Calcium Influx and Signaling in Yeast Stimulated by Intracellular Sphingosine 1-Phosphate Accumulation

Christine J Birchwood,Julie D Saba,Robert C Dickson,Kyle W Cunningham

doi:10.1074/jbc.m010221200

Christine J Birchwood, Julie D Saba + Show 2 more

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https://doi.org/10.1074/jbc.m010221200

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Abstract

In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways. The budding yeast Saccharomyces cerevisiae expresses enzymes that can synthesize and degrade S1P and related molecules, but their possible role in calcium signaling has not yet been tested. Here we examine the effects of S1P accumulation on calcium signaling using a variety of yeast mutants. Treatment of yeast cells with exogenous sphingosine stimulated Ca(2+) accumulation through two distinct pathways. The first pathway required the Cch1p and Mid1p subunits of a Ca(2+) influx channel, depended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P phosphatase (Lcb3p). The biologically inactive stereoisomer of sphingosine did not activate this Ca(2+) influx pathway, suggesting that the active S1P isomer specifically stimulates a calcium-signaling mechanism in yeast. The second Ca(2+) influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and therefore was likely to be nonspecific. Mutants lacking both S1P lyase and phosphatase (dpl1 lcb3 double mutants) exhibited constitutively high Ca(2+) accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases. These results show that endogenous S1P-related molecules can also trigger Ca(2+) accumulation and signaling. Several stimuli previously shown to evoke calcium signaling in wild-type cells were examined in lcb4 lcb5 double mutants. All of the stimuli produced calcium signals independent of sphingosine kinase activity, suggesting that phosphorylated sphingoid bases might serve as messengers of calcium signaling in yeast during an unknown cellular response.

Highlights

In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways
Sphingosine 1-Phosphate Stimulates Calcium Influx and Signaling in Yeast—Exogenous sphingosine added to culture medium can be taken up by yeast cells and phosphorylated to S1P by sphingosine kinases [13]
To determine whether S1P accumulation can evoke calcium signaling in yeast, we first monitored the accumulation of 45Ca2ϩ from the medium into growing yeast cells treated with a wide range of exogenous sphingosine

Summary

Introduction

Intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways. The second Ca2؉ influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and was likely to be nonspecific Mutants lacking both S1P lyase and phosphatase (dpl lcb double mutants) exhibited constitutively high Ca2؉ accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases. These results show that endogenous S1P-related molecules can trigger Ca2؉ accumulation and signaling. MATa leu112 ura his trp rme MATa leu112 ura his trp rme dpl1ϻLEU2 MATa leu112 ura his trp rme lcb3ϻLEU2 MATa leu112 ura his trp rme lcb3ϻLEU2 dpl1ϻleu2ϻTRP1 MAT␣ leu112 ura his trp rme dpl1ϻLEU2 lcb4ϻG418R lcb5ϻG418R MATa leu112 ura his trp rme lcb4ϻG418R lcb5ϻG418R MATa leu112 ura his trp rme dpl1ϻLEU2 cch1ϻTRP1 MAT␣ leu112 ura his trp rme dpl1ϻLEU2 cch1ϻTRP1 lcb4ϻG418R lcb5ϻG418R MAT␣ leu112 ura his trp rme pmr1ϻLEU2 MAT␣ leu112 ura his trp rme pmr1ϻLEU2 lcb4ϻG418R lcb5ϻG418R MATa leu112 ura his trp rme pgm2ϻLEU2 MATa leu112 ura his trp rme pgm2ϻLEU2 lcb4ϻG418R lcb5ϻG418R MATa leu112 ura his trp rme dpl1ϻTRP1 lcb3ϻLEU2 lcb4ϻG418R lcb5ϻG418R MATa leu112 ura his trp rme TPS2-lacZϻURA3 MATa leu112 ura his trp rme lcb3ϻLEU2 TPS2-lacZϻURA3 MATa leu112 ura his trp rme dpl1ϻLEU2 TPS2-lacZϻURA3 MATa leu112 ura his trp rme dpl1ϻleu2ϻTRP1 lcb3ϻLEU2 TPS2-lacZϻURA3

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