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Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and

Radoslav Janoštiak1, Ondřej Tolde, Zuzana Brůhová

  • 1Department of Cell Biology, Charles University, 12843 Prague, Czech Republic.

Molecular Biology of the Cell
|September 23, 2011
PubMed

Insights

Crk-associated substrate (CAS) phosphorylation at Tyr-12 regulates cell adhesion and migration. This finding is crucial for understanding invasiveness in Src-transformed cells and human carcinomas.

Area of Science:

  • Cell Biology
  • Molecular Oncology
  • Biochemistry

Background:

  • Crk-associated substrate (CAS) is a key tyrosine-phosphorylated protein involved in cell invasiveness, particularly in Src-transformed cells.
  • CAS phosphorylation is critical for regulating cellular processes like adhesion and migration.
  • Src and Crk oncogenes are implicated in various cancers, making CAS a relevant target for study.

Purpose of the Study:

  • To investigate the biological significance of a novel CAS phosphorylation site, Tyr-12 (Y12), located within the CAS SH3 domain.
  • To elucidate the role of CAS Y12 phosphorylation in regulating focal adhesion dynamics, cell migration, and invasiveness.

Main Methods:

  • Site-directed mutagenesis to create phosphomimicking (Y12E) and nonphosphorylatable (Y12F) CAS mutants.
  • Biochemical assays to assess protein-protein interactions (CAS SH3 domain with FAK and PTP-PEST) and tyrosine phosphorylation levels.
  • Live-cell imaging using fluorescently tagged CAS mutants to track localization in focal adhesions and podosomes.
  • Functional assays in cas-/- mouse embryonic fibroblasts and Src-transformed cells to evaluate cell migration and invasiveness.

Main Results:

  • CAS Y12 phosphorylation, mimicked by Y12E, reduced the interaction of the CAS SH3 domain with FAK and PTP-PEST, decreasing FAK tyrosine phosphorylation.
  • The Y12E mutant was excluded from focal adhesions but localized to podosome-type adhesions, unlike wild-type or Y12F CAS.
  • Expression of the nonphosphorylatable Y12F mutant in cas-/- cells led to hyperphosphorylation of the CAS substrate domain, slower focal adhesion turnover, and reduced cell migration.
  • Expression of CAS Y12F significantly decreased invasiveness in Src-transformed cells compared to wild-type CAS.

Conclusions:

  • CAS Y12 phosphorylation plays a critical role in regulating focal adhesion assembly and dynamics.
  • Phosphorylation at CAS Y12 is essential for efficient cell migration and invasiveness in Src-transformed cells.
  • Targeting CAS Y12 phosphorylation may offer a therapeutic strategy for inhibiting cancer cell invasion.

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