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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

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.
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pone.0123623.g002: 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.

Mentions: We next sought to determine the molecular mechanism underlying the increased kinase activity of the H878Y isoform. Autophosphorylation of ErbB receptor family members is crucial for the activation of their tyrosine kinase activity. We therefore ask whether the H878Y isoform increases the autophosphorylation ability as compared to the WT isoform. To this end, the cytoplasmic domains of WT and H878Y HER2 were expressed in Sf21 cells using a baculovirus system and were subsequently purified for kinase assays. After onehour treatment with ATP using equal amounts of the purified kinases, the proteins were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the levels of autophosphorylation were assessed by western blot analysis. H878Y isoform exhibited increased levels of autophosphorylation on the Y1221/Y1222 residues. We also detected increased levels of phosphorylated Y877 (Fig 2A, upper panels), indicating that the H878Y mutant is autophosphorylated more efficiently than the wild type HER2. Consistently, Coomassie staining of SDS-polyacrylamide gels showed that after one hour incubation in ATP-containing reaction buffer, the H878Y isoform was phosphorylated to the degree that the corresponding unphosphorylated band was almost invisible (lower band due to faster moving speed in electrophoresis). In contrast, the unphosphorylated band was clearly visible for the WT isoform (bottom panel, Fig 2A). These data demonstrate that H878Y mutation increases the autophosphorylation of HER2.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390223&req=5

pone.0123623.g002: 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.
Mentions: We next sought to determine the molecular mechanism underlying the increased kinase activity of the H878Y isoform. Autophosphorylation of ErbB receptor family members is crucial for the activation of their tyrosine kinase activity. We therefore ask whether the H878Y isoform increases the autophosphorylation ability as compared to the WT isoform. To this end, the cytoplasmic domains of WT and H878Y HER2 were expressed in Sf21 cells using a baculovirus system and were subsequently purified for kinase assays. After onehour treatment with ATP using equal amounts of the purified kinases, the proteins were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the levels of autophosphorylation were assessed by western blot analysis. H878Y isoform exhibited increased levels of autophosphorylation on the Y1221/Y1222 residues. We also detected increased levels of phosphorylated Y877 (Fig 2A, upper panels), indicating that the H878Y mutant is autophosphorylated more efficiently than the wild type HER2. Consistently, Coomassie staining of SDS-polyacrylamide gels showed that after one hour incubation in ATP-containing reaction buffer, the H878Y isoform was phosphorylated to the degree that the corresponding unphosphorylated band was almost invisible (lower band due to faster moving speed in electrophoresis). In contrast, the unphosphorylated band was clearly visible for the WT isoform (bottom panel, Fig 2A). These data demonstrate that H878Y mutation increases the autophosphorylation of HER2.

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