title: Distinct FAK-Src activation events promote alpha5beta1 and alpha4beta1 integrin-stimulated neuroblastoma cell motility.
abstract: Signals from fibronectin-binding integrins promote neural crest cell motility during development in part through protein-tyrosine kinase (PTK) activation. Neuroblastoma (NB) is a neural crest malignancy with high metastatic potential. We find that alpha4 and alpha5 integrins are present in late-stage NB tumors and cell lines derived thereof. To determine the signaling connections promoting either alpha4beta1- or alpha5beta1-initiated NB cell motility, pharmacological, dominant negative and short-hairpin RNA (shRNA) inhibitory approaches were undertaken. shRNA knockdown revealed that alpha5beta1-stimulated NB motility is dependent upon focal adhesion kinase (FAK) PTK, Src PTK and p130Cas adapter protein expression. Cell reconstitution showed that FAK catalytic activity is required for alpha5beta1-stimulated Src activation in part through direct FAK phosphorylation of Src at Tyr-418. Alternatively, alpha4beta1-stimulated NB cell motility is dependent upon Src and p130Cas but FAK is not essential. Catalytically inactive receptor protein-tyrosine phosphatase-alpha overexpression inhibited alpha4beta1-stimulated NB motility and Src activation consistent with alpha4-regulated Src activity occurring through Src Tyr-529 dephosphorylation. In alpha4 shRNA-expressing NB cells, alpha4beta1-stimulated Src activation and NB cell motility were rescued by wild type but not cytoplasmic domain-truncated alpha4 re-expression. These studies, supported by results using reconstituted fibroblasts, reveal that alpha4beta1-mediated Src activation is mechanistically distinct from FAK-mediated Src activation during alpha5beta1-mediated NB migration and support the evaluation of inhibitors to alpha4, Src and FAK in the control of NB tumor progression.
authors.affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: L
authors.initials: L
authors.lastname: Wu
authors.forename: J A
authors.initials: JA
authors.lastname: Bernard-Trifilo
authors.forename: Y
authors.initials: Y
authors.lastname: Lim
authors.forename: S-T
authors.initials: ST
authors.lastname: Lim
authors.forename: S K
authors.initials: SK
authors.lastname: Mitra
authors.forename: S
authors.initials: S
authors.lastname: Uryu
authors.forename: M
authors.initials: M
authors.lastname: Chen
authors.forename: C J
authors.initials: CJ
authors.lastname: Pallen
authors.forename: N-Kv
authors.initials: NK
authors.lastname: Cheung
authors.forename: D
authors.initials: D
authors.lastname: Mikolon
authors.forename: A
authors.initials: A
authors.lastname: Mielgo
authors.forename: D G
authors.initials: DG
authors.lastname: Stupack
authors.forename: D D
authors.initials: DD
authors.lastname: Schlaepfer
mesh_terms: D002465:Cell Movement / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D039041:Integrin alpha4beta1 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D039081:Integrin alpha5beta1 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D017434:Protein Structure, Tertiary / Q000502:physiology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology*; D014407:Tumor Cells, Cultured