ABSTRACT
Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3's function as an allosteric activator and its role in downstream signaling.
Subject(s)
Animals , Cricetinae , Humans , Amino Acid Sequence , CHO Cells , Cell Movement , Cell Proliferation , Cricetulus , Extracellular Signal-Regulated MAP Kinases , Metabolism , Intracellular Space , MAP Kinase Signaling System , Models, Molecular , Molecular Sequence Data , Phosphatidylinositol 3-Kinases , Metabolism , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt , Metabolism , Receptor, ErbB-2 , Chemistry , Receptor, ErbB-3 , Chemistry , Genetics , Metabolism , Recombinant Fusion Proteins , Chemistry , Genetics , Metabolism , Signal TransductionABSTRACT
Background and purpose:PTEN mutation has been found in 20%-40% of malignant gliomas.The common mutant epidermal growth factor receptor(EGFR vⅢ)was reported to coexpress in PTEN-deficient EGFR-expressing tumor.PTEN has been shown to interact directly with FAK and reduce its tyrosine phosphorylation levels to inhibit cell invasion.The invasion of glioma cells with EGFRvⅢ expression and PTEN deficiency is increased.This study was to observe whether PTEN inhibits glioma cell invasion even in the presence of strong pro-invasive signals provided by constitutive EGFR activity.Methods:U87?EGFR cells were transfected with pcDNA3.1 constructs encoding PTEN and the cells invasion levels were detected by transwell invasion assay.The expression of FAK was detected by immunoblotting.FAK expression vector was transfected into U87?EGFR-wtPTEN cells and the change of cells invasion was documented.Results:PTEN and PTEN(G129E)could inhibit cell invasion induced by EGFRvⅢ.PTEN and PTEN(G129E)could decrease the FAK phosphorylation at Tyr397.Over expression of FAK in U87?EGFR-PTEN abrogated PTEN-induced down-regulation of the phosphorylation status of FAK and rescued cell invasion.Conclusions:PTEN could inhibit cell invasion induced by EGFRvⅢ by dephosphorylating FAK.