PXD018324

PXD018324 is an original dataset announced via ProteomeXchange.

Title	Mutant EGFR is a preferred SMURF2 substrate of ubiquitination: role in enhanced receptor stability and TKI sensitivity
Description	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) as a stabilizer of EGFR in a ligase (E3) activity-dependent manner. 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 three lysine (K) residues (K721, 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, disruption of the SMURF2-UBCH5 complex 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
HostingRepository	PRIDE
AnnounceDate	2020-07-29
AnnouncementXML	Submission_2020-07-29_07:31:50.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dipankar Ray
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	ubiquitination signature dipeptidyl lysine; iodoacetamide derivatized residue; deamidated residue
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2020-04-01 08:33:48	ID requested
⏵ 1	2020-07-29 07:31:51	announced

Ray P, Raghunathan K, Ahsan A, Allam US, Shukla S, Basrur V, Veatch S, Lawrence TS, Nyati MK, Ray D, Ubiquitin ligase SMURF2 enhances epidermal growth factor receptor stability and tyrosine-kinase inhibitor resistance. J Biol Chem, 295(36):12661-12673(2020) [pubmed]

submitter keyword: Epidermal growth factor receptor (EGFR)

Smad ubiquitination regulatory factor 2 (SMURF2)

Ubiquitin conjugating enzyme H5 (UBCH5)

protective ubiquitination

tyrosine kinase inhibitor (TKI) resistance

LC-MS

Dipankar Ray
contact affiliation	Dept. of Radiation Oncology, The University of Michigan Medical School, Medical Science Building I, Ann Arbor, MI 48109
contact email	dipray@umich.edu
lab head
Dipankar Ray
contact affiliation	University of Michigan
contact email	dipray@umich.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD018324
     1. Label: PRIDE project
     2. Name: Mutant EGFR is a preferred SMURF2 substrate of ubiquitination: role in enhanced receptor stability and TKI sensitivity