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. 2015 Aug 4;35(17):3083–3102. doi: 10.1128/MCB.00248-15

FIG 5.

FIG 5

The NPXY motif encompassing Tyr 1086 of EGFR is required for Tyr 845 phosphorylation in HEK-293T cells. (A) Flow cytometry analysis of Tyr 845 phosphorylation with EGFR tail deletion mutants, with and without EGF stimulation. Phosphorylation at Tyr 845 is greatly diminished relative to wild-type EGFR in constructs lacking residues 1051 to 1097 (Δtail-5) or residues 1074 to 1120 (Δtail-6). (B) Flow cytometry analysis of Tyr 845 phosphorylation with EGFR mutated either at Tyr 1086 or with the deletion of residues 1083 to 1086 (ΔNPXY). (C) Western blot analysis of Tyr 845 phosphorylation with and without EGF stimulation for Y1086A and ΔNPXY mutants. Mutants lacking an intact phosphotyrosine recognition motif at Tyr 1086 have greatly diminished phosphorylation at Tyr 845. αEGFR, anti-EGFR antibody; αpY845, antibody against phosphorylated tyrosine at position 845. (D) Flow cytometry analysis of Tyr 974 and Tyr 992 phosphorylation for Y1086A and ΔNPXY mutants. Phosphorylation of proximal tail tyrosines is reduced by mutation of the Tyr 1086 site, but to a lesser degree than that of Tyr 845. (E) Flow cytometry analysis of Tyr 845 phosphorylation with fine-scale scanning deletion mutants. Three residues were deleted in each DS construct listed below the x axis, spanning residues 1075 to 1098. Mutants missing residues 1084 to 1086 (DS4) and 1087 to 1098 (DS5) have reduced phosphorylation at Tyr 845 relative to wild-type EGFR upon EGF stimulation.