Figure 1

Asymmetric kinase dimer formation is involved in EGFRvIII activation and ERBB3 phosphorylation. (A) EGFRvIII point mutations were transfected into HEK293 cells either alone or in combinations as indicated by colored circles (lanes 2 to 13). Untransfected cells were taken as negative control (lane 1). Cell lysis was performed 36 hours after transfection followed by SDS-PAGE. Immunoblotting was performed with anti-p-EGFR (Y1068) (upper panel), anti-EGFR (second panel), anti-p-STAT5 (third panel) and anti-STAT5 (lower panel) antibodies. (B) Schematic representation of the most important mechanisms of wild-type and mutant EGFRvIII kinase activation corresponding to the experiments shown in Figure 1A in lanes 2, 7 and 10. Kinases were labeled black (intact active centre) or red (kinase dead) and yellow (N-lobe mutant) or green (C-lobe mutant) circles. (C) Wild-type or mutated EGFRvIII kinase (I706Q or V948R) was co-transfected with wild-type ERBB3 in HEK293 cells. After 36 hours, serum starvation for 12 hours was performed. Cells then were stimulated or not with 50 ng/ml heregulin for 5 minutes before cell lysis was performed. Immunoprecipitates with anti ERBB3 (upper 2 panels) or whole cell lysates (lower four panels) were resolved by SDS-PAGE and western blotting was performed with the antibodies indicated. (D) Schematic representation of key kinase domain interactions in receptor homo- and hetero-dimers corresponding to the experiments shown in Figure 1C in lanes 7, 8, 11 and 12.