The goal of this work is the elucidation of the mechanisms controlling the rate of protein synthesis at the cytoplasmic level in mammalian cells and the differences between these mechanisms in normal and malignant cells. We have demonstrated that, upon nutrient deprivation (essential amino acid, glucose, or serum) of the Ehrlich ascites tumor cell in suspension culture, one such mechanism modulates chain initiation by inhibiting the formation of the complex between the 40s ribosomal subunit and the Met-tRNA?f??.?eIF-2?.?GTP ternary complex (i.e., the 40s initiation complex). We are now examining eIF-2 function. During the initiation reactions, GTP in the ternary complex is hydrolyzed and eIF-2 is released as, or rapidly forms, eIF-2?.?GDP. We have found a GDP/GTP exchange factor (GEF) that allows GTP to replace GDP in the binary complex. This is a new initiation factor, since without GEF the half-time of dissociation of the eIF-2?.?GDP complex is greater than 30 min. Phosphorylation of the alpha subunit of eIF-2 prevents the GEF-catalyzed GDP exchange and thus can stop eIF-2 cycling and chain initiation. We have measured the alterations in the extent of phosphorylation of eIF-2 in cells deprived of an essential amino acid. We find less than 10% change in extent of phosphorylation of total eIF-2 and of the subfraction of eIF-2 bound to ribosomes, bound to free 40s ribosomal subunits, and soluble eIF-2. We are presently developing approaches to determine whether these changes can account for the large changes seen in the rate of protein synthesis. Over the past year, we have found that small changes in ADP:ATP and GDP:GTP ratios, and not the absolute amount of any of these nucleotides, result in large changes in the rate of polypeptide chain initiation in Ehrlich cell-free protein-synthesizing systems. Currently, we are investigating the effect of altering these nucleotide ratios in the cell-free system on eIF-2 function and phosphorylation. We have also found that defective initiation--in cell-free protein-synthesizing systems prepared from heat-shocked Ehrlich cells--is completely restored by the addition of eIF-4F (mRNA cap binding protein). (F)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA021663-11
Application #
3165608
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-04-01
Project End
1988-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
11
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Rochester
Department
Type
Schools of Medicine
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Olmsted, E A; O'Brien, L; Henshaw, E C et al. (1993) Purification and characterization of eukaryotic initiation factor (eIF)-2 alpha kinases from Ehrlich ascites tumor cells. J Biol Chem 268:12552-9
Kumar, R V; Panniers, R; Wolfman, A et al. (1991) Inhibition of protein synthesis by antagonists of calmodulin in Ehrlich ascites tumor cells. Eur J Biochem 195:313-9
Cripps-Wolfman, J; Henshaw, E C; Bambara, R A (1989) Alterations in the phosphorylation and activity of DNA polymerase alpha correlate with the change in replicative DNA synthesis as quiescent cells re-enter the cell cycle. J Biol Chem 264:19478-86
Montine, K S; Henshaw, E C (1989) Serum growth factors cause rapid stimulation of protein synthesis and dephosphorylation of eIF-2 in serum deprived Ehrlich cells. Biochim Biophys Acta 1014:282-8
Kumar, R V; Wolfman, A; Panniers, R et al. (1989) Mechanism of inhibition of polypeptide chain initiation in calcium-depleted Ehrlich ascites tumor cells. J Cell Biol 108:2107-15
Rowlands, A G; Montine, K S; Henshaw, E C et al. (1988) Physiological stresses inhibit guanine-nucleotide-exchange factor in Ehrlich cells. Eur J Biochem 175:93-9
Panniers, R; Rowlands, A G; Henshaw, E C (1988) The effect of Mg2+ and guanine nucleotide exchange factor on the binding of guanine nucleotides to eukaryotic initiation factor 2. J Biol Chem 263:5519-25
Rowlands, A G; Panniers, R; Henshaw, E C (1988) The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by phosphorylated eukaryotic initiation factor 2. J Biol Chem 263:5526-33
Scorsone, K A; Panniers, R; Rowlands, A G et al. (1987) Phosphorylation of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis. J Biol Chem 262:14538-43
Clemens, M J; Galpine, A; Austin, S A et al. (1987) Regulation of polypeptide chain initiation in Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor GEF. J Biol Chem 262:767-71

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