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

这是一项研究H-RAS的POWRE拨款，H-RAS是一种小的GTP酶，它激活几个信号转导级联，通过改变细胞周期机制、基因表达的变化和肌动蛋白细胞骨架的重组来控制细胞的生长和形状。HRAS蛋白与多个靶蛋白相互作用，但目前尚不清楚对HRAS激活的特殊反应是如何产生的。HRAS蛋白必须附着(系)在细胞的质膜上，才能与它们在信号传递中的伙伴蛋白有效地相互作用。HRAS蛋白C-末端与脂类结合的结果是，HRAS有两种脂结合在半胱氨酸残基上--类异戊二烯的法尼基和棕榈酸酯脂肪酸。最近的证据表明，这些脂质也可能影响HRA的功能。这位研究者用6个赖氨酸残基取代了C末端的法尼化位点。这种名为Ext61L的蛋白质具有增强的诱导PC 12细胞分化的能力，并增加了磷脂酰肌醇(PI)-3激酶。本研究旨在确定这种通过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，并在完整的PC12细胞中使用PI3-K途径的抑制剂，来确定附加蛋白中特定的C末端变化是否在物理上或功能上改变了附加蛋白与PI3K之间的相互作用。第二个目的是确定能够支持棕榈酰化和膜结合的最小C-末端结构，并确定能够增强细胞HRA分化功能的残基的位置和类型。将构建、表达和监测新的附加蛋白的C末端突变体，其棕榈酰化位点数量减少，带电残基的分布发生变化，从而丧失导致PC12细胞神经分化的能力。这个项目的长期目标是了解HRAS蛋白如何选择他们的伴侣，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.