Plasma Membrane Targeting of the Yck2 Casein Kinase 1 Isoform

Yck2 酪蛋白激酶 1 同工型的质膜靶向

• 批准号: 9974459
• 负责人: Lucy Robinson
• 金额: $ 39万
• 依托单位: LSU Health Sciences Center -Shreveport
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9974459&HistoricalAwards=false
• 关键词: Plasma; Membrane; Targeting; Yck2; Casein

The Yck1 and Yck2 protein kinases are together essential for viability in Saccharomyces cerevisiae. The two proteins are members of an abundant protein kinase family that is highly conserved in eukaryotic cells, whose properties have been well-studied but whose functions are not yet clear in most organisms. The plasma membrane-associated Yck protein kinases are required for cellular morphogenesis and cytokinesis in yeast, and are enriched at specific sites within the membrane during the cell cycle. Studies with a green fluorescent protein fusion to Yck2p allowed determination that the route of delivery of Yck2p to the plasma membrane requires a functioning secretory pathway. This was unexpected because the Yck proteins carry a C-terminal sequence necessary for membrane localization that could direct modification by a prenyltransferase that heretofore was only known to prenylate members of the Rab family of ras-like GTPases. This research is directed toward understanding the targeting mechanisms of the Yck2 protein by determining the cis-acting elements on Yck2p and the trans-acting factors that are required for modification and localization. Also, the question of whether prenylation has functional consequences, either to the targeting mechanism or to the protein's interaction with targets, will be tested. Finally, evidence that the Yck2 kinase cycles on and off the plasma membrane in association with vesicular structures will be explored, to characterize the apparent cycling and to determine whether this is an integral part of the essential function of Yck in yeast. The results of these studies will be important not only in understanding the localization mechanism of an essential protein kinase, but will address important questions in the area of protein prenylation -- especially the question of whether prenylation can determine targeting of these proteins to particular membranes and the functional consequences of this modification. The project addresses a fundamental question in cell biology, namely, how do specific proteins, that need to be located in specific places inside the cell in order to perform their function, get to where they need to be? The protein under investigation in this project, Yck, is a protein kinase, a type of protein that functions in intracellular signal transduction events by activating or inactivating other specific proteins by phosphorylating them. The project will explore the role of protein prenylation in subcellular localization of proteins. The biochemistry of prenylation (an important post-translational modification of a protein that results from the covalent addition of a specific fatty acyl group to the protein; such a modification allows a protein, that would otherwise not interact with membranes, to interact with membranes) is reasonably well understood. However, the specific cell biological consequences of prenylation are not so well understood. The study will be done in a model organism, the yeast Saccharomyces cerevisiae, that historically has richly provided cell biologists with fundamental information about cellular processes that are applicable to a broad range of other types of organisms, including plants and mammals.

Yck 1和Yck 2蛋白激酶共同对酿酒酵母中的活力至关重要。这两种蛋白是一个丰富的蛋白激酶家族的成员，在真核细胞中高度保守，其性质已被充分研究，但其功能在大多数生物体中尚不清楚。质膜相关的Yck蛋白激酶是酵母细胞形态发生和胞质分裂所必需的，并且在细胞周期期间在膜内的特定位点富集。用绿色荧光蛋白融合到Yck 2 p的研究允许确定Yck 2 p递送到质膜的途径需要功能分泌途径。这是出乎意料的，因为Yck蛋白携带膜定位所必需的C-末端序列，其可以通过异戊二烯基转移酶指导修饰，迄今为止仅已知异戊二烯基转移酶对ras样GTP酶的Rab家族的成员进行异戊二烯基化。本研究旨在通过确定Yck 2 p上的顺式作用元件和修饰和定位所需的反式作用因子来了解Yck 2蛋白的靶向机制。此外，异戊烯化是否具有功能性后果的问题，无论是靶向机制或蛋白质与目标的相互作用，将进行测试。最后，证据表明，YCK 2激酶周期上和关闭质膜与囊泡结构将被探索，表征明显的循环，并确定这是否是一个不可分割的一部分，YCK在酵母中的基本功能。这些研究的结果将是重要的，不仅在了解一个必要的蛋白激酶的定位机制，但将解决重要的问题，在该地区的蛋白质异戊烯化-特别是异戊烯化是否可以确定这些蛋白质的靶向特定的膜和这种修饰的功能后果的问题。该项目解决了细胞生物学中的一个基本问题，即需要位于细胞内特定位置以执行其功能的特定蛋白质如何到达它们需要的位置？ 该项目中研究的蛋白质Yck是一种蛋白激酶，是一种通过磷酸化激活或灭活其他特定蛋白质而在细胞内信号转导事件中发挥作用的蛋白质。 该项目将探索蛋白质异戊二烯化在蛋白质亚细胞定位中的作用。 异戊烯化的生物化学（一种重要的蛋白质翻译后修饰，由特定脂肪酰基与蛋白质的共价加成产生;这种修饰允许蛋白质，否则不会与膜相互作用，与膜相互作用）是合理理解的。 然而，异戊烯化的具体细胞生物学后果还没有得到很好的理解。 这项研究将在一种模式生物--酿酒酵母中进行，该酵母在历史上为细胞生物学家提供了丰富的细胞过程的基本信息，这些信息适用于广泛的其他类型的生物体，包括植物和哺乳动物。