Mouse sphingosine kinase isoforms SPHK1a and SPHK1b differ in enzymatic traits including stability, localization, modification, and oligomerization
Mouse sphingosine kinase isoforms SPHK1a and SPHK1b differ in enzymatic traits including stability, localization, modification, and oligomerization
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DOI：
10.1074/jbc.m510308200
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发表时间：
2006-02-17
影响因子：
4.8
通讯作者：
Igarashi, Y
中科院分区：
文献类型：
--
作者：
Kihara, A;Anada, Y;Igarashi, Y
鞘氨醇激酶催化生物活性脂质分子鞘氨醇1-磷酸的产生。小鼠有两种鞘氨醇激酶1型异构体SPHK1a和SPHK1b。除了先前报道的酶活性差异外，我们还发现这些同工异构体在几个酶特性上存在差异。首先，SPHK1b是不稳定的，而SPHK1a是高度稳定的。SPHK1b的降解发生在膜上，并被蛋白酶体抑制剂抑制。其次，只有SPHK1b在SDS-PAGE上表现出异常迁移，这可能与SPHK1b抗sds结构有关。第三，当降解受到抑制时，SPHK1a和SPHK1b分别主要存在于可溶性组分和膜组分中。第四，只有SPHK1b在其独特的Cys残基（Cys-4和Cys-5）上被脂质修饰。这些Cys残基的定点突变导致鞘氨酸激酶活性增加，表明这种修饰对该酶具有抑制作用。最后，SPHK1b倾向于形成同质低聚物，而大多数SPHK1a以单体形式存在。我们还确定了SPHK1b的脂质修饰参与了其同质寡聚化。因此，尽管这两种蛋白质仅在少数n端氨基酸残基上有所不同，但它们的酶特性却截然不同。
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.