FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy

Santos, Aline Mara dos; Schechtman, Deborah; Cardoso, Alisson C.; Clemente, Carolina F.M.Z.; Silva, Julio C.; Fioramonte, Mariana; Pereira, Michelle B. M.; Marin, Talita Miguel; Oliveira, Paulo Sérgio Lopes de; Figueira, Ana Carolina M.; Oliveira, Saulo H P; Torriani, Íris L.; Gozzo, Fábio C.; Neto, José Xavier; Franchini, Kleber G.

doi:10.1038/nchembio.717

Cited by 18 publications

(21 citation statements)

References 38 publications

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“…A complete understanding of how an interplay with aB-crystallin results in regulation of FAK function requires knowledge of structural features of the aB-crystallin:FAK complex. In this study, we used protein chemical crosslinking combined with mass spectrometry and mutational analyses 26,27 to show that a surface-exposed patch sited between a1 and a4 helices of the FAK FAT domain binds to the b4-b8 hydrophobic groove of ACD. The hydrophobic nature of the FAK FAT a1-a4 ARTICLE patch has been reported to favour its sequestration within intermolecular interfaces of FAK protein partners.…”

Section: Discussionmentioning

confidence: 99%

“…To gather information on the structural features of the FAKcTERM-CryAB complex, we used crosslinking in combination with mass spectrometry (CXMS) analysis and computational modelling 26,27 . Given the size and high flexibility of the multidomain full-length FAK, we decided to use the truncated FAK-cTERM in the structural and computational studies.…”

Section: Structural Basis Of the Fak-cterm-ab-crystallin Interactionmentioning

confidence: 99%

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αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes

Pereira¹,

Santos²,

Gonçalves³

et al. 2014

Nat Commun

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Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that aB-crystallin interacts with and confers protection to FAK against calpainmediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the b4-b8 groove of aB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. aB-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 aB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of aB-crystallin by RNA interference. These studies define a role for aB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.

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Section: Discussionmentioning

confidence: 99%

Section: Structural Basis Of the Fak-cterm-ab-crystallin Interactionmentioning

confidence: 99%

αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes

Pereira¹,

Santos²,

Gonçalves³

et al. 2014

Nat Commun

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“…It has been recently reported that the interaction of FERM with myosin negatively regulates FAK activity by promoting the autoinhibited FAK conformation (43). This recent study provided strong evidence of direct interaction between myosin heavy chain Integrin α5β1 can be fully activated in the tensioned state where both RGD peptide (yellow circle) and synergy site (red circle) bind to α5 and β1 subunits, respectively.…”

Section: Distinct Biophysical Mechanism Of Fak Mechanoactivation Detementioning

confidence: 83%

“…The key residues of FERM F2 domain for myosin binding, E158/D161/Q162 (EDQ) (43), are positioned proximal to the FERM F2 basic patch KAKTLRK (SI Appendix, Fig. S14A).…”

Section: Distinct Biophysical Mechanism Of Fak Mechanoactivation Detementioning

confidence: 99%

“…and the FERM domain of FAK by extensive biochemical assays including GST pull-down assays as well as coimmunoprecipitation experiment (43). The key residues of FERM F2 domain for myosin binding, E158/D161/Q162 (EDQ) (43), are positioned proximal to the FERM F2 basic patch KAKTLRK (SI Appendix, Fig.…”

Section: Distinct Biophysical Mechanism Of Fak Mechanoactivation Detementioning

confidence: 99%

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Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins

Seong

Tajik²,

Sun

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

155

150

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Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.FRET biosensor | intracellular tension | substrate rigidity C ells can sense and respond to the mechanical microenvironment by converting forces into biochemical signals inside the cells; that is, mechanotransduction (1). Focal adhesions (FAs) are the major sites of interaction between a cell and its extracellular matrix (ECM) microenvironment, and thus, outside mechanical signals can be sensed at FAs through transmembrane receptor integrins. In particular, it has been shown that matrix elasticity can control the cell fate (2) by modulating the interactions between ECM proteins and their receptor integrins (3, 4). The modifications in ECM-integrin bonds can be further translated into biochemical signals through FA proteins (5). For example, mechanical stretching of Crk-associated substrate (p130 CAS ) promotes its phosphorylation by Src family kinases (6). Unfolding purified talin rod domain by mechanical stretching allows the recruitment of vinculin, potentially reinforcing the connection between integrin and actin (7). Myosin II-dependent endogenous tension has also been shown to directly unfold vinculin in living cells (8). However, it remains unclear how focal adhesion kinase (FAK), a major downstream molecule of integrin signaling, is regulated by mechanical signals.Various ECM proteins including fibronectin (FN) and type 1 collagen (Col I) are specifically recognized by integrin subtypes with different combinations of α and β subunits, allowing diver...

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Analysis of secondary structure in proteins by chemical cross‐linking coupled to MS

et al. 2012

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Chemical cross-linking is an attractive technique for the study of the structure of protein complexes due to its low sample consumption and short analysis time. Furthermore, distance constraints obtained from the identification of cross-linked peptides by mass spectrometry can be used to construct and validate protein models. If a sufficient number of distance constraints are obtained, then determining the secondary structure of a protein can allow inference of the protein’s fold. In this work, we show how the distance constraints obtained from cross-linking experiments can identify secondary structures within the protein sequence. Molecular modeling of alpha helices and beta sheets indicate cross-linking patterns based on the topological distances between reactive residues. DSS[1] cross-linking experiments with model alpha helix containing proteins corroborated the molecular modeling predictions. The patterns established here can be extended to other cross-linkers with known spacing lengths.

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FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy

Cited by 18 publications

References 38 publications

αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes

αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes

Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins

Analysis of secondary structure in proteins by chemical cross‐linking coupled to MS

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