Two protein kinases have been identified that have the capacity to affect translational control of protein synthesis by phosphorylation of the alpha subunit of peptide initiation factor 2, eIF-2. One is induced by interferon and activated by double-stranded RNA. A different kinase is activated by heme deficiency in reticulocytes. Both kinases phosphorylate the same site in a 4 kilodalton, terminal segment of eIF-2-alpha with concomitant inhibition of protein synthesis, thereby exerting control through a common mechanism. A crucial question for understanding translational control involves the mechanism by which these kinases are activated and inactivated. Phosphorylation of both kinases appears to be an essential step in their activation. A 90,000 dalton peptide shown previously to be associated with the heme-regulated kinase has been identified as derived from the beta subunit of spectrin. Potentially, the state of phosphorylation of either eIF-2 or the kinases can be regulated by a cognate phosphatase. A 55 kilodalton, Mn?2+?-dependent protein phosphatase has been purified to homogeneity from reticulocytes. Its activity is modulated by a 230 kilodalton protein that appears to be associated with spectrin in the membrane skeleton of reticulocytes. We have named this protein """"""""regulin."""""""" It is extremely sensitive to proteolysis, and at least some of the larger peptides derived from it are active in regulating the phosphatase. The interaction of regulin and its peptides with the phosphatase has been studied using fluorescence techniques after labeling of the phosphatase with a maleidyl derivative of coumarin. Mn?2+? is required for efficient interaction of regulin with the phosphatase, and it appears that both Mn?2+? and regulin cause conformational changes in the enzyme. The results indicate that both the phosphatase and kinase are associated with elements of the membrane skeleton that are very sensitive to proteases. (F)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA016608-13S1
Application #
3164434
Study Section
Molecular Biology Study Section (MBY)
Project Start
1977-12-01
Project End
1988-07-31
Budget Start
1988-04-01
Budget End
1988-07-31
Support Year
13
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Type
Schools of Arts and Sciences
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78713
Rose, D W; Welch, W J; Kramer, G et al. (1989) Possible involvement of the 90-kDa heat shock protein in the regulation of protein synthesis. J Biol Chem 264:6239-44
Szyszka, R; Kramer, G; Hardesty, B (1989) The phosphorylation state of the reticulocyte 90-kDa heat shock protein affects its ability to increase phosphorylation of peptide initiation factor 2 alpha subunit by the heme-sensitive kinase. Biochemistry 28:1435-8
Szyszka, R; Kudlicki, W; Kramer, G et al. (1989) A type 1 phosphoprotein phosphatase active with phosphorylated Mr = 68,000 initiation factor 2 kinase. J Biol Chem 264:3827-31
Zardeneta, G; Kramer, G; Hardesty, B (1988) Quantification and characterization of regulin, a Mr-230,000 highly elongated protein of rabbit reticulocytes. Eur J Biochem 178:267-76
Kramer, G; Kudlicki, W; Fullilove, S et al. (1987) Association of the heme-controlled eIF-2 alpha kinase with spectrin-derived peptides. Haematol Blood Transfus 31:265-7
Kudlicki, W; Fullilove, S; Read, R et al. (1987) Identification of spectrin-related peptides associated with the reticulocyte heme-controlled alpha subunit of eukaryotic translational initiation factor 2 kinase and of Mr 95,000 peptide that appears to be the catalytic subunit. J Biol Chem 262:9695-701
Kudlicki, W; Wettenhall, R E; Kemp, B E et al. (1987) Evidence for a second phosphorylation site on eIF-2 alpha from rabbit reticulocytes. FEBS Lett 215:16-20
Rose, D W; Wettenhall, R E; Kudlicki, W et al. (1987) The 90-kilodalton peptide of the heme-regulated eIF-2 alpha kinase has sequence similarity with the 90-kilodalton heat shock protein. Biochemistry 26:6583-7
Hardesty, B; Kudlicki, W; Chen, S C et al. (1987) Involvement of the membrane skeleton in the regulation of the cAMP-independent protein kinase and a protein phosphatase that control protein synthesis. Haematol Blood Transfus 31:268-73
Kudlicki, W; Kramer, G; Hardesty, B (1986) Inhibition of protein synthesis by the beta-subunit of spectrin. FEBS Lett 200:271-4

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