The long-term goals of this proposal are to understand the physiologic regulation of phosphorylation of eIF-4E and the p25 subunit of eIF-4F and how this may effect translational control in vivo. Published studies have indicated that this protein is dephosphorylated in HeLa cells during the decrease in protein synthesis occurring after heat shock and during the mitotic phase of the cell cycle. Recent studies provide several lines of evidence for the existence of a novel nonabundant protein kinase in rabbit reticulocytes that phosphorylates eIF-4E in vitro at the same serine residue that in phosphorylated in intact reticulocytes and HeLa cells. The hypothesis to be tested is that a previously uncharacterized mammalian protein kinase (tentatively designated 4E kinase) exists which phosphorylates eIF-4E at serine-53 in vivo and that this phosphorylation event selectively modulates the translation of different mRNAs. In order to test this hypothesis the following immediate objectives are proposed: 1) purify the 4E kinase to enzymatic purity from rabbit reticulocytes using standard chromatography and HPLC methods and then characterize its substrate specificity and in vitro regulation; 2) prepare mouse monoclonal antibodies against the 4E kinase, immunohistochemical studies of cells and tissues to study the distribution of the enzyme and enzyme inhibition controls in cell-free translations and possibly intact cells; and 3) determine if 4E kinase has a mRNA-selective effect on translation in the reticulocyte lysate system. These studies may have implications for understanding the translational control mechanisms that occur in thermotolerance, the stress response and/or viral effects on host cells. Understanding the basic mechanisms of translational control may provide insight into the molecular mechanisms of diseases where a specific protein is synthesized in excess as in liver or pulmonary fibrosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040219-03
Application #
3297624
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1989-04-01
Project End
1992-06-30
Budget Start
1991-04-01
Budget End
1992-06-30
Support Year
3
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Hagedorn, C H; Spivak-Kroizman, T; Friedland, D E et al. (1997) Expression of functional eIF-4Ehuman: purification, detailed characterization, and its use in isolating eIF-4E binding proteins. Protein Expr Purif 9:53-60
Bu, X; Bondurant, M C; Hagedorn, C H (1996) Erythropoietin stimulates phosphorylation of eIF-4E and identification of a 37-kD phosphoprotein that binds mRNA caps in erythroblasts. Exp Hematol 24:423-8
Graves, P R; Haas, D W; Hagedorn, C H et al. (1993) Molecular cloning, expression, and characterization of a 49-kilodalton casein kinase I isoform from rat testis. J Biol Chem 268:6394-401
Bu, X; Haas, D W; Hagedorn, C H (1993) Novel phosphorylation sites of eukaryotic initiation factor-4F and evidence that phosphorylation stabilizes interactions of the p25 and p220 subunits. J Biol Chem 268:4975-8
Baker, B F; Miraglia, L; Hagedorn, C H (1992) Modulation of eucaryotic initiation factor-4E binding to 5'-capped oligoribonucleotides by modified anti-sense oligonucleotides. J Biol Chem 267:11495-9
Haas, D W; Shepherd, V L; Hagedorn, C H (1992) Lipopolysaccharide stimulates phosphorylation of eukaryotic initiation factor-4F in macrophages and tumor necrosis factor participates in this event. Second Messengers Phosphoproteins 14:163-71
Haas, D W; Hagedorn, C H (1992) Protein kinase C phosphorylates both serine and threonine residues of the mRNA cap binding protein eIF-4E. Second Messengers Phosphoproteins 14:55-63
Bu, X; Hagedorn, C H (1992) Phosphoprotein phosphatase 2A dephosphorylates eIF-4E and does not alter binding to the mRNA cap. FEBS Lett 301:15-8
Bu, X; Hagedorn, C H (1991) Platelet-derived growth factor stimulates phosphorylation of the 25 kDa mRNA cap binding protein (eIF-4E) in human lung fibroblasts. FEBS Lett 283:219-22
Haas, D W; Hagedorn, C H (1991) Casein kinase I phosphorylates the 25-kDa mRNA cap-binding protein. Arch Biochem Biophys 284:84-9

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