RESUMO
Focal adhesion kinase (Fak) has been implicated as a signaling molecule involved in the early response of cardiac myocytes to mechanical stress. The mechanism of Fak activation by mechanical stimuli is not clear. In this study, we report the load-induced Fak activation and its association with myosin heavy chain in cardiac myocytes. Pressure overload lasting from 3 to 60 minutes was shown to induce Fak phosphorylation at Tyr-397, -576/7, -861, and -925 as detected by phosphospecific antibodies. This was paralleled by increases of Fak/Src association and Src activity (Tyr-418 phosphorylation). Yeast two-hybrid screening of an adult rat cDNA library revealed an interaction between Fak and C-terminal coiled-coil region of alpha-myosin heavy chain. This was confirmed by pulldown assay with GST-C-terminal myosin fragment and native Fak from rat left ventricle. Such interaction was confirmed by coimmunoprecipitation assay with anti-Fak and anti-heavy chain cardiac myosin antibodies, confocal microscopy of double-labeled isolated cardiac myocytes and immunoelectron microscopy with anti-Fak antibody. Fak activation by mechanical stress was accompanied by a reduction of Fak/myosin heavy chain association and its relocation at subcellular sites such as costameres, Z-discs, and nuclei. Thus, our present data identify Fak interaction with C-terminal region of myosin heavy chain adding comprehensive data on Fak activation by mechanical stress and mechanotransduction in cardiac myocytes.
Assuntos
Mecanotransdução Celular/fisiologia , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/fisiologia , Motivos de Aminoácidos , Animais , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Masculino , Microscopia Confocal , Microscopia Imunoeletrônica , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/ultraestrutura , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/química , Fosforilação , Pressão , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Tirosina Quinases/análise , Ratos , Ratos Wistar , Sarcômeros/enzimologia , Estresse Mecânico , Frações Subcelulares/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Crk-associated substrate (CAS) is a tyrosine kinase substrate implicated in integrin control of cell behavior. Phosphorylation, by Src family kinases, of multiple tyrosine residues in the CAS substrate domain (SD) is a major integrin signaling event that promotes cell motility. In this study, novel phosphospecific antibodies directed against CAS SD phosphotyrosine sites ("pCAS" antibodies) were characterized and employed to investigate the cellular regulation and localization of CAS SD tyrosine phosphorylation. An analysis of CAS and focal adhesion kinase (FAK) variants expressed in CAS- and FAK-deficient cell lines, respectively, indicated that CAS SD tyrosine phosphorylation is substantially achieved by Src family kinases brought into association with CAS through two distinct mechanisms: direct binding to the CAS Src-binding domain and indirect association through a FAK bridge. Cell immunostaining with pCAS antibodies revealed that CAS SD tyrosine phosphorylation occurs exclusively at sites of integrin adhesion including both nascent focal complexes formed at the edges of extending lamellipodia as well as mature focal adhesions underlying the cell body. These findings further document a role for FAK as an important upstream regulator of CAS SD tyrosine phosphorylation and implicate CAS-mediated signaling events in promoting membrane protrusion/lamellipodium extension during cell motility.
Assuntos
Proteína de Suscetibilidade a Apoptose Celular/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Animais , Anticorpos/imunologia , Proteína de Suscetibilidade a Apoptose Celular/imunologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Camundongos , Fosforilação , Fosfotirosina/imunologia , Fosfotirosina/metabolismo , Estrutura Terciária de Proteína/fisiologia , Proteínas Tirosina Quinases/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologia , Especificidade por Substrato , Vinculina/imunologia , Vinculina/metabolismoRESUMO
We investigated the influence of stretch and contractile activity on load-induced activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 in isolated rat hearts. Increases of diastolic pressure from approximately 0 to approximately 15 mmHg rapidly increased FAK tyrosine phosphorylation (maximum: 2.3-fold) and binding to c-Src (maximum: 2.8-fold) and Grb2 (maximum: 3.6-fold). This was paralleled by activation (maximum: 2.8-fold) and binding of ERK1/2 to FAK. FAK and ERK1/2 were immunolocalized at sarcolemmal sites of cardiac myocytes and in the nuclei, in the case of ERK1/2. Balloon inflation to raise ventricular pressure in hearts perfused with cardioplegic solution also activated FAK and ERK1/2. However, increases in contractile activity induced by increasing calcium concentration in the perfusate (from 0.5 to 5 mM) did not activate the FAK multicomponent signaling complex or ERK1/2 in the myocardium. These results indicate that stretch rather than contractile activity induces FAK and ERK1/2 activation in the myocardium. In addition, the activation and binding of ERK1/2 to FAK suggest that FAK drives the load-induced activation of ERK1/2.
Assuntos
Diástole/fisiologia , Miocárdio/enzimologia , Proteínas Tirosina Quinases/metabolismo , Animais , Pressão Sanguínea/fisiologia , Citoesqueleto/fisiologia , Ativação Enzimática/fisiologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fibras Musculares Esqueléticas/enzimologia , Miocárdio/citologia , Ratos , Ratos Wistar , Estresse MecânicoRESUMO
We investigated the effects of acute pressure overload on activation of p160(ROCK) in rat myocardium. Constriction of transverse aorta, controlled to increase peak systolic pressure of ascending aorta by approximately 40 mmHg, induced a rapid association of RhoA with Dbl-3 and p160(ROCK). The binding of p160(ROCK) to RhoA was rapidly increased, peaking at 30 min (approximately 3.5-fold), but reduced to lower levels (approximately 1.9-fold) by 60 min of pressure overload. The activity of immunoprecipitated p160(ROCK) toward myosin light chain increased approximately 2.5-fold within 10 min but decreased to lower levels (approximately 1.6-fold) after 60 min of pressure overload. Confocal microscopic analysis indicated that pressure overload induced the formation of aggregates of p160(ROCK) and RhoA along the longitudinal axis of cardiac myocytes. Immunoelectron microscopic analysis showed that pressure overload induced the association of p160(ROCK) and RhoA to Z-line, T-tubule, and subsarcolemmal areas. The rapid activation of p160(ROCK) by pressure overload and its aggregation in subcellular structures involved in transmission of mechanical force suggest a role for this enzyme in the mechanobiochemical transduction in the myocardium.