Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor

Oncogene. 1997 Oct 9;15(15):1823-32. doi: 10.1038/sj.onc.1201351.


The SH3-SH3-SH3-SH2 adapter protein Nck links receptor tyrosine kinases, such as EGF and PDGF receptors, to downstream signaling pathways, among which p21cdc42/rac-activated kinase cascade, Sos-activated Ras signaling and the human Wiskott-Aldrich Syndrome protein (WASp)-mediated actin cytoskeleton changes, have been implicated. In EGF stimulated cells, Nck co-immunoprecipitates with a number of phosphotyrosine proteins including the EGF receptor (Li et al., 1992 Mol. Cell. Biol. 12: 5824-2833). To identify the phosphotyrosine protein(s) that directly interacts with Nck and to distinguish it from indirectly associated proteins, preexisting phosphoytrosine protein complexes in the cell lysate were dissociated by heat and SDS prior to the test for binding to Nck. We found that Nck does not directly bind to EGF receptor, instead it binds via its SH2 domain to a 62 kDa phosphotyrosine protein. We present evidence demonstrating that the Nck-bound p62 is related to the previously identified GTPase-activating protein (GAP)-associated phosphotyrosine protein p62. (1) The Nck-bound and the GAP-bound p62 proteins co-migrate with each other in SDS-PAGE. (2) SH2 domains from Nck and GAP compete for binding to p62 in vitro. (3) Purified GST-Nck-SH2 binds directly to the GAP-associated p62. Under these conditions, SH2 domains from PLCgamma, PI-3 kinase, SHC, and Grb2 did not bind p62. (4) Tryptic phosphopeptide maps of the Nck- and the GAP-associated p62 proteins are identical. However, Nck and GAP do not co-immunoprecipitate with each other and apparently bind to different pools of p62. This study suggests that the GAP-associated p62 acts as an SH2 domain docking protein and mediates the interaction between Nck and EGF receptor in response to EGF stimulation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Adaptor Proteins, Signal Transducing
  • Animals
  • Binding, Competitive
  • Electrophoresis, Polyacrylamide Gel
  • Epidermal Growth Factor / pharmacology*
  • GTPase-Activating Proteins
  • Humans
  • Mice
  • Oncogene Proteins / metabolism*
  • Peptide Mapping
  • Platelet-Derived Growth Factor / pharmacology
  • Protein Binding
  • Proteins / metabolism*
  • Recombinant Proteins / pharmacology
  • Signal Transduction
  • ras GTPase-Activating Proteins
  • src Homology Domains


  • Adaptor Proteins, Signal Transducing
  • GTPase-Activating Proteins
  • Nck protein
  • Oncogene Proteins
  • Platelet-Derived Growth Factor
  • Proteins
  • Recombinant Proteins
  • ras GTPase-Activating Proteins
  • Epidermal Growth Factor