Distinct FAK-Src activation events promote alpha5beta1 and alpha4beta1 integrin-stimulated neuroblastoma cell motility.
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.
Wu
L
L
Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA.
Bernard-Trifilo
J A
JA
Lim
Y
Y
Lim
S-T
ST
Mitra
S K
SK
Uryu
S
S
Chen
M
M
Pallen
C J
CJ
Cheung
N-Kv
NK
Mikolon
D
D
Mielgo
A
A
Stupack
D G
DG
Schlaepfer
D D
DD
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