Analysis of Phosphorylation of Elongation Factor 1
伸长因子 1 磷酸化分析
基本信息
- 批准号:9316900
- 负责人:
- 金额:$ 25.9万
- 依托单位:
- 依托单位国家:美国
- 项目类别:Continuing Grant
- 财政年份:1994
- 资助国家:美国
- 起止时间:1994-02-01 至 1998-01-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
9316900 Traugh A. Specific Objectives of the Proposed Work Elongation factor 1 (EF-1) is found in multiple forms in the cytosol as EF-1, EF-1.valyl-tRNA synthetase, and EF-1a. In rabbit reticulocytes, all of the valyl-tRNA synthetase is associated with about 15% of the EF-1. Recently, all four subunits of EF-1 have been shown to the phosphorylated. The and subunits are phosphorylated b y casein kinase II in vitro and in reticulocytes and Artemia salina (1-4). EF-1 , , , and are phosphorylated by protein kinase C in reticulocytes treated with phorbol ester and in vitro (1-5). The subunit is phosphorylated in Xenopus oocytes and in vitro by the cell division control kinases p34cdc2 (4,6). The subunit has also been shown to be modified by methylation and addition of glycerolphosphorylethanolamine (7). Phosphorylation of EF-1 by protein kinase C, both in vivo and in vitro, stimulates the rate of elongation by 2-3 fold (1,5). In these studies, we propose to analyze, at the molecular level, the two complexes of EF-1 with regard to structure, function, and regulation. The role of phosphorylation by protein kinase C and casein kinase II on complex formation and individual subunits of EF-1 expressed in E. coli. The latter will enable us to determine the effects of site-specific mutations of the individual phosphorylation sites on GDP/GTP exchange activity and on the rate of elongation. The following specific aims will be carried out. 1. Analyze the effects of phosphorylation of EF-1 valyl-tRNA synthetase an EF-1 by protein kinase C and casein kinase II on elongation activity and on GDP/GTP exchange activity. 2. The , , and subunits of EF-1 from rabbit have been cloned and expressed in E. coli. Site specific mutations of the individual phosphorylation sites for protein kinase C and casein kinase II sites will be prepared. 3. EF-1 will be reconstituted from the subunits expressed in E. coli and the effects of the site-specific mutations on activity will be analyzed. %%% Elongation factor 1 (EF-1) is one of two proteins factors involved in the elongation phase of protein synthesis. EF-1 consists of four subunits. EF-1 forms a ternary complex with GTP and aminoacyl- tRNA and correctly positions the tRNA on the ribosome. GTP is the energy source for this step and is cleaved to GDP and phosphate; the GDP remains bound to EF-1 and must be exchanged for GTP by EF- 1 for EF-1 to function again. All four subunits of EF-1 have been shown to be phosphorylated. Three different protein kinases have been shown to phosphorylate EF-1; casein kinase II, protein kinase C, and the cell division control kinase p34cdc2. The and subunits by protein kinase C and the subunit by the cell division control kinase. In these studies, we propose to examine phosphorylation of EF-1 by casein kinase II and protein kinase C. The effects of phosphorylation on elongation activity and on GDP/GTP exchange activity will be examined. The subunits of EF-1 from rabbit have been cloned and expressed in E. coli. These subunits will be reconstituted and the effects of phosphorylation on individual subunits will be determined. Site specific mutations of the individual phosphorylation sites will be prepared. Subunits containing these mutations will be reconstituted and analyzed and the effects of the site-specific mutations on activity will be determined. *** .
工作延伸因子1 (EF-1)在细胞质中以多种形式存在,如EF-1、EF-1。谷氨酸trna合成酶和EF-1a。在兔网织红细胞中,所有谷氨酸trna合成酶与约15%的EF-1相关。最近,EF-1的四个亚基都被证明是磷酸化的。酪蛋白激酶II在体外、网织红细胞和盐蒿中磷酸化和亚基(1-4)。EF-1 , , , 和磷酸化的蛋白激酶C网织红细胞体外接受佛波醇酯和(1 - 5)。该亚基在爪蟾卵母细胞和体外通过细胞分裂控制激酶p34cdc2磷酸化(4,6)。该亚基也被证明可以通过甲基化和添加甘油磷酸乙醇胺来修饰(7)。蛋白激酶C对EF-1的磷酸化,无论在体内还是体外,都能刺激2-3倍的伸长率(1,5)。在这些研究中,我们建议在分子水平上分析EF-1的两个复合物的结构、功能和调控。蛋白激酶C和酪蛋白激酶II磷酸化对大肠杆菌中EF-1复合物形成和单个亚基表达的作用。后者将使我们能够确定单个磷酸化位点的位点特异性突变对GDP/GTP交换活性和延伸率的影响。具体目标如下:1. 分析蛋白激酶C和酪蛋白激酶II磷酸化EF-1谷氨酸trna合成酶和EF-1对延长活性和GDP/GTP交换活性的影响。2. 从家兔中克隆了EF-1的、和亚基,并在大肠杆菌中表达。将制备蛋白激酶C和酪蛋白激酶II位点的个别磷酸化位点的位点特异性突变。3. EF-1将从大肠杆菌中表达的亚基重组,并分析位点特异性突变对活性的影响。%%%伸长因子1 (EF-1)是参与蛋白质合成的伸长期的两个蛋白质因子之一。EF-1由四个亚基组成。EF-1与GTP和氨基酰基tRNA形成三元配合物,并将tRNA正确定位在核糖体上。GTP是这一步骤的能量来源,与GDP和磷酸盐结合;GDP仍然与EF-1绑定,必须由EF-1交换GTP,以使EF-1再次发挥作用。EF-1的所有四个亚基都被证明是磷酸化的。三种不同的蛋白激酶已被证明磷酸化EF-1;酪蛋白激酶II、蛋白激酶C和细胞分裂控制激酶p34cdc2。蛋白激酶C的和亚基和细胞分裂的亚基控制激酶。在这些研究中,我们建议检测酪蛋白激酶II和蛋白激酶c对EF-1的磷酸化作用,并研究磷酸化对延伸活性和GDP/GTP交换活性的影响。已克隆家兔EF-1亚基并在大肠杆菌中表达。这些亚基将被重组,磷酸化对单个亚基的影响将被确定。将制备单个磷酸化位点的位点特异性突变。包含这些突变的亚基将被重组和分析,并确定位点特异性突变对活性的影响。*** .
项目成果
期刊论文数量(0)
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Jolinda Traugh其他文献
Jolinda Traugh的其他文献
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{{ truncateString('Jolinda Traugh', 18)}}的其他基金
Long & Medium Term Research: Cyclin Destruction in Meiotic and Early Mitotic Cell Cycles of Xenopus Laevis
长的
- 批准号:
9007777 - 财政年份:1990
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$ 25.9万 - 项目类别:
Standard Grant
Immuno-Biochemical Studies of E. Coli Recbc Enzyme
大肠杆菌 Recbc 酶的免疫生化研究
- 批准号:
7922987 - 财政年份:1980
- 资助金额:
$ 25.9万 - 项目类别:
Continuing Grant
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