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. 1982 Dec;79(23):7092–7096. doi: 10.1073/pnas.79.23.7092

Effects of skeletal muscle protein phosphatase inhibitor-2 on protein synthesis and protein phosphorylation in rabbit reticulocyte lysates*

Vivian Ernst †, Daniel H Levin ‡, J Gordon Foulkes §,, Irving M London ‡
PMCID: PMC347284  PMID: 6296814

Abstract

Reticulocyte lysates contain two major classes of protein phosphatase activities, designated type 1 and type 2. These designations are based on criteria derived from the analyses of protein phosphatase species in other tissues. The criteria include (i) chromatographic elution profiles on DEAE-cellulose; (ii) specificity of lysate phosphatases toward [32P]phosphorylase a and [32P]phosphorylase kinase; (iii) sensitivity of lysate phosphatases to Mg2+ ATP; and (iv) sensitivity to the heat-stable protein phosphatase inhibitor-2. The lysate phosphatase species are similar to those described in rabbit skeletal muscle and rabbit liver. Reticulocyte protein phosphatase type 1, but not type 2, is inhibited by heat-stable protein phosphatase inhibitor-1 and -2 which have been characterized from rabbit skeletal muscle. We have initiated a study on the function and specificity of lysate protein phosphatase activities involved in the regulation of protein synthesis by examining the effects of protein phosphatase inhibitor-2 on reticulocyte protein synthesis and protein phosphorylation. Our findings are as follows. (a) Protein phosphatase inhibitor-2 inhibits protein chain initiation in hemin-supplemented lysates. (b) Inhibition is characterized by biphasic kinetics and is reversed by the delayed addition of purified reticulocyte eukaryotic initiation factor 2 (eIF-2). (c) Inhibition of protein synthesis by inhibitor-2 is accompanied by the phosphorylation of the α-subunit (38,000 daltons) of eIF-2 (eIF-2α) and of two heat-stable polypeptides of 29,000 and 44,000 daltons. (d) The 29,000-dalton component is phosphorylated in lysates under conditions of protein synthesis and appears to be inhibitor-2, but the physiological significance of this modification of inhibitor-2 is not clear. (e) Inhibitor-2 has no effect on the activation in vitro of isolated heme-regulated or double-stranded RNA-dependent eIF-2α kinases. We propose that the inhibition of protein synthesis in hemin-supplemented lysates by added inhibitor-2 is due at least in part to the inhibition of a type 1 eIF-2α phosphatase activity, which permits a basal eIF-2α kinase activity to be expressed leading to the accumulation of phosphorylated eIF-2α and an inhibition of protein synthesis.

Keywords: protein chain initiation, translational control, eukaryotic initiation factor 2α kinase, phosphorylation of eukaryotic initiation factor 2α

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Antoniw J. F., Nimmo H. G., Yeaman S. J., Cohen P. Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle. Biochem J. 1977 Feb 15;162(2):423–433. doi: 10.1042/bj1620423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Crouch D., Safer B. Purification and properties of eIF-2 phosphatase. J Biol Chem. 1980 Aug 25;255(16):7918–7924. [PubMed] [Google Scholar]
  3. Ernst V., Levin D. H., Leroux A., London I. M. Site-specific phosphorylation of the alpha subunit of eukaryotic initiation factor eIF-2 by the heme-regulated and double-stranded RNA-activated eIF-2 alpha kinases from rabbit reticulocyte lysates. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1286–1290. doi: 10.1073/pnas.77.3.1286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ernst V., Levin D. H., London I. M. Evidence that glucose 6-phosphate regulates protein synthesis initiation in reticulocyte lysates. J Biol Chem. 1978 Oct 25;253(20):7163–7172. [PubMed] [Google Scholar]
  5. Ernst V., Levin D. H., London I. M. In situ phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in reticulocyte lysates inhibited by heme deficiency, double-stranded RNA, oxidized glutathione, or the heme-regulated protein kinase. Proc Natl Acad Sci U S A. 1979 May;76(5):2118–2122. doi: 10.1073/pnas.76.5.2118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Farrell P. J., Balkow K., Hunt T., Jackson R. J., Trachsel H. Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis. Cell. 1977 May;11(1):187–200. doi: 10.1016/0092-8674(77)90330-0. [DOI] [PubMed] [Google Scholar]
  7. Farrell P. J., Hunt T., Jackson R. J. Analysis of phosphorylation of protein synthesis initiation factor eIF-2 by two-dimensional gel electrophoresis. Eur J Biochem. 1978 Sep 1;89(2):517–521. doi: 10.1111/j.1432-1033.1978.tb12556.x. [DOI] [PubMed] [Google Scholar]
  8. Floyd G. A., Traugh J. A. Heme deficiency and phosphorylation of ribosome-associated proteins. Eur J Biochem. 1980 May;106(1):269–277. doi: 10.1111/j.1432-1033.1980.tb06019.x. [DOI] [PubMed] [Google Scholar]
  9. Foulkes J. G., Cohen P. The regulation of glycogen metabolism. Purification and properties of protein phosphatase inhibitor-2 from rabbit skeletal muscle. Eur J Biochem. 1980 Mar;105(1):195–203. doi: 10.1111/j.1432-1033.1980.tb04489.x. [DOI] [PubMed] [Google Scholar]
  10. Gross M., Mendelewski J. Additional evidence that the hemin-controlled translational repressor from rabbit reticulocytes is a protein kinase. Biochem Biophys Res Commun. 1977 Jan 24;74(2):559–569. doi: 10.1016/0006-291x(77)90340-0. [DOI] [PubMed] [Google Scholar]
  11. Henderson A. B., Miller A. H., Hardesty B. Multistep regulatory system for activation of a cyclic AMP-independent eukaryotic initiation factor 2 kinase. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2605–2609. doi: 10.1073/pnas.76.6.2605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Huang F. L., Glinsmann W. H. Separation and characterization of two phosphorylase phosphatase inhibitors from rabbit skeletal muscle. Eur J Biochem. 1976 Nov 15;70(2):419–426. doi: 10.1111/j.1432-1033.1976.tb11032.x. [DOI] [PubMed] [Google Scholar]
  13. Hunt T., Vanderhoff G., London I. M. Control of globin synthesis: the role of heme. J Mol Biol. 1972 May 28;66(3):471–481. doi: 10.1016/0022-2836(72)90427-5. [DOI] [PubMed] [Google Scholar]
  14. Jackson R. J., Hunt T. The use of hexose phosphates to support protein synthesis and generate [gamma-32P]ATP in reticulocyte lysates. FEBS Lett. 1978 Sep 15;93(2):235–238. doi: 10.1016/0014-5793(78)81111-9. [DOI] [PubMed] [Google Scholar]
  15. Kramer G., Cimadevilla J. M., Hardesty B. Specificity of the protein kinase activity associated with the hemin-controlled repressor of rabbit reticulocyte. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3078–3082. doi: 10.1073/pnas.73.9.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leroux A., London I. M. Regulation of protein synthesis by phosphorylation of eukaryotic initiation factor 2 alpha in intact reticulocytes and reticulocyte lysates. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2147–2151. doi: 10.1073/pnas.79.7.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Levin D., Ernst V., London I. M. Effects of the catalytic subunit of cAMP-dependent protein kinase (type II) from reticulocytes and bovine heart muscle on protein phosphorylation and protein synthesis in reticulocyte lysates. J Biol Chem. 1979 Aug 25;254(16):7935–7941. [PubMed] [Google Scholar]
  18. Levin D., Ranu R. S., Ernst V., London I. M. Regulation of protein synthesis in reticulocyte lysates: phosphorylation of methionyl-tRNAf binding factor by protein kinase activity of translational inhibitor isolated from hemedeficient lysates. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3112–3116. doi: 10.1073/pnas.73.9.3112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nimmo G. A., Cohen P. The regulation of glycogen metabolism. Purification and characterisation of protein phosphatase inhibitor-1 from rabbit skeletal muscle. Eur J Biochem. 1978 Jun 15;87(2):341–351. doi: 10.1111/j.1432-1033.1978.tb12383.x. [DOI] [PubMed] [Google Scholar]
  20. Nimmo H. G., Cohen P. Hormonal control of protein phosphorylation. Adv Cyclic Nucleotide Res. 1977;8:145–266. [PubMed] [Google Scholar]
  21. Petryshyn R., Levin D. H., London I. M. Purification and characterization of a latent precursor of a double-stranded RNA dependent protein kinase from reticulocyte lysates. Biochem Biophys Res Commun. 1980 Jun 30;94(4):1190–1198. doi: 10.1016/0006-291x(80)90545-8. [DOI] [PubMed] [Google Scholar]
  22. Safer B., Jagus R. Control of eIF-2 phosphatase activity in rabbit reticulocyte lysate. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1094–1098. doi: 10.1073/pnas.76.3.1094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stewart A. A., Crouch D., Cohen P., Safer B. Classification of an eIF-2phosphatase as a type-2 protein phosphatase. FEBS Lett. 1980 Sep 22;119(1):16–19. doi: 10.1016/0014-5793(80)80988-4. [DOI] [PubMed] [Google Scholar]
  24. Tahara S. M., Traugh J. A., Sharp S. B., Lundak T. S., Safer B., Merrick W. C. Effect of hemin on site-specific phosphorylation of eukaryotic initiation factor 2. Proc Natl Acad Sci U S A. 1978 Feb;75(2):789–793. doi: 10.1073/pnas.75.2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Trachsel H., Ranu R. S., London I. M. Purification of the heme-reversible form of the translational inhibitory protein kinase. Methods Enzymol. 1979;60:485–495. doi: 10.1016/s0076-6879(79)60046-0. [DOI] [PubMed] [Google Scholar]
  26. de Haro C., Manne V., de Herreros A. G., Ochoa S. Heat-stable inhibitor of translation in reticulocyte lysates. Proc Natl Acad Sci U S A. 1982 May;79(10):3134–3137. doi: 10.1073/pnas.79.10.3134. [DOI] [PMC free article] [PubMed] [Google Scholar]

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