Avaliação das interações proteicas da quinase de adesão focal (FAK) em miocitos cardiacos : caracterização estrutural e funcional da interação da FAK com a cadeia pesada de miosina sarcomerica

AUTOR(ES)
DATA DE PUBLICAÇÃO

2005

RESUMO

Mechanical stress is a major factor involved in the development of myocardial adaptive and maladaptive changes in heart diseases. Local mechanical forces activate signaling mechanisms in cardiac myocytes inducing the expression of specific genetic programs linked to myocardial structural and functional remodeling. Although mechanical forces might directly trigger signaling mechanisms in cardiac myocytes the mechanism by which they are sensed and convertedinto biochernicalsignals remains elusive. 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 c1ear.In this study we performed a yeast twohybrid screening in order to search for novel protein binding partners with FAK. We screened a N-Terminal FAK construction containing the FERM and kinase domain (bait) against a cDNA library constructed from a 6h pressure overloaded adult rat left ventricle. We found an interaction between FAK and C-terminal coiled-coil region of a-myosin heavy chain. We confrnned this interaction with pulldown assay with the hybrid protein GST-Myosin and myocardium rat extracts. Laser confocal rnicroscopy confrnned the colocalization of FAK and MHC in the sarcomericregion corresponding to the A band in cultured neonatal rat ventricular myocytes (NRVMs). We also demonstrated FAK and MHC interaction in purified myosin extracts of NRVMs in buffer with high salt concentration indicating that FAK-MHCis a strong interaction. Pulldown assay with GST-FERM showed that the N-Terminal domain is enough for FAK-MHC interaction. In coimmunoprecipitationassays with NRVMs we found out that cyclic stretch reduces FAK-MHC interaction and this effect is parallel to FAK phosphorylationin tyr397. Treatmentwith PP2 inhibitedFAKphosphorylation and blocked FAK-MHC dissociation showing the importance of FAK phosphorylation in FAK-MHC interaction. Laser confocal microscopy of NRMV treated with PP2 confirmed the colocalization of FAK-MHC afier 60 minutes of cyc1ic stretch. GST-FERM pulldown assay with NMRVs extracts presented reduction in FAK-MHC association suggesting that the cyc1ic stretch produces structural and/or functional modification in the C-terminal coiled-coil region of a-MHC responsible for FAK-MHCinteraction. We found out that FAKlMHC interaction presents distinct characteristics compatible with a biomechanical transducer system, responsive to cyc1ic stretch. Our results are compatible with the idea that the mechano-biochemical transduction involving FAK in cardiomyocytes depends on the stretch of MHC. In this case we could consider FAK as a sensor of mechanical stress in cardiomyocytes.

ASSUNTO(S)

miocardio hipertrofia

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