Figure 3

Activated EGFR/ERBB2 kinase phosphorylates ERBB3 independent of asymmetric kinase dimer formation. (A) Schematic representation of two hypothetic mechanisms potentially involved in activator function-defective EGFR/ERBB3 phosphorylation by activated EGFR: The activated EGFR dissociates from the activator kinase to phosphorylate another inactive (activator function-defective) EGFR (i) or ERBB3 (ii) receptor. Alternatively, activated EGFR may form higher-order oligomer or hetero-tetramer complexes to cause phosphorylation of activator function-defective ERBB receptors (iii). (B) HEK293 cells were transfected with wild-type ERBB2 either alone or together with WT or mutant ERBB3 (V945R). After 36 hours, cells were serum starved for 12 hours followed by stimulation with 50 ng/ml heregulin for 5 minutes as indicated. Cells were then lysed in TMNSV buffer [16]. Cells lysates were subjected to immunoprecipitation using rabbit anti-ERBB3 antibody and immunoblotting was performed using mouse anti-phospho-tyrosine and mouse anti-ERBB3 antibodies (upper 2 panels). Western blotting on whole cell lysates (input) was performed with anti-pERBB2 and anti-ERBB2 antibodies (lower 2 panels).