The tyrosine kinase Src acts as a key regulator of cell motility by phosphorylating multiple protein substrates that control cytoskeletal and adhesion dynamics. In an earlier phosphotyrosine proteomics study, we identified a novel Rho-GTPase activating protein, now known as ARHGAP42, as a likely biologically relevant Src substrate. ARHGAP42 is a member of a family of RhoGAPs distinguished by tandem BAR-PH domains lying N-terminal to the GAP domain. Like other family members, ARHGAP42 acts preferentially as a GAP for RhoA. We show that Src principally phosphorylates ARHGAP42 on tyrosine 376 (Tyr-376) in the short linker between the BAR-PH and GAP domains. The expression of ARHGAP42 variants in mammalian cells was used to elucidate its regulation. We found that the BAR domain is inhibitory toward the GAP activity of ARHGAP42, such that BAR domain deletion resulted in decreased active GTP-bound RhoA and increased cell motility. With the BAR domain intact, ARHGAP42 GAP activity could be activated by phosphorylation of Tyr-376 to promote motile cell behavior. Thus, phosphorylation of ARHGAP42 Tyr-376 is revealed as a novel regulatory event by which Src can affect actin dynamics through RhoA inhibition.

• MeSH
• fokální adheze metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• myši MeSH
• pohyb buněk fyziologie   MeSH
• proteiny aktivující GTPasu genetika   metabolismus   MeSH
• rho proteiny vázající GTP antagonisté a inhibitory   metabolismus   MeSH
• rhoA protein vázající GTP antagonisté a inhibitory   metabolismus   MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• tyrosin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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