Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate.
Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397-transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC, 20007, USA.
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Wang
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Nohara
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Olivera
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Thompson
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Spiegel
D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology*; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002633:Chemotaxis / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004338:Drug Combinations; D004789:Enzyme Activation / Q000187:drug effects; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D018093:Gelatinases / Q000037:antagonists & inhibitors; D006801:Humans; D007797:Laminin / Q000378:metabolism; D008246:Lysophospholipids; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011509:Proteoglycans / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000502:physiology; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D004338:Drug Combinations; D007797:Laminin; D008246:Lysophospholipids; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011509:Proteoglycans; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; C059485:matrigel; C060506:sphingosine 1-phosphate; D014443:Tyrosine; D003094:Collagen; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D013110:Sphingosine