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Figure 3 | Molecular Cancer

Figure 3

From: EGFR and EGFRvIII undergo stress- and EGFR kinase inhibitor-induced mitochondrial translocalization: A potential mechanism of EGFR-driven antagonism of apoptosis

Figure 3

Amino acid substitutions of the N-terminal conserved motifs enhance mitochondrial targeting of both EGFR and EGFRvIII. A, Structures of the two N-terminal conserved motifs within EGFR and EGFRvIII. The two motifs are homologous to the L/I/V/F/M-rich sequence that has been shown to involve in protein intracellular trafficking [29–31]. Notably, the first motif is present in EGFR, EGFRvIII and HER2 while the second motif is found in all members of the EGFR family of receptors. B, Simplified structures of EGFR/EGFRvIII show that both sequences are present in the extracellular region of both receptors. TM, transmembrane domain. N, nuclear-localization signal. I, internalization domain. C, Both motifs are conserved among EGFR proteins in different mammalian species. D, EGFRvIII mutants with amino acid substitutions within the conserved motifs. To determine the role of the motifs in modulating EGFR/EGFRvIII intracellular trafficking, we performed site-directed mutagenesis to substitute leucine/isoleucine residues to alanines, thereby altering the L/I-rich property of the motifs. E, EGFR-MTS and EGFRvIII-MTS mutants demonstrate enhanced degrees of mitochondrial localization. Using immunofluorescence staining/confocal microscopy, we examined subcellular location of EGFR/EGFRvIII and all four mutants in EGFR/EGFRvIII-null CHO cells. The yellow signals indicate mitochondrially localized receptors, merged products of the green fluorescence (receptors) and red fluorescence (mitochondrion labeled by mitotracker). F,G, The EGFR-MTS1 mutant did not display enhanced nuclear accumulation as shown by immunofluorescence staining and confocal microscopy (Panel F) and nuclear fractionation followed by western blotting (Panel G). Transfected CHO cells were serum-starved and treated with and without EGF (100 ng/ml) for 10 min to induce EGFR nuclear import. The results showed that the EGFR MTS mutants did not have enhanced nuclear accumulation, in contrast to their ability to richly localize in the mitochondria (EGFR-MTS2 data not shown).

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Molecular Cancer

ISSN: 1476-4598