Protein p130Cas constitutes an adaptor protein mainly involved in integrin signaling downstream of Src kinase. Owing to its modular structure, p130Cas acts as a general regulator of cancer cell growth and invasiveness induced by different oncogenes. However, other mechanisms of p130Cas signaling leading to malignant progression are poorly understood. Here, we show a novel interaction of p130Cas with Ser/Thr kinase PKN3, which is implicated in prostate and breast cancer growth downstream of phosphoinositide 3-kinase. This direct interaction is mediated by the p130Cas SH3 domain and the centrally located PKN3 polyproline sequence. PKN3 is the first identified Ser/Thr kinase to bind and phosphorylate p130Cas and to colocalize with p130Cas in cell structures that have a pro-invasive function. Moreover, the PKN3-p130Cas interaction is important for mouse embryonic fibroblast growth and invasiveness independent of Src transformation, indicating a mechanism distinct from that previously characterized for p130Cas. Together, our results suggest that the PKN3-p130Cas complex represents an attractive therapeutic target in late-stage malignancies.

• Klíčová slova
• CAS, BCAR1, PKN3, SH3, Src, p130Cas,
• MeSH
• fibroblasty metabolismus   MeSH
• fosforylace MeSH
• fosfothreonin metabolismus   MeSH
• invazivní růst nádoru MeSH
• kontraktilní svazky metabolismus   MeSH
• lidé MeSH
• myši nahé MeSH
• nádory metabolismus   patologie   MeSH
• podozomy metabolismus   MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• proteinkinasa C metabolismus   MeSH
• pseudopodia metabolismus   MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• substrátový protein asociovaný s Crk metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfothreonin MeSH
• protein kinase N MeSH Prohlížeč
• proteinkinasa C MeSH
• skupina kinas odvozených od src-genu MeSH
• substrátový protein asociovaný s Crk 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

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

Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligand-binding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein-tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas-/- mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells.

• MeSH
• fokální adheze metabolismus   MeSH
• fokální adhezní tyrosinkinasy metabolismus   MeSH
• fosforylace MeSH
• invazivní růst nádoru MeSH
• lidé MeSH
• molekuly buněčné adheze metabolismus   MeSH
• mutace MeSH
• myši MeSH
• nádorová transformace buněk MeSH
• nádorové buněčné linie MeSH
• pohyb buněk MeSH
• signální transdukce MeSH
• src homologní domény MeSH
• substrátový protein asociovaný s Crk chemie   genetika   metabolismus   MeSH
• transformované buněčné linie MeSH
• tyrosin metabolismus   MeSH
• tyrosinfosfatasa nereceptorového typu 12 metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fokální adhezní tyrosinkinasy MeSH
• molekuly buněčné adheze MeSH
• PTPN12 protein, human MeSH Prohlížeč
• substrátový protein asociovaný s Crk MeSH
• tyrosin MeSH
• tyrosinfosfatasa nereceptorového typu 12 MeSH
• zelené fluorescenční proteiny MeSH