Kobayashi Scott D, Nagiec Marek M
Pharmacia Corporation, Kalamazoo, Michigan 49001, USA.
Eukaryot Cell. 2003 Apr;2(2):284-94. doi: 10.1128/EC.2.2.284-294.2003.
Sphingolipid precursors, namely, ceramide and long-chain base phosphates (LCBPs), are important growth regulators with often opposite effects on mammalian cells. A set of enzymes that regulate the levels of these precursors, referred to as a ceramide/LCBP rheostat, is conserved in all eukaryotes. In order to gain further insight into the function of the rheostat in Saccharomyces cerevisiae, we searched for mutants that are synthetically lethal with a deletion of the LCB3 gene encoding LCBP phosphatase. In addition to acquiring expected mutants lacking the LCBP lyase, the screen revealed elo3 (sur4) mutants that were defective in fatty acid elongation and cka2 mutants lacking the alpha' subunit of the protein kinase CK2 (casein kinase). Both mutations affected the in vivo activity of the acyl coenzyme A (acyl-CoA)-dependent and fumonisin B(1)-sensitive ceramide synthase (CS). The Elo3 protein is necessary for synthesis of C(26)-CoA, which in wild-type yeast is a source of C(26) fatty acyls found in the ceramide moieties of all sphingolipids. In the in vitro assay, CS had a strong preference for acyl-CoAs containing longer acyl chains. This finding suggests that a block in the formation of C(26)-CoA in yeast may cause a reduction in the conversion of LCBs into ceramides and lead to an overaccumulation of LCBPs that is lethal in strains lacking the Lcb3 phosphatase. In fact, elo3 mutants were found to accumulate high levels of LCBs and LCBPs. The cka2 mutants, on the other hand, exhibited only 25 to 30% of the in vitro CS activity found in wild-type membranes, indicating that the alpha' subunit of CK2 kinase is necessary for full activation of CS. The cka2 mutants also accumulated high levels of LCBs and had elevated levels of LCBPs. In addition, both the elo3 and cka2 mutants showed increased sensitivity to the CS inhibitors australifungin and fumonisin B(1). Together, our data demonstrate that the levels of LCBPs in yeast are regulated by the rate of ceramide synthesis, which depends on CK2 kinase activity and is also strongly affected by the supply of C(26)-CoA. This is the first evidence indicating the involvement of protein kinase in the regulation of de novo sphingolipid synthesis in any organism.
鞘脂前体，即神经酰胺和长链碱基磷酸盐（LCBPs），是重要的生长调节因子，对哺乳动物细胞往往具有相反的作用。一组调节这些前体水平的酶，被称为神经酰胺/LCBP变阻器，在所有真核生物中都是保守的。为了进一步深入了解酿酒酵母中变阻器的功能，我们筛选了与编码LCBP磷酸酶的LCB3基因缺失合成致死的突变体。除了获得预期的缺乏LCBP裂解酶的突变体外，筛选还发现了脂肪酸延长缺陷的elo3（sur4）突变体和缺乏蛋白激酶CK2（酪蛋白激酶）α'亚基的cka2突变体。这两种突变都影响了酰基辅酶A（acyl-CoA）依赖性和伏马菌素B1敏感的神经酰胺合酶（CS）的体内活性。Elo3蛋白是合成C(26)-CoA所必需的，在野生型酵母中，C(26)-CoA是所有鞘脂神经酰胺部分中C(26)脂肪酰基的来源。在体外试验中，CS对含有较长酰基链的酰基辅酶A有强烈偏好。这一发现表明，酵母中C(26)-CoA形成的阻断可能导致LCB向神经酰胺转化的减少，并导致LCBPs的过度积累，这在缺乏Lcb3磷酸酶的菌株中是致命的。事实上，发现elo3突变体积累了高水平的LCB和LCBP。另一方面，cka2突变体在体外CS活性方面仅表现出野生型膜中活性的25%至30%，这表明CK2激酶的α'亚基是CS完全激活所必需的。cka2突变体也积累了高水平的LCB，并且LCBP水平升高。此外，elo3和cka2突变体对CS抑制剂澳大利亚菌素和伏马菌素B1均表现出增加的敏感性。总之，我们的数据表明，酵母中LCBP的水平受神经酰胺合成速率的调节，神经酰胺合成速率取决于CK2激酶活性，并且也受到C(26)-CoA供应的强烈影响。这是首次有证据表明蛋白激酶参与了任何生物体中从头合成鞘脂的调节。
