Abstract 187: αB-crystallin Interacts And Prevents Stress-activated Proteolysis Of Focal Adhesion Kinase In Cardiomyocytes

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Aline M dos Santos ◽  
Michelle B Pereira ◽  
Danieli C Gonçalves ◽  
Alisson C Cardoso ◽  
Silvio R Consonni ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Mechanical Stress ◽  
Focal Adhesion ◽  
Resonance Energy Transfer ◽  
Pressure Overload ◽  
Resonance Energy ◽  
Calpain Inhibitor ◽  
Biochemical Assays ◽  
Αb Crystallin ◽  
Response To Stress

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here, we show that αB-crystallin confers protection to FAK against calpain-mediated proteolysis under mechanical stress in cardiomyocytes. Biochemical assays, chemical cross-linking coupled to mass spectrometry experiments, mutational analyses and Förster resonance energy transfer (FRET) were combined to investigate the basis of FAK and αB-crystallin interaction. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation by mechanical stress. αB-crystallin silencing results in calpain-dependent FAK depletion and in increased apoptosis of cardiomyocytes. The overexpression of a myc-FAK construct or treatment with a calpain inhibitor (E64) restored the survival of cardiomyocytes depleted of αB-crystallin. The association between FAK and αB-crystallin was also demonstrated to occur in response to pressure overload in rat left ventricle. The myocardial depletion of αB-crystallin by siRNA results in enhanced apoptosis of cardiomyocytes and myocardial fibrosis in overloaded hearts. These alterations were markedly attenuated in the overloaded left ventricles of transgenic mice with cardiac specific FAK expression. These findings define a novel mechanism by which αB-crystallin controls FAK function, with impact on cardiomyocyte survival and cardiac remodelling in response to stress.

scholarly journals Spatial and Temporal Regulation of Focal Adhesion Kinase Activity in Living Cells

2007 ◽  
Vol 28 (1) ◽  
pp. 201-214 ◽  
Author(s):  
Xinming Cai ◽  
Daniel Lietha ◽  
Derek F. Ceccarelli ◽  
Andrei V. Karginov ◽  
Zenon Rajfur ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Conformational Change ◽  
Focal Adhesion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Live Cells ◽  
Major Mechanism

ABSTRACT Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.

A role for focal adhesion kinase in cardiac mitochondrial biogenesis induced by mechanical stress

2011 ◽  
Vol 300 (3) ◽  
pp. H902-H912 ◽  
Author(s):  
Thais F. Tornatore ◽  
Ana Paula Dalla Costa ◽  
Carolina F. M. Z. Clemente ◽  
Carla Judice ◽  
Silvana A. Rocco ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Mechanical Stress ◽  
Focal Adhesion ◽  
Mitochondrial Biogenesis ◽  
Pressure Overload ◽  
Left Ventricular ◽  
Neonatal Rat ◽  
Peroxisome Proliferator ◽  
Mitochondrial Transcription ◽  
Hypertrophic Growth

We studied the implication of focal adhesion kinase (FAK) in cardiac mitochondrial biogenesis induced by mechanical stress. Prolonged stretching (2–12 h) of neonatal rat ventricular myocytes (NRVM) upregulated the main components of mitochondrial transcription cascade [peroxisome proliferator-activated receptor coactivator-1 (PGC-1α), nuclear respiratory factor (NRF-1), and mitochondrial transcription factor A]. Concomitantly, prolonged stretching enhanced mitochondrial biogenesis [copy number of mitochondrial DNA (mtDNA), content of the subunit IV of cytochrome oxidase, and mitochondrial staining-green fluorescence intensity of Mitotracker green] and induced the hypertrophic growth (cell size and atrial natriuretic peptide transcripts) of NRVM. Furthermore, the stretching of NRVM enhanced phosphorylation, nuclear localization, and association of FAK with PGC-1α. Recombinant FAK COOH-terminal, but not the NH2-terminal or kinase domain, precipitated PGC-1α from nuclear extracts of NRVM. Depletion of FAK by RNA interference suppressed the upregulation of PGC-1α and NRF-1 and markedly attenuated the enhanced mitochondrial biogenesis and hypertrophic growth of stretched NRVM. In the context of energy metabolism, FAK depletion became manifest by a reduction of ATP levels in stretched NRVM. Complementary studies in adult mice left ventricle demonstrated that pressure overload upregulated PGC-1α, NRF-1, and mtDNA. In vivo FAK silencing transiently attenuated the upregulation of PGC-1α, NRF-1, and mtDNA, as well as the left ventricular hypertrophy induced by pressure overload. In conclusion, activation of FAK signaling seems to be important for conferring enhanced mitochondrial biogenesis coupled to the hypertrophic growth of cardiomyocytes in response to mechanical stress, via control of mitochondrial transcription cascade.

scholarly journals Cardiac Hypertrophy Changes Compartmentation of cAMP in Non-Raft Membrane Microdomains

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 535
Author(s):  
Nikoleta Pavlaki ◽  
Kirstie A. De Jong ◽  
Birgit Geertz ◽  
Viacheslav O. Nikolaev ◽  
Alexander Froese
Keyword(s):  
Cardiac Hypertrophy ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Pressure Overload ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Differential Regulation ◽  
Membrane Microdomains ◽  
Transgenic Mouse Line

3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which plays critical roles in cardiac function and disease. In adult mouse ventricular myocytes (AMVMs), several distinct functionally relevant microdomains with tightly compartmentalized cAMP signaling have been described. At least two types of microdomains reside in AMVM plasma membrane which are associated with caveolin-rich raft and non-raft sarcolemma, each with distinct cAMP dynamics and their differential regulation by receptors and cAMP degrading enzymes phosphodiesterases (PDEs). However, it is still unclear how cardiac disease such as hypertrophy leading to heart failure affects cAMP signals specifically in the non-raft membrane microdomains. To answer this question, we generated a novel transgenic mouse line expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor E1-CAAX targeted to non-lipid raft membrane microdomains of AMVMs and subjected these mice to pressure overload induced cardiac hypertrophy. We could detect specific changes in PDE3-dependent compartmentation of β-adrenergic receptor induced cAMP in non-raft membrane microdomains which were clearly different from those occurring in caveolin-rich sarcolemma. This indicates differential regulation and distinct responses of these membrane microdomains to cardiac remodeling.

Dynamic localization of αB-crystallin at the microtubule cytoskeleton network in beating heart cells

2020 ◽  
Vol 168 (2) ◽  
pp. 125-137 ◽  
Author(s):  
Eri Ohto-Fujita ◽  
Saaya Hayasaki ◽  
Aya Atomi ◽  
Soichiro Fujiki ◽  
Toshiyuki Watanabe ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Heart Cells ◽  
Striated Muscles ◽  
Slow Skeletal Muscle ◽  
Αb Crystallin ◽  
Cardiac Muscles ◽  
Myoblast Cells

Abstract αB-crystallin is highly expressed in the heart and slow skeletal muscle; however, the roles of αB-crystallin in the muscle are obscure. Previously, we showed that αB-crystallin localizes at the sarcomere Z-bands, corresponding to the focal adhesions of cultured cells. In myoblast cells, αB-crystallin completely colocalizes with microtubules and maintains cell shape and adhesion. In this study, we show that in beating cardiomyocytes α-tubulin and αB-crystallin colocalize at the I- and Z-bands of the myocardium, where it may function as a molecular chaperone for tubulin/microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the striated patterns of GFP-αB-crystallin fluorescence recovered quickly at 37°C. FRAP mobility assay also showed αB-crystallin to be associated with nocodazole-treated free tubulin dimers but not with taxol-treated microtubules. The interaction of αB-crystallin and free tubulin was further confirmed by immunoprecipitation and microtubule sedimentation assay in the presence of 1–100 μM calcium, which destabilizes microtubules. Förster resonance energy transfer analysis showed that αB-crystallin and tubulin were at 1–10 nm apart from each other in the presence of colchicine. These results suggested that αB-crystallin may play an essential role in microtubule dynamics by maintaining free tubulin in striated muscles, such as the soleus or cardiac muscles.

scholarly journals The Scripps Molecular Screening Center and Translational Research Institute

2019 ◽  
Vol 24 (3) ◽  
pp. 386-397 ◽  
Author(s):  
Pierre Baillargeon ◽  
Virneliz Fernandez-Vega ◽  
Banu Priya Sridharan ◽  
Steven Brown ◽  
Patrick R. Griffin ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Translational Research ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Screening ◽  
Time Resolved ◽  
Biochemical Assays ◽  
Automated Screening ◽  
La Jolla ◽  
Research Institute

The Scripps Research Molecular Screening Center (SRMSC) was founded in 2004 and comprises more than $22 million of specialized automation. As part of the Translational Research Institute (TRI), it comprises early drug discovery labs and medicinal chemistry. Together with Scripps Research at the La Jolla, California, campus, this represents one of the most competitive academic industrial screening centers worldwide. The SRMSC uses automated platforms, one a screening cell and the other a cherry-picking platform. Matched technologies are available throughout Scripps to allow scientists to develop assays and prepare them for automated screening. The library comprises more than 1 million drug-like compounds, including a proprietary collection of >665,000 molecules. Internal chemistry has included ~40,000 unique compounds that are not found elsewhere. These collections are screened against a myriad of disease targets, including cell-based and biochemical assays that are provided by Scripps faculty or from global investigators. Scripps has proven competence in all detection formats, including high-content analysis, fluorescence, bioluminescence resonance energy transfer (BRET), time-resolved fluorescence resonance energy transfer (TR-FRET), fluorescence polarization (FP), luminescence, absorbance, AlphaScreen, and Ca++ signaling. These technologies are applied to NIH-derived collaborations as well as biotech and pharma initiatives. The SRMSC and TRI are recognized for discovering multiple leads, including Ozanimod.

scholarly journals Real-time observation of flow-induced cytoskeletal stress in living cells

2011 ◽  
Vol 301 (3) ◽  
pp. C646-C652 ◽  
Author(s):  
Jason Rahimzadeh ◽  
Fanjie Meng ◽  
Fredrick Sachs ◽  
Jianbin Wang ◽  
Deepika Verma ◽  
...  
Keyword(s):  
Shear Stress ◽  
Mechanical Stress ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Host Protein ◽  
Apical Surface ◽  
Intracellular Ca ◽  
In Series ◽  
Time Observation

The mechanical stress due to shear flow has profound effects on cell proliferation, transport, gene expression, and apoptosis. The mechanisms for flow sensing and transduction are unclear, but it is postulated that fluid flow pulls upon the apical surface, and the resulting stress is eventually transmitted through the cytoskeleton to adhesion plaques on the basal surface. Here we report a direct observation of this flow-induced stress in the cytoskeleton in living cells using a parallel plate microfluidic chip with a fluorescence resonance energy transfer (FRET)-based mechanical stress sensor in actinin. The sensing cassette was genetically inserted into the cytoskeletal host protein and transfected into Madin-Darby canine kidney cells. A shear stress of 10 dyn/cm2 resulted in a rapid increase in the FRET ratio indicating a decrease in stress across actinin with flow. The effect was reversible, and cells were able to respond to repeated stimulation and showed adaptive changes in the cytoskeleton. Flow-induced Ca2+ elevation did not affect the response, suggesting that flow-induced changes in actinin stress are insensitive to intracellular Ca2+ level. The reduction in FRET ratio suggests actin filaments are under normal compression in the presence of flow shear stress due to changes in cell shape, and/or actinin is not in series with actin. Treatment with cytochalasin-D that disrupts F-actin reduced prestress and the response to flow. The FRET/flow method is capable of resolving changes of stress in multiple proteins with optical spatial resolution and time resolution >1 Hz. This promises to provide insight into the force distribution and transduction in all cells.

scholarly journals Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain

1996 ◽  
Vol 318 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Prasad COORAY ◽  
Yuping YUAN ◽  
Simone M SCHOENWAELDER ◽  
Christina A MITCHELL ◽  
Hatem H SALEM ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Receptor Tyrosine Kinase ◽  
Human Platelets ◽  
Calpain Inhibitor ◽  
Ionophore A23187 ◽  
Dramatic Reduction ◽  
Calpain Inhibitors ◽  
Autokinase Activity

Focal adhesion kinase (125 kDa form; pp125FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signal transduction. We have identified a novel means of pp125FAK regulation in human platelets, in which this kinase undergoes sequential proteolytic modification from the native 125 kDa form to 90, 45 and 40 kDa fragments in thrombin-, collagen- and ionophore A23187-stimulated platelets. The proteolysis of pp125FAK was prevented by pretreating platelets with the calpain inhibitors calpeptin or calpain inhibitor-1, and was reproduced in vitro by incubating immunoprecipitated pp125FAK with purified calpain. Proteolysis of pp125FAK resulted in a dramatic reduction in its autokinase activity and led to its dissociation from the cytoskeletal fraction of platelets. These studies define a novel signal-terminating role for calpain, wherein proteolytic modification of pp125FAK attenuates its autokinase activity and induces its subcellular relocation within the cell.

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