title: Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis.
abstract: 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.
authors.affiliation: Laboratory of Molecular Oncology, The Rockefeller University, New York, New York 10021, USA.
authors.forename: M
authors.initials: M
authors.lastname: Escalante
authors.forename: J
authors.initials: J
authors.lastname: Courtney
authors.forename: W G
authors.initials: WG
authors.lastname: Chin
authors.forename: K K
authors.initials: KK
authors.lastname: Teng
authors.forename: J I
authors.initials: JI
authors.lastname: Kim
authors.forename: J E
authors.initials: JE
authors.lastname: Fajardo
authors.forename: B J
authors.initials: BJ
authors.lastname: Mayer
authors.forename: B L
authors.initials: BL
authors.lastname: Hempstead
authors.forename: R B
authors.initials: RB
authors.lastname: Birge
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016313:Genes, abl; D006801:Humans; D007668:Kidney; D051379:Mice; D018345:Mice, Knockout; D009024:Morphogenesis; D016297:Mutagenesis, Site-Directed; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016716:PC12 Cells; D051419:Paxillin; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016315:Proto-Oncogene Proteins c-abl / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine
chemical_list: C493872:Crk protein, rat; D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; C496062:Pxn protein, mouse; C496063:Pxn protein, rat; D011994:Recombinant Proteins; D014443:Tyrosine; D020932:Nerve Growth Factor; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D016315:Proto-Oncogene Proteins c-abl; C496051:Ptk2 protein, mouse; C496052:Ptk2 protein, rat