ABSTRACT
The small heat shock protein αB-Crystallin (CryAB, HspB5) and SH2 domain-containing tyrosine phosphatase 2 (Shp2) are important molecules in heart response to pathophysiological stress. Here we show that CryAB interacts with and potentially regulates Shp2 catalytic activity in stretched cardiomyocytes. Such an interaction requires CryAB oligomer to attenuate Shp2 activation. Stretched cardiomyocytes show a robust CryAB/Shp2 association accompanied by a reduction in the Shp2 phosphatase activity. Accordingly, CryAB knock-down in cardiomyocytes enhances Shp2 activity induced by mechanical stress. These results revealed a new role for CryAB, as a modulator of Shp2 phosphatase activity during a functionally relevant stimulus in cardiomyocytes.
Subject(s)
Crystallins/metabolism , Microtubule-Associated Proteins/metabolism , Myocytes, Cardiac/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Stress, Physiological/physiology , Animals , Cells, Cultured , Crystallins/genetics , Enzyme Activation/physiology , Gene Knockdown Techniques , Microtubule-Associated Proteins/genetics , Myocytes, Cardiac/cytology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Rats , Rats, Wistar , Stress, MechanicalABSTRACT
Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the ß4-ß8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.
Subject(s)
Calpain/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gene Expression Regulation, Enzymologic , Myocytes, Cardiac/cytology , alpha-Crystallin B Chain/chemistry , Animals , Aorta/metabolism , Apoptosis , Cell Survival , Fluorescence Resonance Energy Transfer , Gene Silencing , Homeostasis , Male , Mice , Mice, Transgenic , Microscopy, Fluorescence , Models, Molecular , Myocardium/metabolism , Phosphorylation , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Rats, Wistar , Stress, Mechanical , src-Family Kinases/metabolismABSTRACT
Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 Å(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.
