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Phosphorylation of mutationally introduced tyrosine in the activation loop of HER2 confers gain-of-function activity.

Hu Z, Wan X, Hao R, Zhang H, Li L, Li L, Xie Q, Wang P, Gao Y, Chen S, Wei M, Luan Z, Zhang A, Huang N, Chen L - PLoS ONE (2015)

Bottom Line: Y878 represents a phosphorylation site, and phospho-Y878 interacts with R898 residue to stabilize the active conformation of HER2, thereby enhancing its kinase activity.H878Y mutant is transforming and the transformed cells are sensitive to HER2 kinase inhibitors.Thus, our study reveals the following novel mechanism underlying the tumorigenic function of the HER2 H878Y mutation: the introduction of a tyrosine residue into the kinase activation loop via mutagenesis modulates the conformation of the kinase, thereby enhancing its activity.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, Beijing, 100875, China; National Institute of Biological Sciences, Beijing. Beijing, 102206, China.

ABSTRACT
Amplification, overexpression, and somatic mutation of the HER2 gene have been reported to play a critical role in tumorigenesis of various cancers. The HER2 H878Y mutation was recently reported in 11% of hepatocellular carcinoma (HCC) patients. However, its functional impact on the HER2 protein and its role in tumorigenesis has not been determined. Here, we show that HER2 H878Y is a gain-of-function mutation. Y878 represents a phosphorylation site, and phospho-Y878 interacts with R898 residue to stabilize the active conformation of HER2, thereby enhancing its kinase activity. H878Y mutant is transforming and the transformed cells are sensitive to HER2 kinase inhibitors. Thus, our study reveals the following novel mechanism underlying the tumorigenic function of the HER2 H878Y mutation: the introduction of a tyrosine residue into the kinase activation loop via mutagenesis modulates the conformation of the kinase, thereby enhancing its activity.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0123623.g004: 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.

Mentions: Currently multiple HER2 inhibitors are being evaluated in various phases of clinical trial, and we ask whether these HER2 inhibitors (AEE788, BIBW2992, CP-724714, CI-1033 and HKI-272) are able to efficiently block H878Y mediated transformation capability. We first overexpressed the H878Y HER2 mutant in Ba/F3 cells and found that the transformants were no longer dependent on IL-3 for survival. Interestingly, the second-generation irreversible pan-ErbB inhibitors exhibited significant toxicity against the HER2-driven Ba/F3 cells (Fig 4A and S2 Fig). We next chose to work on HKI-272 as it demonstrated the highest efficacy and least toxicity to parental Ba/F3 cells, implicating that HKI-272 shows less side effect in vivo. HKI-272 efficiently eliminated phosphorylation of HER2 proteins and induced cleaved PARP (Fig 4B and S2 Fig) in these Ba/f3 cells. Importantly, HKI-272 efficiently eliminated colony forming ability of 3T3, Beas-2B, and AML12 cells transfected with either WT or H878Y HER2 (Fig 4C and S2 Fig), clearly indicating that HKI-272 was toxic to H878Y transformed cells.


Phosphorylation of mutationally introduced tyrosine in the activation loop of HER2 confers gain-of-function activity.

Hu Z, Wan X, Hao R, Zhang H, Li L, Li L, Xie Q, Wang P, Gao Y, Chen S, Wei M, Luan Z, Zhang A, Huang N, Chen L - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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pone.0123623.g004: 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.
Mentions: Currently multiple HER2 inhibitors are being evaluated in various phases of clinical trial, and we ask whether these HER2 inhibitors (AEE788, BIBW2992, CP-724714, CI-1033 and HKI-272) are able to efficiently block H878Y mediated transformation capability. We first overexpressed the H878Y HER2 mutant in Ba/F3 cells and found that the transformants were no longer dependent on IL-3 for survival. Interestingly, the second-generation irreversible pan-ErbB inhibitors exhibited significant toxicity against the HER2-driven Ba/F3 cells (Fig 4A and S2 Fig). We next chose to work on HKI-272 as it demonstrated the highest efficacy and least toxicity to parental Ba/F3 cells, implicating that HKI-272 shows less side effect in vivo. HKI-272 efficiently eliminated phosphorylation of HER2 proteins and induced cleaved PARP (Fig 4B and S2 Fig) in these Ba/f3 cells. Importantly, HKI-272 efficiently eliminated colony forming ability of 3T3, Beas-2B, and AML12 cells transfected with either WT or H878Y HER2 (Fig 4C and S2 Fig), clearly indicating that HKI-272 was toxic to H878Y transformed cells.

Bottom Line: Y878 represents a phosphorylation site, and phospho-Y878 interacts with R898 residue to stabilize the active conformation of HER2, thereby enhancing its kinase activity.H878Y mutant is transforming and the transformed cells are sensitive to HER2 kinase inhibitors.Thus, our study reveals the following novel mechanism underlying the tumorigenic function of the HER2 H878Y mutation: the introduction of a tyrosine residue into the kinase activation loop via mutagenesis modulates the conformation of the kinase, thereby enhancing its activity.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, Beijing, 100875, China; National Institute of Biological Sciences, Beijing. Beijing, 102206, China.

ABSTRACT
Amplification, overexpression, and somatic mutation of the HER2 gene have been reported to play a critical role in tumorigenesis of various cancers. The HER2 H878Y mutation was recently reported in 11% of hepatocellular carcinoma (HCC) patients. However, its functional impact on the HER2 protein and its role in tumorigenesis has not been determined. Here, we show that HER2 H878Y is a gain-of-function mutation. Y878 represents a phosphorylation site, and phospho-Y878 interacts with R898 residue to stabilize the active conformation of HER2, thereby enhancing its kinase activity. H878Y mutant is transforming and the transformed cells are sensitive to HER2 kinase inhibitors. Thus, our study reveals the following novel mechanism underlying the tumorigenic function of the HER2 H878Y mutation: the introduction of a tyrosine residue into the kinase activation loop via mutagenesis modulates the conformation of the kinase, thereby enhancing its activity.

No MeSH data available.


Related in: MedlinePlus