Ras protein signaling and the control of cell growth

描述(申请人提供)：对真核细胞的类G0休眠状态的彻底描述对于全面了解控制细胞增殖的机制是必不可少的。为此，我们一直在研究Ras/cAMP依赖的蛋白激酶(PKA)信号通路如何调控酿酒酵母中一种特定的休眠状态-稳定期的生物学。有趣的是，我们已经发现，RAS/PKA信号活性调节自噬，这是一种高度保守的降解途径，是静止细胞生存所必需的。自噬对多细胞生物体的正常发育很重要，最近的研究将这一过程与衰老、细胞程序性死亡、癌症、亨廷顿氏病和细胞对病原体的反应联系在一起。我们最近的数据表明，这个RAS/PKA通路直接针对自噬的关键调节因子--ATg1蛋白激酶。有趣的是，其他工作表明，Atg1还受到另外两条信号通路的调控，这两条通路在控制真核细胞生长方面发挥着核心作用。后两条途径涉及高度保守的Tor和AMP激活的蛋白激酶(Snf1)。这里的实验将研究Ras/PKA、Tor和Snf1通路如何共同作用来调节Atg1的活性，从而诱导真核细胞中的自噬。此外，我们将使用自噬和其他活动作为读数，试图更好地理解这三个信号通路的活动是如何协调的，以实现对细胞生长的适当控制。由于自噬机制是高度保守的，这里确定的调控范例很可能将被用于其他真核生物。因此，这些研究可以为这一过程如何在人类身上以临床有用的方式进行操纵提供重要的见解。这一建议的具体目的是：1)确定PKA磷酸化调节Atg1蛋白激酶复合体活性的机制；2)研究在自噬和细胞生长控制过程中，Ras/PKA、Tor和Snf1信号通路之间的相互作用。

DESCRIPTION (provided by applicant): A thorough description of the G0-like resting states of eukaryotic cells is essential for a complete understanding of the mechanisms controlling cell proliferation. Towards this end, we have been studying how the Ras/cAMP-dependent protein kinase (PKA) signaling pathway regulates the biology of one particular resting state, stationary phase in Saccharomyces cerevisiae. Interestingly, we have found that Ras/PKA signaling activity regulates autophagy, a highly conserved, degradative pathway required for resting cell survival. Autophagy is important for normal development in multicellular organisms and recent studies have linked this process to aging, programmed cell death, cancer, Huntington'.s disease and the cellular response to pathogens. Our recent data indicate that this Ras/PKA pathway directly targets a key regulator of autophagy, the Atg1 protein kinase. Interestingly, other work has shown that Atg1 is also regulated by two additional signaling pathways that have a central role in the control of eukaryotic cell growth. These latter two pathways involve the highly conserved Tor and AMP-activated (Snf1) protein kinases. The experiments here will examine how the Ras/PKA, Tor and Snf1 pathways work together to regulate Atg1 activity, and thus the induction of autophagy in eukaryotic cells. In addition, we will use autophagy, and other activities, as readouts in an attempt to develop a better understanding of how the activities of these three signaling pathways are coordinated to bring about the proper control of cell growth. Since the autophagy machinery is highly conserved, it is likely that the regulatory paradigms identified here will be used in other eukaryotes. Thus, these studies could provide important insights into how this process might be manipulated in clinically useful ways in humans. The Specific Aims of this proposal are: 1) to determine the mechanism by which PKA phosphorylation regulates the activity of the Atg1 protein kinase complex; and 2) to examine the interplay between the Ras/PKA, Tor and Snf1 signaling pathways during the control of autophagy and cell growth.