Proteolytic cleavages in the extracellular domain of receptor tyrosine kinases by membrane-associated serine proteases.

Li-Mei Chen,Karl X Chai

doi:10.18632/oncotarget.17009

Abstract

The epithelial extracellular membrane-associated serine proteases matriptase, hepsin, and prostasin are proteolytic modifying enzymes of the extracellular domain (ECD) of the epidermal growth factor receptor (EGFR). Matriptase also cleaves the ECD of the vascular endothelial growth factor receptor 2 (VEGFR2) and the angiopoietin receptor Tie2. In this study we tested the hypothesis that these serine proteases may cleave the ECD of additional receptor tyrosine kinases (RTKs). We co-expressed the proteases in an epithelial cell line with Her2, Her3, Her4, insulin receptor (INSR), insulin-like growth factor I receptor (IGF-1R), the platelet-derived growth factor receptors (PDGFRs) α and β, or nerve growth factor receptor A (TrkA). Western blot analysis was performed to detect the carboxyl-terminal fragments (CTFs) of the RTKs. Matriptase and hepsin were found to cleave the ECD of all RTKs tested, while TMPRSS6/matriptase-2 cleaves the ECD of Her4, INSR, and PDGFR α and β. Prostasin was able to cleave the ECD of Her3 and PDGFRα. Matriptase cleaves phosphorylated Her2 at Arg558 and Arg599 and the Arg599 cleavage produces a CTF not recognized by the monoclonal antibody trastuzumab/Herceptin. Her2 cleavages by matriptase can be inhibited by the hepatocyte growth factor activator inhibitor 1 (HAI-1) in the MDA-MB-231 human breast cancer cells. Matriptase silencing in the Her2, matriptase, and HAI-1 triple-positive SKBR3 human breast cancer cells enhanced Her2 protein down-regulation induced by a sustained exposure to phorbol 12-myristate 13-acetate (PMA), which down-regulated matriptase protein. The novel Her2 cleavage and expression regulation mechanisms mediated by matriptase may have potential impacts in Her2-targeting therapies.

Highlights

We have previously reported that the extracellular domain (ECD) of the epidermal growth factor receptor (EGFR, aka, Her1/ErbB1) is proteolytically modified by membrane-associated serine proteases, matriptase, prostasin, and hepsin in cultured epithelial cells [1, 2]
The type-II transmembrane serine proteases matriptase and hepsin and the GPI-anchored serine protease prostasin are all expressed as extracellular proteolytic enzymes, proteolytic cleavages on any receptor tyrosine kinase are expected to occur only in the receptor’s ECD
We can rule out membrane localization by the GPI anchor being a factor contributing to prostasin’s inability to cleave the Her2 ECD because the GPI-anchored matriptase readily cleaved Her2 (Lane 9), whereas the GPI-anchored protease-dead mutant matriptase (GPI-Mat-Mut) did not

Summary

Introduction

We have previously reported that the extracellular domain (ECD) of the epidermal growth factor receptor (EGFR, aka, Her1/ErbB1) is proteolytically modified by membrane-associated serine proteases, matriptase (aka, MT-SP1/epithin/ST14/PRSS14), prostasin (aka, CAP1/ PRSS8), and hepsin (aka, TMPRSS1) in cultured epithelial cells [1, 2]. The amino-terminally truncated EGFR fragments produced by matriptase cleavages are tyrosine phosphorylated and activate the extracellular signalregulated kinases (erk1/2) [1]. The amino-terminally truncated EGFR fragments produced by hepsin cleavages are tyrosine phosphorylated but it is unclear what downstream signaling pathways are activated as a result [2]. Matriptase was reported to cleave the ECD of the vascular endothelial growth factor receptor 2 (VEGFR2, aka, KDR), inactivating this RTK and its downstream signaling [3]. Matriptase was further reported to cleave the angiopoietin receptor Tie ECD to activate www.impactjournals.com/oncotarget this RTK upon a membrane translocation mediated by protein kinase C (PKC), under sustained exposure to a high dose of phorbol 12-myristate 13-acetate (PMA) [4]

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Conclusion