Brckstmmer for critical reading of the manuscript; and all users of G
Brckstmmer for critical reading of the manuscript; and all users of G.S.-F.’s group for help and discussions. signaling pathways. Quantitative proteomics analysis showed that tyrosine kinase inhibitors lead to a disruption of this network. Particular parts still appear to interact with Bcr-Abl inside a phosphotyrosine-independent manner. We propose that Bcr-Abl and additional drug focuses on, rather than becoming considered as solitary polypeptides, can be considered as complex protein assemblies that remodel upon drug action. nodes, where each node is definitely linked to at least additional nodes. For example, inside a 4-core (we.e., = 4) each node is definitely connected to at least 4 additional members of the protein network constituting the 4-core network. Generation of randomized networks demonstrates the 4-core (consisting of Bcr-Abl, SHIP-2, c-Cbl, p85/, Sts-1, Shc1, Grb2, and Crk-I as nodes) is definitely significant in the 10?5 level. Interestingly, if Sts-1 is definitely excluded, the closer Bcr-Abl interactors form an even more connected 5-core (Fig. 3, reddish halo). The 2 2 Faucet baits of the AP2 adaptor complex (Eps15 and AP21) display 3 (AP21) and only 1 1 edge (Eps15) to the members of the 5-core and are consequently positioned more distantly in the network (Fig. 3, blue halo). Open in a separate windows Fig. 3. The Bcr-Abl protein network. (value of a Fonadelpar for = 1, Fonadelpar 2, 3, 4, 5. Consequently, the experimentally-observed 4-core and 5-core networks are statistically highly significant. To limit the possible bias introduced from the a priori selection of the 9 bait proteins, where a protein that was not initially selected as Bcr-Abl interactor could have a reduced connectivity with the complex around Bcr-Abl, we symbolize the TAP-MS results as an undirected graph, meaning that the edges between the nodes in the network do not consist of directionality information within the relationships. Therefore, a link created from a bait protein is also valid starting from its prey. To assess the significance of our getting of a 4- and 5-core, we performed network randomization simulations. Generating 105 different randomized networks resulted only in 2- and 3-cores in approximately equal proportion (Fig. 3 3. Consequently, the observed 4- and 5-core networks are statistically highly significant ( 10?5; Fig. 3and Fig. S2and and and for details). Tyrosine Kinase Inhibitor Treatment. Dasatinib (Sprycel; BMS-354825) and nilotinib (Tasigna; AMN107) were dissolved in DMSO and used at final concentrations of 100 nM and 1 M, respectively. K562 cells were mock-, dasatinib-, or nilotinib-treated in the indicated concentrations for 3 h. Quantitative iTRAQ MS Analysis of Bcr-Abl and Grb2 Complexes in the Presence of Tyrosine Kinase Inhibitors. Bcr-Abl and Grb2 complexes were immunoprecipitated from mock-, dasatinib-, or nilotinib-treated K562 IGLC1 cells. Upon tryptic in-solution digestion each sample was split in half, labeled with iTRAQ reagents (2 channels per sample after splitting), separated with RP-HPLC, and analyzed by MALDI-TOF/TOF tandem MS. Medians of all 4 iTRAQ ratios were determined for each protein followed by statistical analysis of their significance (observe for details). Supplementary Material Supporting Info: Click here to view. Acknowledgments. We say thanks to S. Decker for SHIP-2 cDNA; B. Mayer for c-Cbl Fonadelpar cDNA; D. Wisniewski for SHIP-2 antibody; A. C. Gavin, F. Grebien, C. Baumann, and T. Brckstmmer for crucial reading of the manuscript; and all users of G.S.-F.’s group for help and discussions. This work was supported by Austrian Technology Account Give P18737, the Austrian Proteomics Platform, and the Austrian Academy of Sciences. Footnotes The authors declare no discord of interest. This short article is definitely a PNAS Direct Submission. This short article contains supporting info on-line at www.pnas.org/cgi/content/full/0900653106/DCSupplemental..