Intracellular localization of sphingosine kinase 1 alters access to substrate pools but does not affect the degradative fate of sphingosine-1-phosphate

Deanna L Siow,Charles D Anderson,Evgeny V Berdyshev,Anastasia Skobeleva,Stuart M Pitson,Binks W Wattenberg

doi:10.1194/jlr.m004374

Deanna L Siow, Charles D Anderson + Show 4 more

Open Access

https://doi.org/10.1194/jlr.m004374

Copy DOI

Abstract

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.

Highlights

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid
Targeting SK1 to the plasma membrane (PM) (Lck and localized SK1 construct (Lck)-SA) drastically reduced the magnitude of increase seen in the steady-state levels of dihydroS1P (Fig. 3A). We confirmed that this was not due to differences in the intrinsic ability of these constructs to utilize sphingosine and dihydrosphingosine as substrates. These results suggest that intracellular pools of dihydrosphingosine are not accessible to SK1 that is localized to the PM, and they could point to an unexplored consequence of the PM translocation of SK1 that has been shown to follow agonist stimulation
We show that cytosolic and endoplasmic reticulum (ER)-targeted SK1 are able to draw dihydrosphingosine from the sphingolipid biosynthetic pathway to produce dihydrosphingosine-1-phosphate, confirming earlier results for the cytosolic enzyme [42]. This result fits well with a proposed housekeeping role for SK1, whereby basal SK1 activity acts to provide a means for sphingoid bases to be removed from the sphingolipid metabolic network

Summary

Introduction

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. Sphingosine-1-phosphate (S1P) has emerged as a potent bioactive lipid with established roles in essential cellular processes such as vascular maturity, cell proliferation, trafficking of immune cells, cytoskeletal rearrangement, invasion, and angiogenesis [1,2,3]. Key to such diversity of functions lies in the ability of S1P to act as both an intracellular effector molecule and an extracellular ligand for a family of five G protein-coupled receptors, S1PR1-5 [4,5,6,7].

Methods

Results

Conclusion