Figure 7.

Rewiring of Ras-MAPK signaling. (A) Model for rewired signaling via engineered ErbB2 and PDZ-kinase. Wild-type ErbB2 autophosphorylates sites in the C-terminus, leading to recruitment of Src. Src phosphorylates Y877 of ErbB2, recruiting the Grb2/Sos complex and promoting downstream Ras/MAPK signaling. Kd-ErbB2-VKESLV is not phosphorylated on the C-terminus, but PDZ-kinase binds to the PDZ ligand and phosphorylates Y877. Gray circles represent unphosphorylated tyrosines, and red circles represent phosphorylated sites. (B) COS7 cells were transiently transfected with ZK or Hck in the presence or absence of Kd-ErbB2-VKESLV. The cells were harvested 40 h post-transfection and lysates were subjected to immunoprecipitation using anti-ErbB2 antibody. The immunoprecipitates were separated by 10% SDS–PAGE and Western blotting was carried out with anti-pTyr877 antibody. Gel quantification is given below the pY877 blot. The membranes were stripped and reprobed with anti-ErbB2 antibody to ensure equivalent ErbB2 immunoprecipitation. (C) COS7 cell lysates were separated by 10% SDS–PAGE and probed with anti-pErk and anti-Erk antibodies. Gel quantification is given below the pErk blot. Expression of ErbB2 and kinases was assessed by Western blotting (Supplemental Figure 5, Supporting Information).