The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here, we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility, and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signalling hub in a variety of cellular processes.

• Klíčová slova
• SH3 domain, Src, biochemistry, cell biology, cell transformation, chemical biology, invasiveness, mouse, phosphorylation, protein structure,
• MeSH
• fosforylace MeSH
• skupina kinas odvozených od src-genu * metabolismus   MeSH
• src homologní domény * MeSH
• tyrosin metabolismus   MeSH
• tyrosinkinasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• skupina kinas odvozených od src-genu * MeSH
• tyrosin MeSH
• tyrosinkinasy MeSH

CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.

• MeSH
• aminokyseliny metabolismus   MeSH
• fosforylace fyziologie   MeSH
• lidé MeSH
• ligandy MeSH
• protoonkogen Mas MeSH
• sekvence aminokyselin MeSH
• signální transdukce fyziologie   MeSH
• src homologní domény fyziologie   MeSH
• substrátový protein asociovaný s Crk metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• vazebná místa fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• ligandy MeSH
• MAS1 protein, human MeSH Prohlížeč
• protoonkogen Mas MeSH
• substrátový protein asociovaný s Crk MeSH

CAS is a docking protein, which was shown to act as a mechanosensor in focal adhesions. The unique assembly of structural domains in CAS is important for its function as a mechanosensor. The tension within focal adhesions is transmitted to a stretchable substrate domain of CAS by focal adhesion-targeting of SH3 and CCH domain of CAS, which anchor the CAS protein in focal adhesions. Mechanistic models of the stretching biosensor propose equal roles for both anchoring domains. Using deletion mutants and domain replacements, we have analyzed the relative importance of the focal adhesion anchoring domains on CAS localization and dynamics in focal adhesions as well as on CAS-mediated mechanotransduction. We confirmed the predicted prerequisite of the focal adhesion targeting for CAS-dependent mechanosensing and unraveled the critical importance of CAS SH3 domain in mechanosensing. We further show that CAS localizes to the force transduction layer of focal adhesions and that mechanical stress stabilizes CAS in focal adhesions.

• MeSH
• buněčná adheze MeSH
• buněčný převod mechanických signálů * MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fokální adheze metabolismus   MeSH
• mechanický stres MeSH
• mutantní proteiny chemie   MeSH
• myši MeSH
• proteinové domény MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• signální transdukce MeSH
• stabilita proteinů MeSH
• substrátový protein asociovaný s Crk chemie   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Bcar1 protein, mouse MeSH Prohlížeč
• mutantní proteiny MeSH
• rekombinantní fúzní proteiny MeSH
• substrátový protein asociovaný s Crk MeSH
• zelené fluorescenční proteiny MeSH

Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.

• MeSH
• aminokyselinové motivy MeSH
• biomechanika MeSH
• buněčná adheze MeSH
• buněčné linie MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fokální adheze metabolismus   ultrastruktura   MeSH
• fokální adhezní tyrosinkinasy metabolismus   MeSH
• fosforylace MeSH
• mapy interakcí proteinů MeSH
• myši MeSH
• peptidy chemie   metabolismus   MeSH
• src homologní domény MeSH
• substrátový protein asociovaný s Crk analýza   metabolismus   MeSH
• vazba proteinů MeSH
• vinkulin analýza   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fokální adhezní tyrosinkinasy MeSH
• peptidy MeSH
• polyproline MeSH Prohlížeč
• substrátový protein asociovaný s Crk MeSH
• vinkulin MeSH