Ubiquitin ligase SMURF2 enhances epidermal growth factor receptor stability and tyrosine-kinase inhibitor resistance
Journal of Biological Chemistry(2020)
摘要
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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关键词
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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