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Activity and cellular localization of an oncogenic glioblastoma multiforme-associated EGF receptor mutant possessing a duplicated kinase domain

Abstract

A mutation of the epidermal growth factor receptor (EGFR) that results in a tandem kinase domain duplication (TKD-EGFR) has been described in glioblastoma multiforme biopsies and cell lines. Although the TKD-EGFR confers tumorigenicity, little is known about the molecular underpinnings of receptor dysregulation. Therefore, we transfected B82L mouse fibroblast cells devoid of endogenous EGFR to determine the molecular mechanisms of receptor activation when expressed in cells as well as the contribution of each duplicated kinase domain to receptor phosphorylation. The TKD-EGFR displayed chronically elevated basal autophosphorylation at five known phosphotyrosine sites. The chronically phosphorylated TKD-EGFR was also resistant to competitive inhibition of ligand-binding compared with wild-type EGFR (WT-EGFR) and showed undetectable levels of basal dimerization, suggesting the TKD-EGFR escapes known mechanisms of receptor downregulation. Immunofluorescence analyses revealed a substantial portion of the TKD-EGFR resides in the cytosol in an activated state, although surface-localized subsets of the receptor retain ligand responsiveness. Kinase activity-deficient knockouts of the N-terminal or the C-terminal kinase domains generated TKD-EGFRs that recapitulate the autophosphorylation/localization patterns of a constitutively activated receptor versus a WT-like EGFR, respectively. Investigation of the molecular activity of the TKD-EGFR yields evidence for a unique mechanism of constitutive activity and dual kinase domain activation.

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Abbreviations

EGFR:

epidermal growth factor receptor

EV:

empty vector

WT:

wild type

TKD-EGFR:

tandem kinase domain duplication mutant

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Acknowledgements

We thank MJ Ciesielski and RA Fenstermaker for generously providing the pLXIN plasmids of the TKD-EGFR mutant. In addition, we thank CR Peet for her helpful discussions and assistance in reviewing the paper and TL Becker for her advice on statistical analyses. This work was supported in part by grants from the NIH (R33 CA122892, R01 CA108467), and from the NSF (MRSEC Award DMR-0520527).

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Correspondence to P J Bertics.

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Ozer, B., Wiepz, G. & Bertics, P. Activity and cellular localization of an oncogenic glioblastoma multiforme-associated EGF receptor mutant possessing a duplicated kinase domain. Oncogene 29, 855–864 (2010). https://doi.org/10.1038/onc.2009.385

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