A new model for regulation of sphingosine kinase 1 translocation to the plasma membrane in breast cancer cells

Ryan D.R Brown,Ben E.P Veerman,Jeongah Oh,Rothwelle J Tate,Federico Torta,Margaret R Cunningham,David R Adams,Susan Pyne,Nigel J Pyne

doi:10.1016/j.jbc.2021.100674

Abstract

The translocation of sphingosine kinase 1 (SK1) to the plasma membrane (PM) is crucial in promoting oncogenesis. We have previously proposed that SK1 exists as both a monomer and dimer in equilibrium, although it is unclear whether these species translocate to the PM via the same or different mechanisms. We therefore investigated the structural determinants involved to better understand how translocation might potentially be targeted for therapeutic intervention. We report here that monomeric WT mouse SK1 (GFP-mSK1) translocates to the PM of MCF-7L cells stimulated with carbachol or phorbol 12-myristate 13-acetate, whereas the dimer translocates to the PM in response to sphingosine-1-phosphate; thus, the equilibrium between the monomer and dimer is sensitive to cellular stimulus. In addition, carbachol and phorbol 12-myristate 13-acetate induced translocation of monomeric GFP-mSK1 to lamellipodia, whereas sphingosine-1-phosphate induced translocation of dimeric GFP-mSK1 to filopodia, suggesting that SK1 regulates different cell biological processes dependent on dimerization. GFP-mSK1 mutants designed to modulate dimerization confirmed this difference in localization. Regulation by the C-terminal tail of SK1 was investigated using GFP-mSK1 truncations. Removal of the last five amino acids (PPEEP) prevented translocation of the enzyme to the PM, whereas removal of the last ten amino acids restored translocation. This suggests that the penultimate five amino acids (SRRGP) function as a translocation brake, which can be released by sequestration of the PPEEP sequence. We propose that these determinants alter the arrangement of N-terminal and C-terminal domains in SK1, leading to unique surfaces that promote differential translocation to the PM.

Highlights

Exists as both a monomer and dimer in equilibrium, receptors (S1P1-S1P5), on cells or can act on it is unclear whether these species intracellular targets, such as histone deacetylase 1/2, translocate to the plasma membrane (PM) via the same or different to induce cellular responses [1]
We investigated the for sphingosine 1-phosphate (S1P) in cancer is evident from studies showing structural determinants involved to better understand that high expression of sphingosine kinase 1 (SK1) and S1P receptors in Journal Pre-proof how translocation might potentially be targeted for therapeutic intervention
We report here that monomeric wild type (WT) mouse SK1 (GFPmSK1) translocates to the PM of MCF-7L cells stimulated with carbachol or phorbol myristate acetate (PMA), whereas the dimer translocates to the PM in response to sphingosine 1-phosphate (S1P); the equilibrium between monomer and dimer is sensitive to cellular stimulus

Summary

Introduction

Sphingosine 1-phosphate (S1P) is a bioactive lipid transformation, as evidenced by data showing that a that is formed by the phosphorylation of sphingosine neutralising anti-TFR1 antibody blocks oncogenesis by sphingosine kinase, of which there are two [10]. These findings suggest that the loss of one of the two symmetry-related salt bridges involving K49 at the modelled mSK1 dimerisation interface (Fig. 6B) is sufficient to substantially weaken dimerization but not to completely ablate formation of a WT Myc-tagged mSK1/K49E GFP-mSK1 heterodimer The loss of both salt bridges and introduction of greater charge opposition is likely to weaken affinity of the protomers for each other yet further, such that homodimer formation with the K49E GFPmSK1 mutant would be even more severely compromised, resulting in a species that is essentially monomeric when expressed in MCF-7L cells and which we show here to be localised exclusively to lamellipodia in response to carbachol or PMA challenge (Fig. 7A-C). T2-T5 mutants lacking 10-25 amino acids all Characterization of translocation properties for translocated to the PM in response carbachol, S1P or

PMA and these translocations were inhibited by

Mat e ri als

Pearson correlation coefficients

Findings

Fi g u r es