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Fig. 2 | Breast Cancer Research

Fig. 2

From: CUB domain-containing protein 1 and the epidermal growth factor receptor cooperate to induce cell detachment

Fig. 2

CDCP1 forms ternary complexes with EGFR and Src. a Immunoprecipitation of FLAG-tagged CDCP1 from MDA-MB-468 and BT474 cells shows that CDCP1 associates with EGFR and HER2. b CDCP1 association with EGFR, Src, and PKCδ are significantly reduced either by mutation of Tyr734 to phenylalanine, or by a 24-hour treatment with 20 μM DDA NSC624203. c DDA treatment of cells dissociates EGFR/CDCP1 complexes in a concentration-dependent manner. d EGFR and Src form ternary complexes, as indicated by sequential CDCP1 and Src co-immunoprecipitation, and complex formation is reduced by treatment of the cells for 24 hours with 20 μM DDA NSC624203. e MDA-MB-468 cells transduced with adenoviruses encoding GFP, CDCP1 or PKCδ were stimulated with either 20 μM EGF or 100 μM sodium pervanadate for 20 minutes as indicated. Cell extracts were subjected to sequential anti-FLAG (CDCP1), anti-Src immunoprecipitation. Immunoprecipitates and the corresponding crude cell lysates were analyzed by immunoblot. f Lapatinib treatment (20 μM) for 24 hours reduces EGFR association with CDCP1. Dasatinib treatment (100 nM) for 24 hours does not alter EGFR association with CDCP1, but reduces overall tyrosine phosphorylation and EGFR phosphorylation on the Src site, Tyr845. In the MDA-MB-468 cell background, complex formation is not significantly altered by stimulation with 10 ng/ml EGF for 20 minutes. g Subjection of affinity purified CDCP1-containing complexes to in vitro kinase assays demonstrated ATP-dependent increases in overall tyrosine phosphorylation, EGFR phosphorylation on Tyr845 and Src phosphorylation on Tyr416. These phosphorylation events were blocked by the addition of 100 nM dasatinib to the kinase assays, but unaffected by 20 μM lapatinib

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