The C terminus of FLNa interacts with SphK1. (A) Diagram of full-length FLNa, with gray boxes representing immunoglobulin-like repeats. Filled box, FLNa dimerization domain; ABD, actin binding domain; H1 and H2, hinge regions 1 and 2. Bars indicate regions of FLNa that have been found to bind SphK1, calcium sensing receptor (CaR), β-arrestin (β-Arr), dopamine receptor 2 and 3 (DopR), human μ opioid receptor (hMOP), and PAK1. (B) The Ct-FLNa (aa 2380 to 2647), identified as a SphK1 interactor in a yeast two-hybrid screen, was cloned into pcDNA3.1-HA and transcribed and translated in vitro in the presence of l-[4,5-³H]leucine. Equal portions of labeled proteins were then incubated with GST or GST-SphK1, pulled down with glutathione-Sepharose beads, washed, resolved by SDS-PAGE, and autoradiographed. (C) HEK 293 cells were transfected with vector, V5-His-SphK1, or with Ct-FLNa or with both, as indicated. After 48 h, cells were lysed, and proteins were resolved by SDS-PAGE, transblotted, and probed with anti-V5 and anti-HA for SphK1 and Ct-FLNa, respectively (upper panel). Equal amounts of cell lysates were also incubated with Ni-NTA-agarose to pull down SphK1, and bound proteins were examined by immunoblotting with V5 (middle panel) or HA antibodies (lower panel). Note that Ct-FLNa was pulled down only in the presence of V5-His-SphK1. (D) Equal amounts of lysates from HEK 293 cells transfected with vector or V5-SphK1 were incubated with Ni-NTA-agarose to pull down SphK1, and bound proteins were resolved by SDS-PAGE and blotted with anti-FLNa for endogenous FLNa (upper panel) or anti-V5 for SphK1 (lower panel). (E) Lysates from naïve HEK 293 cells were incubated with preimmune serum or anti-SphK1 antibody. Immune complexes were isolated with protein A/G agarose, washed, resolved by SDS-PAGE, and immunoblotted with anti-FLNa antibodies.

SphK1 is required for Hrg-induced cell migration. (A and B) M2 and A7 cells were transfected with control siRNA (siControl) or siRNA targeted to SphK1 (siSphK1) as indicated. (A) Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted with anti-SphK1 antibody. Blots were reprobed for tubulin to ensure equal loading and transfer. SphK1 activity was determined in lysates from A7 cells (bottom panel). (B) Cells were placed into a modified Boyden chamber and allowed to migrate toward serum-free medium (vehicle), Hrg (30 ng/ml), or 10% serum for 3 h as indicated. Data are expressed as the number of migrating cells per field. Similar results were obtained for three independent experiments. *, P < 0.001. (C) A7 cells were transfected with ON-TARGETplus SMARTpool siRNA targeted to SphK1 (filled bars) or control siRNA 2 (open bars), and cells were allowed to migrate toward Hrg (10 ng/ml) for 3 h. Data are expressed as the number of migrating cells per field. *, P < 0.001. (D) S1P (1 nM) was added to either the bottom chamber or to both the top and bottom chambers, and the migration of A7 cells was determined. *, P < 0.001; **, P < 0.05. (E) M2 and A7 cells were transfected with vector or with Ct-FLNa, and chemotaxis toward serum-free medium (vehicle) or Hrg (30 ng/ml) was measured in a modified Boyden chamber. Data are expressed as the number of migrating cells per field and are representative of three independent experiments.

SphK1 is required for Hrg- and sphingosine-induced phosphorylation of FLNa and PAK. A7 cells were transfected with control siRNA (siControl) or siRNA targeted to SphK1 (siSphK1), starved overnight, and then stimulated with Hrg (30 ng/ml), sphingosine (5 μM), or S1P (100 nM) for the indicated times. Equal amounts of lysates were resolved by SDS-PAGE and immunoblotted with the phospho-specific antibodies p-PAK1/2 (Thr432/Thr402) (A to C) or p-Ser2152 FLNa (D to F). Blots were reprobed for total PAK or FLNa to ensure equal loading and transfer. The upper band in panel B is PAK2 phosphorylated at Thr402. Densitometry was used to determine the change in stimulation of the samples compared to that of the untreated siControl, and values are expressed as the means ± standard errors of the means from three independent experiments.

Colocalization of SphK1 and S1P₁, but not S1P₂, induced by Hrg at lamellipodia. A7 cells grown on glass coverslips were transfected with V5-SphK1 and green fluorescent protein (GFP)-S1P₁ (A) or GFP-S1P₂ (B). After 48 h, the cells were serum starved overnight and then treated with vehicle or Hrg (30 ng/ml) for 30 min. Cells were then fixed; stained for SphK1 (red), FLNa (blue), and either S1P₁ or S1P₂ (green); and visualized by confocal microscopy. Bar, 10 μm. (C) Fields in panels A and B were scored for the number of nontransfected (untrans) and neighboring transfected cells (trans) exhibiting actin-positive lamellipodia, and the lamellipodia index was expressed as the mean ± standard error of the mean (SEM). *, P < 0.01. Data are representative of at least three independent experiments. (D) A7 cells were serum starved; pretreated with vehicle, VPC 23019 (10 μM), or JTE013 (1 μM) for 30 min; and then exposed to vehicle, Hrg (30 ng/ml), or sphingosine (5 μM) for an additional 30 min. Cells were fixed, stained for actin, and visualized by fluorescent microscopy. The percentage of lamellipodia-positive cells was calculated, and the lamellipodia index was expressed as the mean ± SEM. *, P < 0.01. Data are representative of at least three independent experiments.

Hrg induces colocalization of SphK1 and FLNa to lamellipodia. FLNa-expressing A7 (A) and FLNa-deficient M2 (B) cells were transfected with vector, V5-SphK1, or FLNa, as indicated. After 48 h, cells were serum starved overnight and then treated with vehicle or Hrg (30 ng/ml) for 30 min; fixed; stained with Alexa 488-labeled phalloidin for F-actin (green), polyclonal antibody against V5 to visualize SphK1 (red), or monoclonal antibody against FLNa (blue); and visualized by confocal microscopy. White areas indicate colocalization of SphK1, FLNa, and actin. Arrows indicate colocalization of actin, FLNa, and SphK1 in lamellipodia, defined as membrane ruffles at the edge of the cell, visualized by DIC that also stain strongly for actin. Bar, 10 μm. Results are representative of at least four independent experiments. (C) The percentage of lamellipodia-positive cells was calculated, and the lamellipodia index was expressed as the mean ± standard error of the mean (SEM). *, P < 0.005. A minimum of 300 cells was scored in a double-blind manner. (D) The percent of cells in which SphK1 and FLNa were colocalized to lamellipodia is expressed as the mean ± SEM. *, P < 0.005. (E) FLNa is essential for the activation of SphK1 by Hrg. M2 and A7 cells were serum starved overnight and then treated with Hrg (30 ng/ml). At the indicated times, cells were washed, lysed, and assayed for SphK1 activity. (F) Hrg stimulates ERK1/2 in both M2 and A7 cells. M2 and A7 cells were treated with 30 ng/ml Hrg for the indicated times. Equal amounts of cell lysates were immunoblotted with anti-phospho-ERK1/2 antibodies. Anti-ERK2 was used as a loading control.

SphK1 is required for Hrg- and sphingosine-induced lamellipodia formation. A7 cells were transfected with control siRNA (siControl) or siRNA targeted to SphK1 (siSphK1) as indicated, serum-starved overnight, and then stimulated with vehicle, Hrg (30 ng/ml), or sphingosine (Sph; 5 μM) for 30 min. Cells were then fixed, stained for actin (green) and FLNa (red), and visualized by confocal microscopy. (A) Yellow areas in merged images indicate colocalization of FLNa and actin. Arrows indicate regions of colocalization of FLNa and actin in lamellipodia. Bar, 10 μm. (B) The percentage of lamellipodia-positive cells was calculated, and the lamellipodia index is expressed as the mean ± standard error of the mean. *, P < 0.05; **, P < 0.01. Data are representative of at least three independent experiments.

S1P stimulates in vitro PAK1 activity. (A) Recombinant PAK1 was incubated with vehicle, sphingosine (2 μM), or S1P (2 μM) in the presence of ATP and MBP as substrates. Reactions were stopped at the indicated times, and phosphorylation of MBP was determined by immunoblotting with anti-phospho-threonine. Blots were stripped and reprobed with anti-PAK1 as a loading control. (B) Recombinant PAK1 activity was measured in duplicate samples with [γ-³²P]ATP in the absence or presence of vehicle, sphingosine, S1P, or 1 μg of GTPγS-loaded recombinant Cdc42 for 30 min. ³²P-labeled MBP and autophosphorylated PAK1 were determined with a phosphorimager after the proteins were resolved by SDS-PAGE and transblotted to nitrocellulose. Data are expressed as the change in stimulation of the vehicle compared to that of the untreated controls and are expressed as the means ± standard deviations from four independent experiments. *, P < 0.005.

Proposed roles for the concerted actions of SphK1, S1P, and S1P₁ with FLNa and PAK1 at lamellipodia to promote motility. We propose that the activation of growth factor receptors induces the FLNa-dependent recruitment of SphK1 and PAK1 to nascent lamellipodia and also initiates the signaling pathways leading to their phosphorylation. Activated SphK1 produces S1P that can further enhance PAK1 phosphorylation by direct stimulation. In addition, S1P can also act in an autocrine manner to activate S1P₁, and this autocrine loop potentiates motility as G_i-coupled S1P₁ can activate Rac, which in turn activates PAK1. The FLNa-dependent localization of S1P₁, but not S1P₂, to lamellipodia further amplifies motility signaling and averts the locally released S1P from inhibiting cell migration through S1P₂ (see Discussion for more details). Note that other components associated with FLNa that are relevant to actin filament remodeling are not shown.