CAAX Processing and Cell Signaling in Dictyostelium

描述（由申请人提供）：健康和患病细胞的中心问题是细胞如何感知来自外部的信号，以及这些信号如何用于组织细胞反应。小的GT3家族Ras和Rho密切参与这些过程，并且它们中的突变通常导致疾病状态。因此，多年来已经知道Ras中的激活突变在癌细胞中是常见的，并且广泛认为Ras突变对于哺乳动物肿瘤的发生是必需的。大多数小GTP酶在其C末端携带CAAX氨基酸基序，其中C是半胱氨酸，AA通常是两个脂肪族氨基酸，X指定蛋白质是否将被15碳法尼基脂质或20碳香叶基香叶基脂质修饰。Ras在细胞中的功能需要CAAX序列，并且对于活化的Ras是致瘤的，CAAX序列必须通过异戊烯化途径修饰。因此，该途径是抗癌药物治疗的潜在靶点。最近的药物开发工作已经验证了这一假设，目前有几种有前途的新抑制剂正在临床试验中。关于异戊烯化途径，还有很多东西有待了解：哪些小G蛋白必须被甲基化才能被调节，甲基化水平如何被细胞外和细胞内信号控制，异戊二烯化途径中的三个伴侣如何被调节，当各种异戊二烯化步骤减少或不存在时，细胞形态和对信号的响应的后果，以及甲基化在G-偶联配体反应的最早阶段中所起的作用，是本提案中提出的一些悬而未决的问题。本研究的目的是使用网骨藻作为模型系统来理解这些重要的问题。初步实验表明，甲基化基因的缺失阻断了早期信号传导和对cAMP的反应，但不阻断细胞生长。各种信号蛋白在这种缺失中被错误定位，趋化性和cAMP反应被破坏，细胞发育成连贯组织的能力被阻断。这些观察结果将使用将细胞行为与分子变化相关联的测定法，并通过开发一组异戊二烯化途径中每一步缺陷的突变体来扩展。

DESCRIPTION (provided by applicant): A central issue in healthy and diseased cells is how cells sense signals from the outside, and how these signals are used to organize cellular responses. The small GTPase families Ras and Rho are intimately involved in these processes, and mutations in them often lead to the disease state. Thus it has been known for many years that activating mutations in Ras are common in cancer cells, and it is widely believed that Ras mutations are essential for the genesis of mammalian tumors. Most small GTPases carry a CAAX amino acid motif at their C terminus, where C is cysteine, AA are usually two aliphatic amino acids, and X specifies whether the protein will be modified with the 15 carbon farnesyl lipid or the 20 carbon geranylgeranyl lipid. Ras function in the cell requires the CAAX sequence, and for activated Ras to be tumorigenic, the CAAX sequence must be modified by a prenylation pathway. For this reason the pathway is a potential target for anticancer drug therapy. Recent drug development efforts have validated this assumption, with several promising new inhibitors of the pathway now in clinical trials. Much remains to be known about many the prenylation pathway: which of the small G proteins must be methylated for them to be modulated, how methylation levels are controlled by extra- and intracellular signals, how the three partners in the prenylation pathway are regulated if they are, the consequences for cell morphology and response to signals when various of the prenylation steps are reduced or absent, and the role that methylation plays in the very earliest steps of G-coupled response to ligand, are some of the outstanding questions addressed in this proposal. The aim of this research is to use Dictyostelium as a model system to understand these important questions. Preliminary experiments reveal that deletions in the methylation gene block early signaling and response to cAMP, but not cell growth. Various signaling proteins are mislocalized in this deletion, chemotaxis and cAMP responses are disrupted, and the ability of cells to develop into a coherent tissue is blocked. These observations will be extended using assays that will correlate cellular behavior with molecular changes, and by developing a panel of mutants deficient in each step in the prenylation pathway.