Megidish T, Takio K, Titani K, Iwabuchi K, Hamaguchi A, Igarashi Y, Hakomori S
Pacific Northwest Research Institute, Seattle, Washington 98122, USA.
Biochemistry. 1999 Mar 16;38(11):3369-78. doi: 10.1021/bi982548c.
Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N'-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al. (1995) Biochem. Biophys. Res. Commun. 216, 739-747), have been termed "sphingosine-dependent kinases" (SDKs). We showed previously that a purified SDK (termed "SDK1") phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms beta, eta, and zeta but not tau or sigma (Megidish, T., et al. (1998) J. Biol. Chem. 273, 21834-45). In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. (ii) The activation of these SDKs is specific to D-erythro-Sph and its N-methyl derivatives, the effect of L-threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. An SDK separated as fractions "TN31-33" phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed "SDK2". Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin); another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. An SDK activity separated and present in "fraction 42" from Q-Sepharose chromatography specifically phosphorylated GRP105 (or GRP94) and HSP68 but did not phosphorylate PDI or 14-3-3. This SDK is clearly different from other SDKs in its substrate specificity and is tentatively termed "SDK3". Interestingly, substrates of all these SDKs so far identified are molecular chaperones or adapters capable of binding to enzymes and key molecules involved in signal transduction, maintaining tertiary structure of bioactive molecules, or maintaining cellular homeostasis in response to environmental stress. Thus, the essential role of Sph and DMS is to activate molecular chaperones, thereby providing a link to the mechanism by which SDK activity regulates cellular homeostasis and signal transduction.
其活性仅在存在鞘氨醇（Sph）或N，N'-二甲基鞘氨醇（DMS）时可检测到，而在测试的其他15种鞘脂、磷脂和甘油脂存在时则无法检测到的蛋白激酶（Megidish，T.等人（1995年）《生物化学与生物物理研究通讯》216，739 - 747），已被称为“鞘氨醇依赖性激酶”（SDKs）。我们之前表明，一种纯化的SDK（称为“SDK1”）可磷酸化衔接蛋白/伴侣蛋白14 - 3 - 3同工型β、η和ζ的特定丝氨酸位点，但不能磷酸化tau或sigma（Megidish，T.等人（1998年）《生物化学杂志》273，21834 - 45）。在本研究中我们发现如下：（i）具有不同底物特异性的其他SDKs存在于小鼠Balb/c 3T3（A31）成纤维细胞的胞质和膜提取物中。（ii）这些SDKs的激活对D - 赤藓糖鞘氨醇及其N - 甲基衍生物具有特异性，L - 苏阿糖鞘氨醇或其N - 甲基衍生物的作用最小，而非特异性阳离子两亲物则完全没有作用。作为“TN31 - 33”组分分离出的一种SDK磷酸化了一种50 kDa的底物，该底物被鉴定为钙网蛋白，以及两种分子量分别为58 kDa和55 kDa的内源性底物，两者均被鉴定为蛋白二硫键异构酶（PDI）。这种特异性磷酸化钙网蛋白和PDI的SDK，这两种分子伴侣在内质网中含量很高，暂称为“SDK2”。另一种SDK活性与葡萄糖调节蛋白（GRP）和热休克蛋白（HSP）共纯化。一种GRP底物具有与GRP94相同的氨基酸序列（同义词：内质蛋白）；另一种HSP底物具有与小鼠HSP86或HSP84相同的氨基酸序列，它们是人类HSP90的类似物。从Q - 琼脂糖层析中分离并存在于“42号组分”中的一种SDK活性特异性磷酸化GRP105（或GRP94）和HSP68，但不磷酸化PDI或14 - 3 - 3。这种SDK在底物特异性方面明显不同于其他SDKs，暂称为“SDK3”。有趣的是，到目前为止鉴定出的所有这些SDKs的底物都是分子伴侣或衔接蛋白，它们能够与参与信号转导的酶和关键分子结合，维持生物活性分子的三级结构，或在应对环境应激时维持细胞内稳态。因此，Sph和DMS的重要作用是激活分子伴侣，从而为SDK活性调节细胞内稳态和信号转导的机制提供了一个联系。
