C-Cbl Ubiquitin Ligase Regulates Focal Adhesion Protein Turnover and Myofibril Degeneration Induced by Neutrophil Protease Cathepsin G

Khadija Rafiq,Jianfen Guo,Liudmila Vlasenko,Xinji Guo,Mikhail A Kolpakov,Archana Sanjay,Steven R Houser,Abdelkarim Sabri

doi:10.1074/jbc.m111.307009

Abstract

The neutrophil-derived serine protease, cathepsin G (Cat.G), has been shown to induce myocyte detachment and apoptosis by anoikis through down-regulation of focal adhesion (FA) signaling. However, the mechanisms that control FA protein stability and turnover in myocytes are not well understood. Here, we have shown that the Casitas b-lineage lymphoma (c-Cbl), adaptor protein with an intrinsic E3 ubiquitin ligase activity, is involved in FA and myofibrillar protein stability and turnover in myocytes. Cat.G treatment induced c-Cbl activation and its interaction with FA proteins. Deletion of c-Cbl using c-Cbl knock-out derived myocytes or inhibition of c-Cbl ligase activity significantly reduced FA protein degradation, myofibrillar degeneration, and myocyte apoptosis induced by Cat.G. We also found that inhibition of the proteasome activity, but not the lysosome or the calpain activity, markedly attenuated FA and myofibrillar protein degradation induced by Cat.G. Interestingly, c-Cbl activation induced by Cat.G was mediated through epidermal growth factor receptor (EGFR) transactivation as inhibition of EGFR kinase activity markedly attenuated c-Cbl phosphorylation and FA protein degradation induced by Cat.G. These findings support a model in which neutrophil protease Cat.G promotes c-Cbl interaction with FA proteins, resulting in enhanced c-Cbl-mediated FA protein ubiquitination and degradation, myofibril degradation, and subsequent down-regulation of myocyte survival signaling.

Highlights

The neutrophil protease cathepsin G induces myocyte anoikis
These data together show that the ubiquitin proteasome system (UPS), and not calpains or lysosomes, is involved in focal adhesion (FA) and myofibril degradation induced by Cat.G
We identified a novel role for the E3 ubiquitin ligase and adaptor molecule c-Cbl in mediating myocyte apoptosis induced by neutrophil-derived serine protease Cat.G. c-Cbl phosphorylation was induced in response to Cat.G along with its interaction with FA proteins

Summary

Background

The neutrophil protease cathepsin G induces myocyte anoikis. Results: Cathepsin G promotes c-Cbl activation and interaction with focal adhesion proteins that leads to focal adhesion and myofibril protein degradation and myocyte anoikis. The neutrophil-derived serine protease, cathepsin G (Cat.G), has been shown to induce myocyte detachment and apoptosis by anoikis through down-regulation of focal adhesion (FA) signaling. Neutrophil-derived serine protease, cathepsin G (Cat.G), has been shown to play an important role in tissue remodeling at sites of tissue injury through hydrolysis of a host of protein substrates including chemoattractants, extracellular matrix (ECM), and hormonal factors [3,4,5]. Pathophysiological concentration of Cat.G has been shown to induce endothelial cell and myocyte detachment and apoptosis by anoikis [7, 8] These effects of Cat.G on myocytes do not require PARs but involve MMP activation and epidermal growth factor receptor (EGFR) transactivation [8]. Further studies have shown the role of focal adhesion (FA) signaling down-regulation as a mechanism whereby Cat.G modulates normal cell-cell or cell-ECM interactions necessary for myocyte survival [9].

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION