Activity and cellular localization of an oncogenic glioblastoma multiforme-associated EGF receptor mutant possessing a duplicated kinase domain.
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.
Ozer
B H
BH
Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706, USA.
Wiepz
G J
GJ
Bertics
P J
PJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D002460:Cell Line; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D005909:Glioblastoma / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D051379:Mice; D009154:Mutation; D009857:Oncogenes / Q000235:genetics*; D010766:Phosphorylation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D008024:Ligands; D014443:Tyrosine; D066246:ErbB Receptors