Fig 1.
HER2 H878Yelicited stronger signal to transform cells.
(A), (B) Soft-agar assay on cells transfected with control vector, WT, and H878Y HER2 mutant. (A) Left panel: Quantification and statistics of the soft-agar result. Colonies > 100 μm are counted. Values are mean ± SEM (n = 6). ***P<0.001. Right panel: Western blot was conducted to show the similar expression of HER2 proteins (B) Representative pictures of soft-agar colonies H878Y transformed cells form larger and more colonies on soft-agar assay. Pictures are photographed on day 30, Scale bars, 50μm. (C) Western blot analysis on NIH-3T3, Beas-2B and AML12 cells transfected with control vector, WT or H878Y constructs revealed H878Y could active canonical downstream signal.
Fig 2.
H878Y is a gain-of-function mutation.
(A) H878Y mutant HER2 is more efficiently autophosphorylated. Purified HER2 fragments were autophosphorylated when incubated with ATP for 60 minutes and blotted with antibodies against HER2 pY877 and pY1221/pY1222 (upper panels). Proteins were incubated with ATP for 60 minutes and then separated with SDS-PAGE to visualize phosphorylated (upper band) and unphosphorylated proteins (lower band) (bottom panel). (B) Enzyme titration of wild type (WT) and H878Y isoforms. Enzymatic activity was assayed at 1mM ATP at room temperature for 1 hour. (C) Kinase activity assay using time-resolved fluorescence-resonance energy transfer (TR-FRET) methodology (left panel). WT and H878Y HER2 isoforms were assayed at various concentrations of ATP to determine the constants indicated in right panel.
Fig 3.
Y878 is phosphorylated to interact with R898 to enhance HER2 activity.
(A) Mass spectrometry detection of Y878 phosphorylation. Red arrow indicates the phosphorylated Y878. (B) Molecular simulation on HER2 H878Y. Picture shows the last snapshot of HER2-pY878 mutant system, where pY878 forms salt bridge interactions with residue R898. (C) The distances of atom pairs pY877-NZK762 (colored in red), pY877-CZR844 (orange) and pY878-CZR898 (blue) in HER2-pY878 simulation. (D) Statistics of soft-agar assay on NIH-3T3 cells transfected with WT, R898A, H878Y, and H878Y/R898A HER2. H878Y/R898A double mutant HER2 transformed cells form similar colonies as WT HER2 on soft-agar assay. **P<0.01.
Fig 4.
HKI-272 inhibits H878Y HER2 mutant elicited signaling.
(A) WT and H878Y transformed Ba/f3 and parental cells were treated with 50nM of various HER2 inhibitors for 3 days to determine the viability. (B) WT and H878Y transformed Ba/f3 cells were treated with 50 or 200nM HKI-272 for 24 hours to determine HER2 phosphorylation and cleaved PARP. Western blot were carried out with antibody indicated. (C) HKI-272 efficiently eliminated soft-agar colonies of WT and H878Y transfected NIH-3T3, Beas-2B, and AML12 cells in the presence of 500nM HKI-272. (D) H878Y elicited signals are sensitive to HKI-272 inhibition. Control vector, WT and H878Y constructs transfected NIH-3T3, Beas-2B, and AML12 cells were treated with 500 nM HKI-272 for 1 hour and then subjected to Western blot analysis with antibodies indicated. # p<0.001.