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. *** .

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
9316900
Program Officer
Marcia Steinberg
Project Start
Project End
Budget Start
1994-02-01
Budget End
1998-01-31
Support Year
Fiscal Year
1993
Total Cost
$259,000
Indirect Cost
Name
University of California Riverside
Department
Type
DUNS #
City
Riverside
State
CA
Country
United States
Zip Code
92521