INTERACTION OF FILAMIN WITH SPHINGOSINE KINASE1 REGULATES CELL MIGRATION

ALVAREZ SE; MACEYCKA M; MILSTIEN S; SPIEGEL S

Richmond, VA USA

Congreso; Massey Cancer Center 2004 Research Retreat.; 2004

Sphingosine kinase (SphK) is the key enzyme that catalyzes the phosphorylation of sphingosine to produce sphingosine-1-phosphate (S1P), a pluripotent lipid mediator that regulates diverse biological processes including cell motility, angiogenesis and growth. S1P exerts many of its functions through binding of GPCRs but it also can act intracellularly. Though the regulatory mechanisms governing SphK activation have not been fully characterized, many chemoattractants stimulate SphK1, which in turns, plays a critical role in motility. We have identified filamin A (Fln A), a cytoskeletal protein, as an SphK1-interacting protein by a yeast two-hybrid screen. Importantly, we demonstrated that endogenous SphK1 interacts with endogenous Fln A. To examine the functionality of this interaction, we used established matched melanoma cell lines, one lacking Fln A (M2) and one with Fln A (A7). Heregulin (Hrg) stimulated SphK1 activity and induced translocation of SphK1 to membrane ruffles where it co-localized with Fln A only in A7 cells. Moreover,  downregulation of SphK1 with specific siRNA markedly reduced filamin-dependant migration towards Hrg but not serum. Phosphorylation of Fln A by p21 activated kinase 1 (PAK1) is essential for motility, and we found that while both Hrg and sphingosine (Sph) induced PAK activation, siRNA against SphK1 blocked only Sph-induced activation. Similarly, we demonstrated that SphK1 is required for Sph- but not Hrg-induced phosphorylation of Fln A. Our results suggest that a tri-directional regulatory interaction between Fln A, PAK1, and SphK1 is important not only for translocation of SphK1 to its site of action in membrane ruffles but also for local stimulation of PAK1 and consequently Fln A phosphorylation to influence the dynamics of actin cytoskeletal structures and cell motility. These studies were supported by NIH grant CA61774 to S.S. and Kirchstien-NRSA to M.M.