title: Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and invasiveness.
abstract: 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.
authors.affiliation: Department of Cell Biology, Charles University, 12843 Prague, Czech Republic.
authors.forename: Radoslav
authors.initials: R
authors.lastname: Janoštiak
authors.forename: Ondřej
authors.initials: O
authors.lastname: Tolde
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Brůhová
authors.forename: Marian
authors.initials: M
authors.lastname: Novotný
authors.forename: Steven K
authors.initials: SK
authors.lastname: Hanks
authors.forename: Daniel
authors.initials: D
authors.lastname: Rösel
authors.forename: Jan
authors.initials: J
authors.lastname: Brábek
mesh_terms: D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002465:Cell Movement; D002471:Cell Transformation, Neoplastic; D050719:Crk-Associated Substrate Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D009154:Mutation; D009361:Neoplasm Invasiveness; D010766:Phosphorylation; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12 / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D015815:Cell Adhesion Molecules; D050719:Crk-Associated Substrate Protein; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D051416:Focal Adhesion Protein-Tyrosine Kinases; C516738:PTPN12 protein, human; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12