Targeting to C-terminal myosin heavy chain may explain mechanotransduction involving focal adhesion kinase in cardiac myocytes.
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.
Fonseca
Priscila M
PM
Department of Internal Medicine, School of Medicine, State University of Campinas, Campinas, Brazil.
Inoue
Rosana Y
RY
Kobarg
Claudia B
CB
Crosara-Alberto
Daniella P
DP
Kobarg
Jörg
J
Franchini
Kleber G
KG
D020816:Amino Acid Motifs; D000818:Animals; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D008297:Male; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000502:physiology* / Q000648:ultrastructure; D018995:Myosin Heavy Chains / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011312:Pressure; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D012518:Sarcomeres / Q000201:enzymology; D013314:Stress, Mechanical; D013347:Subcellular Fractions / Q000737:chemistry; D020798:Two-Hybrid System Techniques
D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D018995:Myosin Heavy Chains