partners whose association with Gab2 is disrupted by imatinib and/or dasatinib.

Conclusions

By using MS-based proteomics, we have identified new and confirmed known phosphorylation sites and interaction partners of Gab2, which may play an important role in the regulation of this docking protein. Given the growing importance of Gab2 in several tumour entities we expect that our results will help to understand the complex regulation of Gab2 and how this docking protein can contribute to malignancy.

Keywords:
Chronic myeloid leukaemia; Bcr-Abl; Gab2; Tyrosine kinase inhibitor; Imatinib; Dasatinib; SILAC-based mass spectrometry; Proteomics; Casein kinase; Protein phosphorylation