Filamin A links sphingosine kinase 1 and S1P1 at the leading edge.

ALVAREZ SE; MACEYCKA M; SPIEGEL S

Tucson, AZ USA

Conferencia; FASEB Summer Research Conference. Lysophospholipid mediators in Health and Disease.; 2007

Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to produce sphingosine-1-phosphate (S1P), a potent lipid mediator that regulates diverse biological processes including cell motility, angiogenesis and cell growth. S1P exerts many of its effects through five differentially expressed G protein coupled receptors (S1PRs), named S1P1-5, but it also can act intracellularly. Alhough the regulatory mechanisms governing SphK1 activation have not been fully characterized, many stimuli activate SphK1, which in turn plays a critical role in cell motility. Indeed, some chemoattractants require SphK1-mediated transactivation of S1PRs to induce motility. We identified filamin A (FlnA), an actin crosslinking protein involved in motility, as an SphK1-interacting protein by a yeast two-hybrid screen. Importantly, we demonstrated that endogenous SphK1 co-immunoprecipitates endogenous FlnA. To determine the function(s) of this interaction, we used established matched melanoma cell lines, the FlnA-deficient M2 cells and FlnA-reconstituted subline A7. Modified Boyden chamber cell migration assays demonstrated that FlnA is required for cell migration as only FlnA-containing A7 cells migrated towards the growth factor heregulin (Hrg) and serum. Hrg also stimulated SphK1 activity in a FlnA-dependent manner. Moreover, Hrg induced the translocation and colocalization of SphK1 and FlnA at membrane ruffles. Additionally, siRNA specific for SphK1 inhibited Hrg-induced cell migration, demonstrating that both SphK1 and FlnA are required for motility. Intriguingly, it has been reported that the substrate for SphK1, sphingosine, can induce the activation of the pro-motility Rac-effector PAK, which phosphorylates and activates FlnA. However, SphK1-specific siRNA blocked Hrg- and sphingosine-induced activation of PAK and phosphorylation of FlnA. Indeed, SphK1-specific siRNA also blocked membrane ruffling in response to Hrg and sphingosine, suggesting that the SphK1 product, S1P, not its precursor sphingosine, promotes motility. Consistent with this notion, Hrg induced colocalization to membrane ruffles of FlnA, SphK1, and S1P1, a pro-motility S1P receptor, but not S1P2, which inhibits motility. A requirement for S1P1 was further suggested by the finding that the S1P1 antagonist VPC 23019 inhibited Hrg- and sphingosine-induced membrane ruffling. Together, our data suggest that FlnA physically links SphK1 and S1P1 at the plasma membrane to promote cell motility.  Work supported by Kirchstien-NRSA 5T32 CA085159-04 (MM) and NCI R01CA61774 (SS).