FTY720 analogues as sphingosine kinase 1 inhibitors: enzyme inhibition kinetics, allosterism, proteasomal degradation, and actin rearrangement in MCF-7 breast cancer cells

Abstract

Sphingosine kinase 1 (SK1) catalyzes the conversion of sphingosine to the bioactive lipid sphingosine 1-phosphate. We have previously demonstrated that FTY720 and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 activity. Here, we show that (S)-FTY720 vinylphosphonate binds to a putative allosteric site in SK1 contingent on formation of the enzyme-sphingosine complex. We report that SK1 is an oligomeric protein (minimally a dimer) containing noncooperative catalytic sites and that the allosteric site exerts an autoinhibition of the catalytic site. A model is proposed in which (S)-FTY720 vinylphosphonate binding to and stabilization of the allosteric site might enhance the autoinhibitory effect on SK1 activity. Further evidence for the existence of allosteric site(s) in SK1 was demonstrated by data showing that two new FTY720 analogues (a conjugate of sphingosine with a fluorophore and (S)-FTY720 regioisomer) increased SK1 activity, suggesting relief of autoinhibition of SK1 activity. Comparisons with the SK1 inhibitor, SKi or siRNA knockdown of SK1 indicated that (S)-FTY720 vinylphosphonate and FTY720 behave as typical SK1 inhibitors in preventing sphingosine 1-phosphate-stimulated rearrangement of actin in MCF-7 cells. These findings are discussed in relation to the anticancer properties of SK1 inhibitors.

FIGURE 1.

Structures of FTY720 analogues.

FIGURE 2.

Inhibitor kinetic analysis of SK1 in HEK 293 cells. A and B, V versus S nonlinear regression analysis of stably expressed recombinant SK1 in HEK 293 cells for FTY720 (A) and (S)-FTY720 vinylphosphonate (B). C, effect of varied concentrations of (S)-FTY720 vinylphosphonate on stably expressed recombinant SK1 activity in HEK293 cells assayed with 10 μm sphingosine. D, effect of Bdp-So (50 μm final concentration and varied concentrations, right) and (S)-FTY720 regioisomer (50 μm final concentration) on purified SK1 activity assayed with and without 10 μm sphingosine (left). E, V versus S nonlinear regression analysis for the effect of SKi on recombinant SK1 stably expressed in HEK 293 cells. Results are representative of three independent experiments.

FIGURE 3.

SK1 is an oligomer. HEK 293 cells were transiently transfected with myc-tagged G81D SK1, FLAG-tagged D178N SK1, myc-tagged WT SK1, and/or FLAG-tagged WT SK1 plasmid constructs. A and B, Western blots (WB) of anti-FLAG immunoprecipitates (IP) (A) or anti-myc immunoprecipitates (B) probed with respective anti-myc and anti-FLAG antibodies. Lysate SK1 represents lysates of cells overexpressing both myc- and FLAG-tagged WT SK1. C, Western blot and SK1 activity of HEK 293 cell lysates showing the effect of overexpressing FLAG-tagged D178N SK1 on myc-tagged WT SK1 activity (using micrograms of plasmid constructs shown). FLAG-tagged D178N SK1-myc-tagged G81D SK1 oligomers were catalytically deficient. Results are representative of three experiments. SK1 activity was also measured in immunoprecipitates: anti-myc immunoprecipitates: myc-tagged G81D SK1-transfected cells, 0.23 pmol/min; myc-tagged G81D SK1/FLAG-tagged D178N SK1-transfected cells, 1.65 pmol/min; myc-tagged WT SK1-transfected cells, 33 pmol/min. Anti-FLAG immunoprecipitates: FLAG-tagged D178N SK1-transfected cells, 2.8 pmol/min; FLAG-tagged D178N SK1/myc-tagged G81D SK-transfected cells, 2 pmol/min; FLAG-tagged WT SK1-transfected cells, 20.55 pmol/min. Results are representative of two or three experiments. D, effect of (S)-FTY720 vinylphosphonate or Bdp-So (both at 25 and 50 μm and assayed using 10 μm sphingosine) on WT and G113A SK1 (both transiently over-expressed in HEK293 cells) activity. Results are expressed as % of SK1 activity in the absence of (S)-FTY720 vinylphosphonate or Bdp-So (control = 100% SK1 activity, n = 3 experiments).

FIGURE 4.

Proteasomal degradation of SK1. MCF-7 Neo or MCF-7 HER2 cells were treated with SKi or FTY720, or (S)-FTY720 vinylphosphonate ((S)-vinyl Pn) (all at 10 μm final concentration) for 24 h. MCF-7 HER2 cells were also pretreated with MG132 (10 μm) for 30 min prior to addition of SK1 inhibitors. Western blots were probed with anti-SK1, anti-ERK-2, and anti-actin antibodies. Results are representative of three independent experiments. Bar graph shows quantification of the effect of the SK1 inhibitors on the proteasomal degradation of SK1 in MCF-7 Neo or MCF-7 HER2 cells by calculating the SK1:actin ratio for each treatment (p < 0.05 for control versus SK1 inhibitor-treated cells, n = 3 experiments).

FIGURE 5.

Actin rearrangement in MCF-7 cells. MCF-7 Neo cells were treated with (A) SKi or (S)-FTY720 vinylphosphonate ((S)-vinyl Pn) or (B) FTY720 (both at 10 μm final concentration) for 15 min prior to stimulation with and without S1P (1 μm, 5 min). Actin was detected using phalloidin red staining. Arrows in the panel for S1P treatment identify actin localized to lamellipodia/membrane ruffles, whereas arrows in the other panels identify actin clustered into focal adhesions. Nuclei were stained with DAPI. Results are representative of three independent experiments.

FIGURE 6.

Schematic showing the on or off allosterism model for interaction of SK1 with sphingosine (So), (S)-FTY720 vinylphosphonate ((S)-vinyl Pn), or Bdp-So.