Ubiquitin ligase SMURF2 enhances epidermal growth factor receptor stability and tyrosine-kinase inhibitor resistance

Paramita Ray,Krishnan Raghunathan,Aarif Ahsan,Uday Sankar Allam,Shirish Shukla,Venkatesha Basrur,Sarah Veatch,Theodore S Lawrence,Mukesh K Nyati,Dipankar Ray

doi:10.1074/jbc.ra120.013519

Abstract

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, using in vitro and in vivo ubiquitination 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, whereas SMURF2 knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, whereas SMURF2 knockdown 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.

Highlights

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 demonstrate that L858R/T790M EGFR is preferentially stabilized by SMAD ubiquitination regulatory factor 2 (SMURF2)-UBCH5-mediated polyubiquitination
We propose that SMURF2-mediated polyubiquitination of L858R/ T790M EGFR competes with acetylation-mediated receptor internalization that correlates with enhanced receptor stability; disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance

Summary

Introduction

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. There are two “classical” activating mutations, in-frame deletions in exon 19 and a point mutation in exon 21 (L858R) in EGFR, which drive adenocarcinoma of the lung in the majority of never smokers [1] The presence of these receptor mutations is a marker for sensitivity to treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). Present address for Shirish Shukla: The Janssen Pharmaceutical Company, Spring House, Pennsylvania, USA. These factors together may be only a part of the acquired resistance story

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Conclusion