Tyrosine Y189 in the Substrate Domain of the Adhesion Docking Protein NEDD9 Is Conserved With p130Cas Y253 and Regulates NEDD9-mediated Migration and Focal Adhesion Dynamics

PLoS One. 2013 Jul 9;8(7):e69304. doi: 10.1371/journal.pone.0069304. Print 2013.

Abstract

The focal adhesion docking protein NEDD9/HEF1/Cas-L regulates cell migration and cancer invasion. NEDD9 is a member of the Cas family of proteins that share conserved overall protein-protein interaction domain structure, including a substrate domain that is characterized by extensive tyrosine (Y) phosphorylation. Previous studies have suggested that phosphorylation of Y253 in the substrate domain of the Cas family protein p130Cas is specifically required for p130Cas function in cell migration. While it is clear that tyrosine phosphorylation of the NEDD9 substrate domain is similarly required for the regulation of cell motility, whether individual NEDD9 tyrosine residues have discrete function in regulating motility has not previously been reported. In the present study we have used a global sequence alignment of Cas family proteins to identify a putative NEDD9 equivalent of p130Cas Y253. We find that NEDD9 Y189 aligns with p130Cas Y253 and that it is conserved among NEDD9 vertebrate orthologues. Expression of NEDD9 in which Y189 is mutated to phenylalanine results in increased rates of cell migration and is correlated with increased disassembly of GFP.NEDD9 focal adhesions. Conversely, mutation to Y189D significantly inhibits cell migration. Our previous data has suggested that NEDD9 stabilizes focal adhesions and the present data therefore suggests that phosphorylation of Y189 NEDD9 is required for this function. These findings indicate that the individual tyrosine residues of the NEDD9 substrate domain may serve discrete functional roles. Given the important role of this protein in promoting cancer invasion, greater understanding of the function of the individual tyrosine residues is important for the future design of approaches to target NEDD9 to arrest cancer cell invasion.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Cell Movement* / genetics
  • Consensus Sequence
  • Crk-Associated Substrate Protein / chemistry
  • Crk-Associated Substrate Protein / genetics
  • Crk-Associated Substrate Protein / metabolism*
  • Focal Adhesions* / genetics
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Phosphoproteins / chemistry
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Interaction Domains and Motifs
  • Sequence Alignment
  • Tyrosine / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Crk-Associated Substrate Protein
  • NEDD9 protein, human
  • Phosphoproteins
  • Tyrosine

Grant support

This study was supported by National Health and Medical Research (NHMRC) Australia grants 512251 and 632515 to GO. JBB was supported by an Honours scholarship award from the University of Sydney. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.