Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-Phosphate

Sphingosine l-phosphate (SPP), a bioactive sphingolipid metabolite, inhibit s chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 huma n breast cancer cell line. As in many other cell types, SPP stimulated prol iferation 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 Ma trigel, and only high concentrations of SPP partially inhibited matrix meta lloproteinase-a activation induced by Con A. However, SPP at a concentratio n that strongly inhibited invasiveness also markedly reduced chemotactic mo tility, To investigate the molecular mechanisms by which SPP interferes wit h cell motility, we examined tyrosine phosphorylation of focal adhesion kin ase (FAK) and paxillin, which are important for organization of focal adhes ions and cell motility. SPP rapidly increased tyrosine phosphorylation of F AK 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 in crease in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F3 97) abolished the inhibitory effect of SPP on cell motility. In contrast, t he 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. (C) 1999 Academic Press.