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EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer

ABSTRACT: HCC827 cells were treated with 0.1 microM erlotinib for 0, 2, 6, and 24 hours. Cell lysates were immunoprecipitated with TBK1 antibody. Antibody enriched protein samples were run on SDS-PAGE gels and submitted to UT Southwestern Proteomics Core Facility for Mass Spectrometry. Protein gel pieces were digested overnight with trypsin (Pierce) following reduction and alkylation with DTT and iodoacetamide (Sigma Aldrich. The samples then underwent solid-phase extraction cleanup with Oasis HLB microelution plates (Waters) and the resulting samples were analyzed by LC-MS-MS, using an Orbitrap Fusion Lumos mass spectrometer (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography systems (Dionex).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Amyn A. Habib

PROVIDER: MSV000084654 | MassIVE | Tue Dec 03 12:45:00 GMT 2019

SECONDARY ACCESSION(S): PXD016558

REPOSITORIES: MassIVE

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Identification of the UT-A1 Urea Channel Interactome

Project description:The urea channel Slc14a2 (or UT-A1) mediates vasopressin-regulated urea transport across the inner medullary collecting duct (IMCD). Previously, UT-A1 was found to present in a high molecular weight complex, suggesting UT-A1 is involved in certain protein-protein interactions. The present study sought to identify the proteins that interact with UT-A1 in this complex for a better understanding of how UT-A1 is regulated. Rat IMCD suspensions were treated with or without V2 receptor agonist, dDAVP, followed by in-cell crosslinking using BSOCOES and detergent solubilization. Immunoprecipitation using Dynabeads coated with UT-A1 specific antibody successfully pulled down the UT-A1 proteins. In-gel digestion protocol was carried out to prepare samples for liquid chromatographic mass spectrometry analysis of tryptic peptides using a Velos-Orbitrap mass spectrometer. The peptides passing stringent spectral quality thresholds were quantified (label-free) to identify those with (UTA-1 antibody/preimmune IgG) >4. A total of 128 UT-A1 interacting proteins were identified. Gene Ontology analysis maps the distribution of these proteins throughout major cell compartments: endoplasmic reticulum, Golgi, endosomes, cytosol and plasma membrane. Among them are four protein kinases (Cdc42bpb, Phkb, Camk2d, Mtor) that play roles in vasopressin-regulated phosphorylation of UT-A1. Non-label quantification was also performed to determine the stoichiometry of UT-A3 with UT-A1, the result does not support an oligomeric complex formation of UT-A1/A3. In conclusion, we have provided a refined list of UT-A1 binding proteins which can be useful for further analysis of the vasopressin signaling pathway in regulation of UT-A1 in IMCD.

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TBK1-METTL3 axis facilitates antiviral immunity

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EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer.

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