Transformation potency of ErbB heterodimer signaling is determined by B-Raf kinase
ABSTRACT Cellular transformation occurs only in cells that express both ErbB1 and ErbB4 receptors, but not in cells expressing only one or the other of these receptors. However, when both receptors are coexpressed and ligand-stimulated, they interact with virtually the same adaptor/effector proteins as when expressed singly. To reveal the underlying regulatory mechanism of the kinase/phosphatase network in ErbB homo- and heterodimer receptor signaling, extracellular signal-regulated kinase (ERK) and Akt activities were evaluated in the presence of several enzyme inhibitors in ligand-induced cells expressing ErbB1 (E1), ErbB4 (E4), and ErbB1/ErbB4 (E1/4) receptor. The PP2A inhibitor okadaic acid showed receptor-specific inhibitory profiles for ERK and Akt activities. Moreover, B-Raf isolated only from E1/4 cells could induce in vitro phosphorylation for MEK; this B-Raf kinase activity was abolished by pretreatment of the cells with okadaic acid. Our study further showed that the E1/4 cell-specific B-Raf activity was stimulated by PLC gamma and subsequent Rap1 activation. The present study suggests that B-Raf kinase, which was specifically activated in the cells coexpressing ErbB1 and ErbB4 receptors, elevates total ERK activity within the cell and, therefore, can induce cellular transformation.
SourceAvailable from: Alireza Rafiei[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND AND AIM: Understanding of cellular and molecular mechanisms involved in cell/tumor growth and progression has led to molecular-targeted therapy. HER1 and HER2 are two main oncogenic components of HER-receptor family considered as targets for cancer research and therapy. Reliable results of HER- related research and targeted therapies dependent upon cell line with known HER expression status. But there are no published studies focusing on determination of HER1 and HER2 receptors expression in CHO and HEK-293 cell lines. METHODS: Absolute expressions of HER1 and HER2 in CHO, HEK-293, MDA-MB-468 and SKBR3 cells were evaluated using quantitative real-time PCR. Absolute expression quantification was carried out by serial dilution plasmids harboring HER1 and HER2 to extrapolate standard curve. RESULTS: Real-time PCR amplification was successfully optimized by HER1 and HER2 plasmids with high efficiency and their copy numbers were calculated. The results showed that HER1 was not expressed in CHO and HEK-293 cells but these cells express HER2 up to 9.4 × 102 and 1.1 × 105 copy/µg, respectively. MDA-MB-468 and SKBR3 cells express HER1 up to 8.4 × 105 and 9.7 × 104, respectively. In addition, the expression of HER2 is higher on the surface of SKBR3 cell (6.5 × 106 copy/µg). CONCLUSION: Our study provides a proven insight to HER1 and HER2 expression status in CHO, HEK-293, MDA-MB-468 and SKBR3 cells. Heterologous expression of HER1 and HER2 in cell lines is the first step in structural and functional studies. Since HER1 is not expressed in CHO and HEK-293 endogenously, these cells are ideal for HER receptor related studies.
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ABSTRACT: The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor-stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER(+) HER2(-) breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.Science Signaling 08/2014; 7(339):ra78. DOI:10.1126/scisignal.2005157 · 7.65 Impact Factor