FUNCTION OF LONG CHAIN BASE KINASES AND THEIR PRODUCTS

鞘磷脂的代谢物鞘氨醇-1磷酸(SPP)被认为是 第二信使调节哺乳动物细胞生长和其他 流程。然而，SPP信号的生理意义是 尚待确定。同样，我们也只有一个初步的认识。 SPP的合成和周转是如何调节的，以及什么信号 它控制的转导通路。开始建立生理学的 长链碱基磷酸盐的功能(S)和对SPP的理解 如何调节它们的合成，我们建议研究LCB磷酸 酿酒酵母。选择技术使我们能够分离出两个 编码LCB激酶的基因LCB4和LCB5，这是第一个基因 要隔离的类型。同源Lcb4和Lcb5蛋白没有亲缘关系 与已知蛋白质中的任何一种相比较，从而揭示了一类新的脂蛋白激酶。这个 Lcb4和Lcb5蛋白分别占LCB的95%和5% 酵母细胞中的激酶活性。大约一半的Lcb4蛋白存在于 细胞膜部分，这是一个谜，因为 预测的蛋白质没有膜结合区。这项建议旨在 确定LCB磷酸合成是如何调节的，以及它们的功能是什么 在LCB磷酸盐中。具体目标1是提纯和表征 Lcb4和Lcb5激酶的酶蛋白。具体目标2是 确定这两种蛋白质是否位于细胞中，以及它们的 位置以受监管的方式变化。具体目标3是定位 这两种蛋白质中的底物结合、催化和蛋白质结构域 本地化。具体目标4是确定哪些细胞过程 需要LCB磷酸盐才能发挥作用。这些研究的结果将 为理解这个无处不在的类的功能奠定了基础 哺乳动物中脂质磷酸盐的研究进展，包括它们在生长发育中的作用 调节、细胞凋亡、心率调节、血小板活化 肥大细胞和轴突回缩。

The sphingolipid metabolite sphingosine-1 phosphate (SPP) is thought to be a second messenger for regulating mammalian cell growth and other processes. However, the physiological significance of SPP signaling has yet to be established. Likewise, we have only a rudimentary understanding of how SPP synthesis and turnover are regulated and what signal transduction pathways it controls. To begin to establish the physiological function(s) of long chain base (LCB) phosphates like SPP and to understand how their synthesis is regulated, we propose to study LCB phosphates in Saccharomyces cerevisiae. Selection techniques enabled us to isolate two genes, LCB4 and LCB5, that encode LCB kinases, the first genes of this type to be isolated. The homologous Lcb4 and Lcb5 proteins are not related to any in known protein, thus revealing a new class of lipid kinase. The Lcb4 and Lcb5 proteins account for 95% and 5%, respectively, of the Lcb kinase activity in yeast cells. About half of the Lcb4 protein is in the membrane fraction of the cell, which presents an enigma because the predicted protein has no membrane-binding domain. This proposal seeks to determine how LCB phosphate synthesis is regulated and what the functions of the LCB phosphates are. Specific Aim 1 is to purify and characterize the enzymatic proteins of the Lcb4 and Lcb5 kinases. Specific Aim 2 is to determine whether the two proteins are located in cells and if their location changes in a regulated manner. Specific Aim 3 is to locate domains in the two proteins for substrate binding, catalysis, and protein localization. Specific Aim 4 is to determine which cellular processes require LCB phosphates for function. The results of these studies will form a foundation for understanding the function of this ubiquitous class of lipid phosphates in mammals, including their proposed roles in growth regulation, apoptosis, regulation of heart rate, activation of platelets and mast cells, and neurite retraction.