Analysis of Phosphorylation of Elongation Factor 1

9316900 Traugh A. 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、缬氨酰-tRNA合成酶和EF-1a。 在兔网织红细胞中，所有的缬氨酰-tRNA合成酶都与约15%的EF-1相关。 最近，EF-1的所有四个亚基都被磷酸化。 的 和 在体外以及在网织红细胞和卤虫中，亚基被酪蛋白激酶II磷酸化（1-4）。 在用佛波酯处理的网织红细胞中和体外，EF-1、、和被蛋白激酶C磷酸化（1-5）。 的 亚基在非洲爪蟾卵母细胞中和体外被细胞分裂控制激酶p34 cdc 2磷酸化（4，6）。 的 亚基也被甲基化和添加甘油磷酸乙醇胺修饰（7）。 在体内和体外，蛋白激酶C对EF-1的磷酸化可刺激2-3倍的延长速率（1，5）。 在这些研究中，我们建议在分子水平上分析EF-1的两种复合物的结构、功能和调节。 蛋白激酶C和酪蛋白激酶II的磷酸化对EF-1复合物形成和单个亚基在E.杆菌 后者将使我们能够确定个别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。 将实现以下具体目标。 1. 分析EF-1缬氨酰-tRNA合成酶和EF-1被蛋白激酶C和酪蛋白激酶II磷酸化对延伸活性和GDP/GTP交换活性的影响。 2. The，and，and 克隆了兔EF-1亚基基因，并在E.杆菌 将制备蛋白激酶C和酪蛋白激酶II位点的单个磷酸化位点的位点特异性突变。 3. EF-1将由E. 大肠杆菌中，并分析位点特异性突变对活性的影响。 延伸因子1（EF-1）是参与蛋白质合成的延伸阶段的两种蛋白质因子之一。EF-1由四个亚基组成。 EF-1与GTP和氨酰- tRNA形成三元复合物，并将tRNA正确定位在核糖体上。 GTP是这一步骤的能量来源，并裂解为GDP和磷酸盐; GDP仍然与EF-1结合，必须通过EF- 1交换为GTP 才能让EF-1再次发挥作用 所有四 EF-1的亚基已被磷酸化。 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p34 cdc 2。 的 和 蛋白激酶C亚基和 亚基的细胞分裂控制激酶。 在这些研究中，我们建议检查EF-1的酪蛋白激酶II和蛋白激酶C的磷酸化。 将检查磷酸化对延伸活性和对GDP/GTP交换活性的影响。 克隆了兔EF-1的亚基，并在E.杆菌 这些亚基将被重建，并将确定磷酸化对单个亚基的影响。 将制备单个磷酸化位点的位点特异性突变。 将对含有这些突变的亚基进行复溶和分析，并确定位点特异性突变对活性的影响。 *** .

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. *** .