Limits...
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

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4390223&req=5

pone.0123623.g001: 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.

Mentions: H878Y mutant HER2 was identified in 11% of HCC patients and H878 locates in kinase domain [12], suggesting that it is a gain-of-function mutation. As treatment of HCC is limited by paucity of validated drug targets, it’s therefore highly interesting to study whether H878Y mutant HER2 plays an important role in HCC tumorigenesis. To test whether H878Y is a gain-of-function mutation, we overexpressed H878Y HER2 mutant (H878Y) in NIH-3T3 cells (referred to herein as 3T3 cells) and assayed its soft-agar colony forming capacity as readout of transforming ability side-by-side with wild type HER2 (WT HER2). These cells expressed similar level of HER2 proteins as revealed by Western analysis (right panel, Fig 1A). While control vector transfected cells formed no colonies, the WT isoform exhibited transforming ability in this soft-agar assay, consistent with previous reports [15]. In striking comparison, the H878Y mutant promoted a significant increase in the rate of soft-agar colony formation (p<0.01), and the colonies were larger (left panel, Fig 1A for bar graph and statistics, and 1B for representative pictures).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123623.g001: 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.
Mentions: H878Y mutant HER2 was identified in 11% of HCC patients and H878 locates in kinase domain [12], suggesting that it is a gain-of-function mutation. As treatment of HCC is limited by paucity of validated drug targets, it’s therefore highly interesting to study whether H878Y mutant HER2 plays an important role in HCC tumorigenesis. To test whether H878Y is a gain-of-function mutation, we overexpressed H878Y HER2 mutant (H878Y) in NIH-3T3 cells (referred to herein as 3T3 cells) and assayed its soft-agar colony forming capacity as readout of transforming ability side-by-side with wild type HER2 (WT HER2). These cells expressed similar level of HER2 proteins as revealed by Western analysis (right panel, Fig 1A). While control vector transfected cells formed no colonies, the WT isoform exhibited transforming ability in this soft-agar assay, consistent with previous reports [15]. In striking comparison, the H878Y mutant promoted a significant increase in the rate of soft-agar colony formation (p<0.01), and the colonies were larger (left panel, Fig 1A for bar graph and statistics, and 1B for representative pictures).

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