The fundamental, continuing goal of the proposed research is to investigate the potential importance of Ca2+ as a physiologic regulator of protein synthesis in mammalian tissues and the mechanism by which such regulation may occur. Included in this long range objective is the evaluation of the hypothesis that intracellular Ca2+ functions to co- ordinate the rate of protein synthesis with the stimulus-response status of the cell. The data base now in hand strongly supports the hypothesis that Ca2+ exerts its effects on translational initiation via a cascade of events originating from sites of sequestration external to the translational apparatus. The actual input from this cascade into translation appears to involve the phosphorylation of a 26 kDa ribosomal protein. The primary specific aim of this proposal is to investigate the role of the 26 kDa ribosomal phosphoprotein in translational initiation. The protein will be purified, characterized, and cDNA cloned for subsequent determination of amino acid sequence. The phosphorylation of the protein will be evaluated including the determination of the number and nature of the sites phosphorylated and efforts undertaken to recreate the phosphorylation in cell-free systems in order to define the properties of the protein kinase and phosphatase activities involved in the turnover of the phosphate(s). The effect of phosphorylation of the 26 kDa protein on ribosomal function will be evaluated with respect to ribosomal mRNA content and release of ribosomal bound eIF-2 for subsequent cycling. These experiments will be conducted with GH3 pituitary cells, liver, and reticulocyte lysate systems. Efforts will also be made to ascertain whether the endoplasmic reticulum is the intracellular site of Ca2+ sequestration supporting translational initiation and to understand how differential affinities for the cation are achieved for translation in GH3 versus HeLa cells. The effect of Ca2+ mobilizing hormones on the phosphorylation state of the 26 kDa ribosomal protein of liver cells will be determined. Thermal and chemical stresses, which eliminate the CA2+-supported component of translation in cultured cells, will be examined for effects on phosphorylation of the 26 kDa ribosomal protein and on intracellular Ca2+ storage.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Physiological Chemistry Study Section (PC)
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University of Medicine & Dentistry of NJ
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Gmitter, D; Brostrom, C O; Brostrom, M A (1996) Translational suppression by Ca2+ ionophores: reversibility and roles of Ca2+ mobilization, Ca2+ influx, and nucleotide depletion. Cell Biol Toxicol 12:101-13
Prostko, C R; Dholakia, J N; Brostrom, M A et al. (1995) Activation of the double-stranded RNA-regulated protein kinase by depletion of endoplasmic reticular calcium stores. J Biol Chem 270:6211-5
Brostrom, M A; Prostko, C R; Gmitter, D et al. (1995) Independent signaling of grp78 gene transcription and phosphorylation of eukaryotic initiator factor 2 alpha by the stressed endoplasmic reticulum. J Biol Chem 270:4127-32
Brostrom, M A; Wong Ling, W L; Gmitter, D et al. (1994) Release of Ca2+ from intracellular organelles by peptide analogues: evidence against involvement of metalloendoproteases in Ca2+ sequestration by the endoplasmic reticulum. Biochem J 304 ( Pt 2):499-507
Wong, W L; Brostrom, M A; Kuznetsov, G et al. (1993) Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells. Biochem J 289 ( Pt 1):71-9
Kuznetsov, G; Brostrom, M A; Brostrom, C O (1993) Role of endoplasmic reticular calcium in oligosaccharide processing of alpha 1-antitrypsin. J Biol Chem 268:2001-8
Prostko, C R; Brostrom, M A; Brostrom, C O (1993) Reversible phosphorylation of eukaryotic initiation factor 2 alpha in response to endoplasmic reticular signaling. Mol Cell Biochem 127-128:255-65
Kuznetsov, G; Brostrom, M A; Brostrom, C O (1992) Demonstration of a calcium requirement for secretory protein processing and export. Differential effects of calcium and dithiothreitol. J Biol Chem 267:3932-9
Prostko, C R; Brostrom, M A; Malara, E M et al. (1992) Phosphorylation of eukaryotic initiation factor (eIF) 2 alpha and inhibition of eIF-2B in GH3 pituitary cells by perturbants of early protein processing that induce GRP78. J Biol Chem 267:16751-4
Rotman, E I; Brostrom, M A; Brostrom, C O (1992) Inhibition of protein synthesis in intact mammalian cells by arachidonic acid. Biochem J 282 ( Pt 2):487-94

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