MET receptor variant R970C favors calpain-dependent generation of a fragment promoting epithelial cell scattering

// Remi Montagne 1 , Anne Baranzelli 1, 2 , Ghaffar Muharram 3 , Leroy Catherine 1 , Marie Lesaffre 1 , Audrey Vinchent 1 , Zoulika Kherrouche 1 , Elisabeth Werkmeister 1, 4 , Alexis B. Cortot 1, 2 , David Tulasne 1 1 University Lille, CNRS, Institut Pasteur de Lille, UMR 8161-M3T-Mechanisms of Tumorigenesis and Targeted Therapies, F-59000 Lille, France 2 University Lille, CHU Lille, Thoracic Oncology Department, F-59000 Lille, France 3 University Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France 4 BioImaging Center Lille, Lille 59021, France Correspondence to: David Tulasne, email: david.tulasne@ibl.cnrs.fr Keywords: lung cancer, MET, receptor tyrosine kinase, hepatocyte growth factor/scatter factor, proteolytic cleavages Received: September 02, 2016      Accepted: December 26, 2016      Published: January 04, 2017 ABSTRACT The receptor tyrosine kinase MET and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas deregulation of MET signaling is associated with tumorigenesis leading to various cancers, including lung carcinoma. Mutations in the MET kinase domain lead to constitutive kinase activity and are associated with tumorigenesis. In lung cancer, however, some mutations are found in the juxtamembrane domain, and their functional consequences are unknown. Because the juxtamembrane domain of MET is targeted by several proteolytic cleavages, involved in its degradation during cell death or under steady-state conditions, we evaluated the influence of these mutations on the MET proteolytic cleavages. In stably transfected epithelial cells expressing MET, the juxtamembrane mutations R970C, P991S, and T992I were found not to modify the known caspase or presenilin-dependent regulated intramembrane proteolysis. Yet when overexpressed, the R970C variant caused generation of an as yet undescribed 45-kDa fragment (p45 MET). This fragment was found in the confluent lung cancer cell line NCI-H1437 carrying the R970C mutation and at a lesser extent in cell lines expressing WT MET, suggesting that R970C mutation favors this cleavage. Generation of p45 MET required the activity of the calpain proteases, confirming the involvement of proteolysis. Ectopic expression of reconstituted p45 MET in epithelial cell lines favored cell scattering and invasion indicating active role of this fragment in HGF/SF induced responses. Hence, although the juxtamembrane mutations of MET do not affect its known proteolytic cleavages, the R970C MET variant favors calpain dependent proteolytic cleavage in lung cancer cells.