Fig. 1.
Autophosphorylation of the ErbB3 intracellular domain in vitro. (A) ErbB3-ICD (3 μM) becomes autophosphorylated when incubated with ATP and vesicles containing 10% (mol/mol) NTA-Ni lipid (right two lanes), but not when either ATP or NTA-Ni lipid is absent. The upper panel is antiphosphotyrosine blot, and the lower is antipentahistidine loading control. Lanes 3/4 and 5/6 represent duplicate experiments. (B) ErbB3-ICD harboring a K723M mutation shows little autophosphorylation (lane 2) when treated with ATP and NTA-Ni vesicles as in A, whereas wild-type ErbB3-ICD autophosphorylation is robust. His-tagged ErbB3-TKD665–1001 added at 3 μM (lane 3) causes robust trans-phosphorylation of ErbB3-ICD (K723M), which is diminished by a K723M mutation in the TKD (lane 4). (C) Trans-phosphorylation of ErbB3-ICD by wild-type and mutated forms of ErbB3-TKD665–1001 was compared as described in the text.