Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis

J Biol Chem. 2000 Aug 11;275(32):24787-97. doi: 10.1074/jbc.M000711200.


The Crk family of adaptor proteins participate in diverse signaling pathways that regulate growth factor-induced proliferation, anchorage-dependent DNA synthesis, and cytoskeletal reorganization, important for cell adhesion and motility. Using kidney epithelial 293T cells for transient co-transfection studies and the nerve growth factor (NGF)-responsive PC12 cell line as a model system for neuronal morphogenesis, we demonstrate that the non-receptor tyrosine kinase c-Abl is an intermediary for NGF-inducible c-Crk II phosphorylation on the negative regulatory Tyr(222). Transient expression of a c-Crk II Tyr(222) point mutant (c-Crk Y222F) in 293T cells induces hyperphosphorylation of paxillin on Tyr(31) and enhances complex formation between c-Crk Y222F and paxillin as well as c-Crk Y222F and c-Abl, suggesting that c-Crk II Tyr(222) phosphorylation induces both the dissociation of the Crk SH2 domain from paxillin and the Crk SH3 domain from c-Abl. Interestingly, examination of the early kinetics of NGF stimulation in PC12 cells showed that c-Crk II Tyr(222) phosphorylation preceded paxillin Tyr(31) phosphorylation, followed by a transient initial dissociation of the c-Crk II paxillin complex. PC12 cells overexpressing c-Crk Y222F manifested a defect in cellular adhesion and neuritogenesis that led to detachment of cells from the extracellular matrix, thus demonstrating the biological significance of c-Crk II tyrosine phosphorylation in NGF-dependent morphogenesis. Whereas previous studies have shown that Crk SH2 binding to paxillin is critical for cell adhesion and migration, our data show that the phosphorylation cycle of c-Crk II determines its dynamic interaction with paxillin, thereby regulating turnover of multiprotein complexes, a critical aspect of cytoskeletal plasticity and actin dynamics.

Publication types

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

MeSH terms

  • Animals
  • Cell Adhesion / drug effects
  • Cell Line
  • Cytoskeletal Proteins / chemistry
  • Cytoskeletal Proteins / metabolism*
  • Focal Adhesion Kinase 1
  • Focal Adhesion Protein-Tyrosine Kinases
  • Genes, abl
  • Humans
  • Kidney
  • Mice
  • Mice, Knockout
  • Morphogenesis
  • Mutagenesis, Site-Directed
  • Nerve Growth Factor / pharmacology*
  • PC12 Cells
  • Paxillin
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Point Mutation
  • Protein-Tyrosine Kinases / deficiency
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-abl / deficiency
  • Proto-Oncogene Proteins c-abl / genetics
  • Proto-Oncogene Proteins c-abl / metabolism
  • Proto-Oncogene Proteins c-crk
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Transfection
  • Tyrosine*


  • Crk protein, rat
  • Cytoskeletal Proteins
  • PXN protein, human
  • Paxillin
  • Phosphoproteins
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-crk
  • Pxn protein, mouse
  • Pxn protein, rat
  • Recombinant Proteins
  • Tyrosine
  • Nerve Growth Factor
  • Protein-Tyrosine Kinases
  • Focal Adhesion Kinase 1
  • Focal Adhesion Protein-Tyrosine Kinases
  • PTK2 protein, human
  • Proto-Oncogene Proteins c-abl
  • Ptk2 protein, mouse
  • Ptk2 protein, rat