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

Rémi Montagne,David Tulasne,Elisabeth Werkmeister,Zoulika Kherrouche,Anne Baranzelli,Alexis B. Cortot,Leroy Catherine,Marie Lesaffre,Ghaffar Muharram,Audrey Vinchent

doi:10.18632/oncotarget.14499

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.

Highlights

MET is a receptor tyrosine kinase (RTK) expressed predominantly in cells of epithelial origin
We demonstrate that the R970C, P991S, and T992I variants do not affect the known proteolytic cleavages induced during cell death and by the PS-RIP process
We previously demonstrated that the proteolytic cleavages of MET including those induced during apoptosis and necrosis and by PS-RIP occurred in these cells [14, 20]

Summary

Introduction

MET is a receptor tyrosine kinase (RTK) expressed predominantly in cells of epithelial origin. MET is cleaved by caspases within the juxtamembrane and C-terminal domains These cleavages separate the extracellular ligand-binding domain from the intracellular kinase domain [12,13,14,15], inactivate the receptor, and create a 40-kDa intracellular fragment (p40 METcaspase) that can amplify cell death by promoting mitochondrial permeabilization [16]. Under steady-state conditions, MET is processed by Presenilin-Regulated Intramembrane Proteolysis (PSRIP) This proteolytic process involves a first cleavage by membrane ADAM metalloproteases, leading to generation of a membrane-anchored p55 MET fragment (or MET-C Terminal Fragment, MET-CTF) referred as MET-EC(MET lacking the ectodomain). Ectopic expression of this membrane-anchored fragment is able to promote cellular invasion and tumor growth in experimental models [18]. These cleavages contribute to decreasing the half-life of the receptor and to preventing its accumulation in the membrane through generation of labile intracellular fragments

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