Filamin A Links Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 1 at Lamellipodia To Orchestrate Cell Migration

Abstract

Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to produce the potent lipid mediator sphingosine-1-phosphate (S1P), which plays a critical role in cell motility via its cell surface receptors. Here, we have identified filamin A (FLNa), an actin-cross-linking protein involved in cell movement, as a bona fide SphK1-interacting protein. Heregulin stimulated SphK1 activity only in FLNa-expressing A7 melanoma cells but not in FLNa-deficient cells and induced its translocation and colocalization with FLNa at lamellipodia. SphK1 was required for heregulin-induced migration, lamellipodia formation, activation of PAK1, and subsequent FLNa phosphorylation. S1P directly stimulated PAK1 kinase, suggesting that it may be a target of intracellularly generated S1P. Heregulin also induced colocalization of S1P₁ (promotility S1P receptor) but not S1P₂, with SphK1 and FLNa at membrane ruffles. Moreover, an S1P₁ antagonist inhibited the lamellipodia formation induced by heregulin. Hence, FLNa links SphK1 and S1P₁ to locally influence the dynamics of actin cytoskeletal structures by orchestrating the concerted actions of the triumvirate of SphK1, FLNa, and PAK1, each of which requires and/or regulates the actions of the others, at lamellipodia to promote cell movement.

ACKNOWLEDGMENTS

We thank Y. Ohta and T. Stossel (Brigham and Women's Hospital) for supplying the M2 and A7 cell lines and filamin constructs. We also thank G. Bokoch (Scripps Research Institute) for the generous gift of recombinant PAK1 and Cdc42 and advice on in vitro PAK assays, and we thank Renyan Liu for technical assistance.

This work was supported by NCI grants R01CA61774 (to S.S.) and 5T32 CA085159-04 (to M.M.) and the NIMH Intramural Research Program (S.M.). Confocal microscopy was supported in part by NIH grant P30 CA16059 to the Massey Cancer Center.