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EGFR activity regulates binding of DOK2 to EGFR and DOK2 localization.
(A) Co-immunoprecipitation of DOK2, EGFR, and RASA1. HEK293 cells were co-transfected with pCMV-DOK2 (all conditions) and either pcDNA3.1 empty vector or pcDNA3.1-EGFRWT, pcDNA3.1-EGFRL858R, or pcDNA3.1-HA-HRASG12V. Cells were serum starved overnight in medium containing 0.1% FBS before stimulation with 50 ng/ml EGF for 2-5 minutes. DOK2 and associated proteins were immunoprecipitated from extracts of stimulated and unstimulated cells with an anti-DOK2 antibody and then analyzed by Western blotting. Upper panels show the proteins in the immunoprecipitation fraction; lower panels show proteins from the total lysate. A black arrowhead indicates HA-HRASG12V whereas a white arrowhead indicates endogenous RAS. Data shown is representative of results from at least three independent experiments. (B) Immunofluorescence of NIH3T3 cells stably expressing WT or EGFRL858R. Cells were transfected with pCMV-DOK2, incubated overnight, serum starved, and then either fixed or stimulated with EGF before fixation. The left panels show DOK2 (green) and DAPI (blue). White arrowheads indicate membrane-associated DOK2 staining. A Western blot indicates total levels of EGFR in these cells (right panel). Data shown is representative from at least three independent experiments. (C) Confocal microscopy analysis of colocalization of DOK2 and EGFR. 3T3-EGFRL858R cells were transfected with FLAG-DOK2 and probed with anti-FLAG (red) or anti-EGFR (green) antibodies. Arrowheads indicate colocalization (yellow) in the panel showing the merged signals. Data shown is representative from at least three independent experiments.