Characterization of the Sch9 Longevity Pathway in Yeast

描述(由申请人提供)：包括酿酒酵母在内的模式真核生物正在成为识别调节寿命和衰老的基因以及确定蛋白质在这些过程中作用的分子机制的关键元素。酿酒酵母Sch9蛋白激酶在时序寿命中发挥作用，但它是如何发挥作用的尚不清楚，因为上游激活物和下游底物仍然未知。我们最近发现了第一个Sch9底物Lsp1，它是Pkc1-MAP激酶级联的负调控因子。在具体目标1中，我们将使用LSP1磷酸肽序列(S)来寻找其他可能在时间上发挥作用的底物。我们还将确定我们在体外发现被纯化的Sch9磷酸化的两种蛋白质的身份。最后，我们将使用生化方法来寻找其他Sch9底物。我们的其他新发现是鞘磷脂长链碱基(LCB)在体外激活Sch9激酶活性，随着细胞从对数阶段进入静止期，LCB水平增加100倍以上。基于这些结果，我们试图在特定的目标2中确定LCB是否调节体内的Sch9活性，从而控制时序寿命。在具体目标3中，我们将确定Lsp1是否在按时间顺序排列的寿命中发挥作用，以及它是否调节Sch9的活性。对于目标1中确定的其他Sch9底物也将进行同样的操作。最后，在特定的目标4中，我们将确定Lsp1和相关蛋白Pil1是否在复制寿命中发挥作用。Pil1也是Pkc1-MAP激酶级联的负调节因子，终止于SLT2蛋白激酶。SLT2最近被证明通过Sir3的磷酸化来控制复制寿命，Sir3是一种已知的寿命调节因子。这些研究的结果将确立鞘磷脂在寿命调节中的作用，为Sch9控制时序寿命提供第一个机械性的见解，并为理解Sch9如何调控寿命提供新的线索。由于Sch9是人类Akt/PKB蛋白激酶的同源物，我们的结果可能确定Akts/PKB在人类衰老和长寿中的特定作用。

DESCRIPTION (provided by applicant): Model eucaryotes including Saccharomyces cerevisiae are emerging as key elements for identifying genes that modulate longevity and aging and for determining molecular mechanisms of protein action in these processes. The S. cerevisiae Sch9 protein kinase plays a role in chronological life span but how it does so is unclear, since upstream activators and downstream substrates remain unknown. We recently identified the first Sch9 substrate, Lsp1, a negative regulator of the Pkc1-MAP kinase cascade. In Specific Aim 1 we will use the Lsp1 phosphopeptide sequence(s) to search for other substrates that may play roles in chronological life span. We will also determine the identity of two proteins that we have found to be phosphorylated in vitro by purified Sch9. Finally, we will use biochemical approaches to search for other Sch9 substrates. Our other novel discoveries are that sphingolipid long chain bases (LCBs) activate Sch9 kinase activity in vitro and that LCB levels rise over 100-fold as cells pass from log to stationary phase. Based upon these results we seek to determine in Specific Aim 2 if LCBs regulate Sch9 activity in vivo thereby controlling chronological life span. In Specific Aim 3 we will determine if Lsp1 plays a role in chronological life span and whether or not it regulates the activity of Sch9. The same will be done for other Sch9 substrates identified in Aim 1. Finally, in Specific Aim 4 we will determine if Lsp1 and a related protein Pil1, also a negative regulator of the Pkc1-MAP kinase cascade that terminates with the Slt2 protein kinase, play a role in replicative life span. Slt2 has recently been shown to control replicative life span via phosphorylation of Sir3, a known regulator of life span. The results of these studies will establish a role for sphingolipids in life span regulation, provide the first mechanistic insight into the control of chronological life span by Sch9 and generate new leads for understanding how Sch9 governs life span. Since Sch9 is a homolog of human Akt/PKB protein kinases, our results may identify specific roles for Akts/PKBs in human aging and longevity.