Role of Protein-tyrosine Phosphatase SHP2 in Focal Adhesion Kinase Down-regulation during Neutrophil Cathepsin G-induced Cardiomyocytes Anoikis

Khadija Rafiq,Mikhail A Kolpakov,Malika Abdelfettah,Daniel N Streblow,Aviv Hassid,Louis J Dell'Italia,Abdelkarim Sabri

doi:10.1074/jbc.m513040200

Abstract

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

Highlights

One of the earliest events during the progression of cardiac failure is thought to involve an inflammatory response where inflammatory cells and their proteases orchestrate myocardial repair
The present study demonstrates a role of focal adhesion kinase (FAK) tyrosine dephosphorylation in Cat.G-induced cardiomyocyte anoikis
FAK interacts with SHP2 tyrosine phosphatase that is involved in its tyrosine dephosphorylation

Summary

The abbreviations used are

Cat.G, cathepsin G; ECM, extracellular matrix; NRCM, neonatal rat cardiomyocyte; FAK, focal adhesion kinase; FA, focal adhesion; PTP, protein-tyrosine phosphatase; DMEM, Dulbecco’s modified Eagle’s medium; W T, wild-type; Ad, adenovirus; pfu, plaque-forming units; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; SH2, Src homology 2; ELISA, enzyme-linked immunosorbent assay; TA, transactivator. Several intracellular protein-tyrosine phosphatases (PTPs) have been implicated as positive and negative regulators of integrin-mediated signaling [17, 18]. Key signaling components such as FAK, p130Cas, and Srcprotein-tyrosine kinases appear to be regulated by more than one PTP, probably reflecting the numerous signal inputs that can be integrated by this pathway. We described that treatment of myocytes with Cat.G at concentrations that are likely to occur in area of inflammation in vivo [22,23,24] induces early loss of FAK tyrosine phosphorylation and activity followed by important perturbations in FA organization as well as FAK-mediated myocyte survival signaling. Our data show for the first time a role for FAK and protein-tyrosine phosphatase SHP2 in the control of Cat.G-induced anoikis in cardiomyocytes

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION