{"doi":"10.1101/2020.04.02.022012","title":"Mutant EGFR is a preferred SMURF2 substrate of ubiquitination: role in enhanced receptor stability and TKI sensitivity","abstract":"ABSTRACT We previously reported that differential protein degradation of TKI-sensitive [L858R, del(E746-A750)] and resistant (T790M) epidermal growth factor receptor (EGFR) mutants upon erlotinib treatment correlates with drug sensitivity. However, the molecular mechanism remains unclear. We also reported SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. Here, using in vitro and in vivo ubiquitination assays, mass spectrometry, and super-resolution microscopy, we show SMURF2-EGFR functional interaction is critical in receptor stability and TKI sensitivity. We found that L858R/T790M EGFR is a preferred substrate of SMURF2-UBCH5 (an E3-E2) complex-mediated K63-linked polyubiquitination, which preferentially stabilizes mutant receptor. We identified four lysine (K) residues (K721, 846, 1037 and 1164) as the sites of ubiquitination and replacement of K to acetylation-mimicking asparagine (Q) at K1037 position in L858R/T790M background converts the stable protein sensitive to erlotinib-induced degradation. Using STochastic Optical Reconstruction Microscopy (STORM) imaging, we show that SMURF2 presence allows longer membrane retention of activated EGFR upon EGF treatment, whereas, siRNA-mediated SMURF2 knockdown fastens receptor endocytosis and lysosome enrichment. In an erlotinib-sensitive PC9 cells, SMURF2 overexpression increased EGFR levels with improved erlotinib tolerance, whereas, SMURF2 knockdown decreased EGFR steady state levels in NCI-H1975 and PC9-AR cells to overcome erlotinib and AZD-9291 resistance respectively. Additionally, by genetically altering the SMURF2-UBCH5 complex formation destabilized EGFR. Together, we propose that SMURF2-mediated preferential polyubiquitination of L858R/T790M EGFR may be competing with acetylation-mediated receptor internalization to provide enhanced receptor stability and that disruption of the E3-E2 complex may be an attractive alternate to overcome TKI resistance.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":129278,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9508,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":298133,"name":"Krishnan Raghunathan","orcid":"0000-0003-2228-6313","position":1,"is_corresponding":false},{"id":580758,"name":"Aarif Ahsan","orcid":null,"position":2,"is_corresponding":false},{"id":580506,"name":"Uday Sankar Allam","orcid":"0000-0003-4641-3903","position":3,"is_corresponding":false},{"id":293618,"name":"Shirish Shukla","orcid":null,"position":4,"is_corresponding":false},{"id":253569,"name":"Venkatesha Basrur","orcid":"0000-0002-0853-9655","position":5,"is_corresponding":false},{"id":292925,"name":"Sarah L. Veatch","orcid":"0000-0002-9317-2308","position":6,"is_corresponding":false},{"id":238049,"name":"Theodore S. Lawrence","orcid":"0000-0002-4186-8821","position":7,"is_corresponding":false},{"id":397028,"name":"Mukesh K. Nyati","orcid":"0000-0002-4359-6962","position":8,"is_corresponding":false},{"id":411309,"name":"Dipankar Ray","orcid":"0000-0002-1297-2312","position":9,"is_corresponding":false},{"id":580896,"name":"Paramita Ray","orcid":null,"position":0,"is_corresponding":true}],"reference_count":48,"raw_metadata":null,"created_at":"2026-07-18T23:15:45.920615Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}