Abstract
Sphingosine kinases catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate. Mice have two isoforms of sphingosine kinase type 1, SPHK1a and SPHK1b. In addition to the previously reported difference in their enzyme activities, we have found that these isoforms differ in several enzymatic characteristics. First, SPHK1b is unstable, whereas SPHK1a is highly stable. Degradation of SPHK1b occurs at the membrane and is inhibited by a proteasome inhibitor. Second, only SPHK1b exhibits abnormal mobility on SDS-PAGE, probably due to its SDS-resistant structure. Third, SPHK1a and SPHK1b are predominantly detected in the soluble and membrane fractions, respectively, when their degradation is inhibited. Fourth, only SPHK1b is modified with lipid, on its unique Cys residues (Cys-4 and Cys-5). Site-directed mutagenesis at these Cys residues resulted in increased sphingosine kinase activity, suggesting that the modification is inhibitory to the enzyme. Finally, SPHK1b tends to form homo-oligomers, whereas most SPHK1a is presented as monomers. We have also determined that the lipid modification of SPHK1b is involved in its homo-oligomerization. Thus, although these two proteins differ only in a few N-terminal amino acid residues, their enzymatic traits are extremely different.
Highlights
Sphingosine kinases catalyze the production of S1P from sphingosine and are responsible for the phosphorylation of FTY720 [11, 12]
In the present study we have revealed that two isoforms of SPHK1, SPHK1a and SPHK1b, possess completely different molecular characteristics such as mobility on SDS-PAGE, stability, membrane localization, and oligomerization
Both SPHK1s can form oligomers, SPHK1b exhibits a much higher tendency to oligomerize. This is the first report describing the oligomerization of sphingosine kinases, homo-oligomerization of a related lipid kinase, diacylglycerol kinase, has been reported [43]
Summary
Sphingosine kinases catalyze the production of S1P from sphingosine and are responsible for the phosphorylation of FTY720 [11, 12]. Two mammalian sphingosine kinases are known, SPHK1, which was identified by purification of the enzyme and subsequent sequence determination [13], and SPHK2, which was cloned based on its homology to SPHK1 [16]. Both sphingosine kinases are expressed ubiquitously among tissues, their tissue-specific patterns differ [16]. We found that SPHK1b differs from SPHK1a in several enzymatic characteristics These include structural resistance toward SDS, membrane localization, protein stability, post-translational modification, and oligomer formation. The N terminus plays an important role in the determination of the enzymatic properties of SPHK1
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