SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism.
The potent sphingolipid metabolite sphingosine 1-phosphate is produced by phosphorylation of sphingosine catalyzed by sphingosine kinase (SphK) types 1 and 2. In contrast to pro-survival SphK1, the putative BH3-only protein SphK2 inhibits cell growth and enhances apoptosis. Here we show that SphK2 catalytic activity also contributes to its ability to induce apoptosis. Overexpressed SphK2 also increased cytosolic free calcium induced by serum starvation. Transfer of calcium to mitochondria was required for SphK2-induced apoptosis, as cell death and cytochrome c release was abrogated by inhibition of the mitochondrial Ca(2+) transporter. Serum starvation increased the proportion of SphK2 in the endoplasmic reticulum and targeting SphK1 to the endoplasmic reticulum converted it from anti-apoptotic to pro-apoptotic. Overexpression of SphK2 increased incorporation of [(3)H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide, whereas SphK1 decreased it. Electrospray ionizationmass spectrometry/mass spectrometry also revealed an opposite effect on ceramide mass levels. Importantly, specific down-regulation of SphK2 reduced conversion of sphingosine to ceramide in the recycling pathway and conversely, down-regulation of SphK1 increased it. Our results demonstrate that SphK1 and SphK2 have opposing roles in the regulation of ceramide biosynthesis and suggest that the location of sphingosine 1-phosphate production dictates its functions.
Maceyka
Michael
M
Department of Biochemistry, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, USA.
Sankala
Heidi
H
Hait
Nitai C
NC
Le Stunff
Hervé
H
Liu
Hong
H
Toman
Rachelle
R
Collier
Claiborne
C
Zhang
Min
M
Satin
Leslie S
LS
Merrill
Alfred H
AH
Milstien
Sheldon
S
Spiegel
Sarah
S
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes; D007668:Kidney / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D010168:Palmitates / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013107:Sphingolipids / Q000378:metabolism*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000502:physiology; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000502:physiology; D000099157:Sphingosine Kinase
D002118:Calcium; D002518:Ceramides; D045304:Cytochromes c; D007527:Isoenzymes; D010168:Palmitates; D017853:Phosphotransferases (Alcohol Group Acceptor); D013107:Sphingolipids; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D000099157:Sphingosine Kinase