Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 36, Pages 12661-12673Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA120.013519
Keywords
epidermal growth factor receptor (EGFR); Smad ubiquitination regulatory factor 2 (SMURF2); ubiquitin-conjugating enzyme H5 (UBCH5); protective ubiquitination; tyrosine kinase inhibitor (TKI) resistance; E3 ubiquitin ligase; ubiquitylation (ubiquitination); receptor regulation; tyrosine-protein kinase (tyrosine kinase)
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Funding
- National Institutes of Health [R01CA160981, R01CA131290, R01GM110052]
- M-Cube and the Fast-Forward Innovation Program at the University of Michigan
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The discovery of activating epidermal growth factor receptor (EGFR) mutations spurred the use of EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, as the first-line treatment of lung cancers. We previously reported that differential degradation of TKI-sensitive (e.g.L858R) and resistant (T790M) EGFR mutants upon erlotinib treatment correlates with drug sensitivity. We also reported that SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. However, the molecular mechanisms involved remain unclear. Here, usingin vitroandin vivoubiquitination assays, MS, and superresolution microscopy, we show SMURF2-EGFR functional interaction is important for EGFR stability and response to TKI. We demonstrate that L858R/T790M EGFR is preferentially stabilized by SMURF2-UBCH5 (an E3-E2)-mediated polyubiquitination. We identified four lysine residues as the sites of ubiquitination and showed that replacement of one of them with acetylation-mimicking glutamine increases the sensitivity of mutant EGFR to erlotinib-induced degradation. We show that SMURF2 extends membrane retention of EGF-bound EGFR, whereasSMURF2knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, whereasSMURF2knockdown decreased EGFR steady-state levels and sensitized lung cancer cells. Overall, we propose that SMURF2-mediated polyubiquitination of L858R/T790M EGFR competes with acetylation-mediated receptor internalization that correlates with enhanced receptor stability; therefore, disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance.
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