POWRE: H-Ras C-Terminus and Its Role in Signaling

这是一项power资助研究H-Ras，一种小的GTPase，通过改变细胞周期机制，改变基因表达和重组肌动蛋白细胞骨架，激活控制细胞生长和形状的几个信号转导级联。HRas蛋白与多种靶蛋白相互作用，但目前尚不清楚如何对HRas激活产生特定的反应。HRas蛋白必须附着（系住）在细胞的质膜上，以便在信号传递过程中有效地与任何与其合作的蛋白相互作用。脂质附着于HRas蛋白的c端产生栓系，HRas有两种脂质附着于半胱氨酸残基-类异戊二烯法尼酯和棕榈酸酯脂肪酸。最近的证据表明，这些脂质也可能影响HRas的功能。这个研究者用6个赖氨酸残基取代了C端的法尼基化位点。这种名为Ext61L的蛋白在pc12细胞中具有增强的分化能力，并能增加磷脂酰肌醇(PI) -3激酶。本研究旨在确定通过c端突变激活Ras功能的分子基础。第一个目的是通过直接测量PI3-K的活性，以及在完整的PC12细胞中使用PI3-K途径的抑制剂，确定ExtRas中特定的c端变化是否在物理上或功能上改变了ExtRas与PI3K之间的相互作用。第二个目标是确定能够支持棕榈酰化和膜结合的最小c端结构；并确定可以增强细胞HRas分化功能的残基的位置和类型。新的ExtRas c端突变体，棕榈酰化位点数量减少，带电残基分布改变，将被构建、表达，并监测其导致PC12细胞神经元分化能力的丧失。该项目的长期目标是了解HRas蛋白如何选择它们的伴侣，以及这些选择对HRas功能的影响。

This is a POWRE grant looking at H-Ras, a small GTPase, which activates several signal transduction cascades that control cell growth and shape, through alterations of the cell cycle machinery, changes in gene expression and reorganization of the actin cytoskeleton. HRas proteins interact with multiple target proteins, but at present it is unclear how the particular responses to HRas activation can arise. HRas proteins must be attached (tethered ) to the cell's plasma membrane in order to productively interact with whatever proteins are their partners in signaling. Tethering results from attachment of lipids to the C-terminus of HRas proteins HRas has 2 lipids attached to cysteine residues - the isoprenoid farnesyl and palmitate fatty acids. Recent evidence suggests that these lipids may also affect the function of HRas. This investigator has replaced the farnesylation site at the C terminus with 6 lysine residues. This protein, Ext61L, has an enhanced ability to cause differentiation in PC 12 cells and increases phosphatidylinositol (PI) -3 kinase. This research is aimed at determining the molecular basis for this activation of Ras function by C-terminal mutation. The first aim is to determine if the particular C-terminal changes in ExtRas have physically or functionally altered its interaction between ExtRas and PI3K, by directly measuring the activity of PI3-K, and by using inhibitors of the PI3-K pathway in intact PC12 cells. The second aim is to to define the minimal C-terminal structure that can support palmitoylation and membrane binding; and identify the positions and types of residues that can potentiate the differentiative function of cellular HRas. New C-terminal mutants of ExtRas, with reduced numbers of sites for palmitoylation and an altered distribution of charged residues will be constructed, expressed, and monitored for loss of the ability to cause neuronal differentiation of PC12 cells. The long-term goal of this project is to understand how HRas proteins select their partners, and the impact of those choices on HRas function.