Prostanoid Receptors

Proteins changing significantly interactions with GAB2 by indicated inhibitor treatments are highlighted red

Proteins changing significantly interactions with GAB2 by indicated inhibitor treatments are highlighted red. well as the one mediated by Gab2 and LynY508F. In addition, we exhibited that AX mainly affects the Bcr-Abl/Grb2/Gab2 axis, whereas SF seems to take action independently of the fusion kinase and most likely by blocking signaling pathways up- and downstream of Gab2. Conclusion We demonstrate that SF and AX show potency in various and mechanistically unique scenarios QNZ (EVP4593) of TKI resistance, including Bcr-AblT315I as well as Lyn- and Gab2-mediated resistances. Our data invites for further evaluation und concern of these inhibitors in the treatment of TKI resistant CML. Electronic supplementary material The online version of this article (doi:10.1186/s12964-016-0129-y) contains supplementary material, which is available to authorized users. have been recently linked to Carpenter syndrome subtype 2 associated with defective lateralization [24, 25], whereas SUSD1 with its two Sushi domains represents an almost uncharacterized protein. These interactions invite for further functional studies. However, the contrasting recruitment patterns of the Gab2 conversation partners illustrate the different mode of action of SF and the other TKIs used in this (Fig.?2c) and previous experiments [8]. Open in a separate window Fig. 2 The interactome and phosphorylation status of Gab2 is differentially affected by sorafenib and axitinib. a Differentially SILAC labeled K562tet/Gab2-HA cells were exposed to 1?g/ml doxycycline (to induce Gab2-HA expression) prior to treatment with either 1?M imatinib, 10?M sorafenib or 1?M axitinib, and DMSO as control, respectively for 4?h. Purified Gab2 protein complexes were combined 1:1:1 and analyzed by LC-MS/MS. QNZ (EVP4593) A biological replicate with reversed labels was performed and results of replicates correlated well. Protein interactions dependent on inhibitor sensitive phosphorylation sites will be reduced. b Venn diagram of imatinib, sorafenib and axitinib treatment showing TKI-sensitive Gab2 interactors. c/d TKI-sensitive changes in the Gab2 interactome (c) and the phosphorylation of Gab2 (d). Each bar represents an independent experiment (e) K562tet Vector and Gab2 cells were exposed to 1?g/ml doxycycline prior to the treatment with the indicated inhibitors. Purified Gab2 complexes were analyzed using the indicated antibodies. f Schematic model of TKI action on the Bcr-Abl/Grb2/Gab2 signaling complex. Axitinib acts like imatinib, dasatinb, nilotinib and ponatinib mainly through the Bcr-Abl/Grb2/Gab2 axis, whereas sorafenib seems QNZ (EVP4593) to act independently and most likely by affecting signaling pathways up- and downstream of Gab2. Due to the effects of axitinib on Gab2 mediated resistance, axitinib might act additionally also on other kinases, similar to sorafenib. g Diagram showing the potency of sorafenib and axitinib in all tested TKI resistances We also analyzed the phosphorylation of Gab2 (Fig.?2d; Additional file 7: Table S4). In full agreement with the interactome data, Gab2 phosphorylation sites QNZ (EVP4593) were markedly reduced upon IM and AX but not by SF FHF1 treatment. In addition, an independent Gab2 IP was performed to confirm our MS results and to test the other inhibitors DST, NL and PO (Fig.?2e). As in the MS experiments, SF hardly influenced protein-protein interactions of Gab2, while AX downregulated the its interaction with the PI3K subunit p85, SHP2 and SHC. DST and NL had similar effects as IM. The effects of PO were in most cases more pronounced as for IM, DST and NL, suggesting a stronger inhibition of Bcr-Abl activity. Thus, like IM, DST, NL and PO, AX acts mainly on the Bcr-Abl-Grb2-Gab2 axis, whereas SF seems to act independently and most QNZ (EVP4593) likely by affecting signaling pathways up- and downstream of Gab2. However, as AX is able to break Gab2 mediated resistance, this compound might additionally inhibit other kinases phosphorylating the docking sites on Gab2 and might therefore also cause similar effects as sorafenib (Fig.?2f). Thus, the efficacy of AX in Bcr-AblT315I mutant CML might be explained by its on-target action as a selective inhibitor for this gatekeeper mutant [19].

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