FUNCTION OF LONG CHAIN BASE KINASES AND THEIR PRODUCTS

鞘脂代谢物鞘氨醇-1磷酸（SPP）被认为是 用于调节哺乳动物细胞生长的第二信使和其他 流程.然而，SPP信号传导的生理学意义具有 尚未确定。同样地，我们也只有一个初步的了解， SPP的合成和周转是如何调节的， 它所控制的传导途径。为了开始建立生理 长链碱基（LCB）磷酸盐（如SPP）的功能，并了解 它们的合成是如何调节的，我们建议研究LCB磷酸盐， 酿酒酵母选择技术使我们能够分离出两个 基因，LCB 4和LCB 5，编码LCB激酶，这是第一个基因， 要隔离的类型。同源Lcb 4和Lcb 5蛋白不相关 与任何已知蛋白质相比，从而揭示了一类新的脂质激酶。的 Lcb 4和Lcb 5蛋白分别占Lcb的95%和5 在酵母细胞中的激酶活性。大约一半的Lcb 4蛋白质位于 细胞的膜部分，这是一个谜，因为 预测的蛋白质没有膜结合结构域。这项建议旨在 确定LCB磷酸合成是如何调节的， 的LCB磷酸盐。具体目标1是纯化和表征 Lcb 4和Lcb 5激酶的酶蛋白。具体目标二是 确定这两种蛋白质是否位于细胞中， 以规范的方式改变位置。具体目标3是定位 两种蛋白质中用于底物结合、催化和蛋白质 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 哺乳动物中的脂磷酸盐，包括它们在生长中的作用 调节，细胞凋亡，心率调节，血小板活化 肥大细胞和神经突收缩

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.