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. 1994 Aug 2;91(16):7455–7459. doi: 10.1073/pnas.91.16.7455

Interleukin 3 stimulates protein synthesis by regulating double-stranded RNA-dependent protein kinase.

T Ito 1, R Jagus 1, W S May 1
PMCID: PMC44419  PMID: 7519779

Abstract

In a murine interleukin 3 (IL-3)-dependent cell line, IL-3 deprivation resulted in increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR) that has been reported to inhibit protein synthesis by phosphorylating the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha). Autophosphorylation was characterized by a shift up in mobility of PKR on SDS/PAGE gels from a 60- to a 64-kDa form. In vitro kinase studies comparing the autophosphorylated 64-kDa PKR with the nonphosphorylated 60-kDa PKR confirmed that only the 64-kDa form was active for eIF-2 alpha phosphorylation. PKR activation in vivo was associated with phosphorylation of eIF-2 alpha and inhibition of protein synthesis. Addition of IL-3 to deprived cells elicited a reciprocal response characterized by the rapid dephosphorylation of PKR and eIF-2 alpha, indicating inactivation of PKR. This was rapidly followed by the full recovery of protein synthesis. Furthermore, upon IL-3 addition, a 97-kDa phosphotyrosine-containing protein becomes rapidly and transiently associated with PKR prior to dephosphorylation of PKR and eIF-2 alpha. Genistein, a tyrosine kinase inhibitor, blocks both phosphorylation of the 97-kDa phosphoprotein and protein synthesis after IL-3 addition, suggesting a role for the 97-kDa phosphoprotein in the mechanism of inactivation of PKR and stimulation of protein synthesis. Thus, IL-3 appears to positively regulate protein synthesis by inducing the inactivation of PKR in a growth factor signaling pathway.

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Selected References

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

  1. Akiyama T., Ogawara H. Use and specificity of genistein as inhibitor of protein-tyrosine kinases. Methods Enzymol. 1991;201:362–370. doi: 10.1016/0076-6879(91)01032-w. [DOI] [PubMed] [Google Scholar]
  2. Boswell H. S., Mochizuki D. Y., Burgess G. S., Gillis S., Walker E. B., Anderson D., Williams D. E. A novel mast cell growth factor (MCGF-3) produced by marrow-adherent cells that synergizes with interleukin 3 and interleukin 4. Exp Hematol. 1990 Aug;18(7):794–800. [PubMed] [Google Scholar]
  3. Clark-Lewis I., Aebersold R., Ziltener H., Schrader J. W., Hood L. E., Kent S. B. Automated chemical synthesis of a protein growth factor for hemopoietic cells, interleukin-3. Science. 1986 Jan 10;231(4734):134–139. doi: 10.1126/science.3079915. [DOI] [PubMed] [Google Scholar]
  4. Duclos B., Marcandier S., Cozzone A. J. Chemical properties and separation of phosphoamino acids by thin-layer chromatography and/or electrophoresis. Methods Enzymol. 1991;201:10–21. doi: 10.1016/0076-6879(91)01004-l. [DOI] [PubMed] [Google Scholar]
  5. Duncan R., Hershey J. W. Regulation of initiation factors during translational repression caused by serum depletion. Covalent modification. J Biol Chem. 1985 May 10;260(9):5493–5497. [PubMed] [Google Scholar]
  6. Galabru J., Hovanessian A. Autophosphorylation of the protein kinase dependent on double-stranded RNA. J Biol Chem. 1987 Nov 15;262(32):15538–15544. [PubMed] [Google Scholar]
  7. Hovanessian A. G. The double stranded RNA-activated protein kinase induced by interferon: dsRNA-PK. J Interferon Res. 1989 Dec;9(6):641–647. doi: 10.1089/jir.1989.9.641. [DOI] [PubMed] [Google Scholar]
  8. Hunter T., Karin M. The regulation of transcription by phosphorylation. Cell. 1992 Aug 7;70(3):375–387. doi: 10.1016/0092-8674(92)90162-6. [DOI] [PubMed] [Google Scholar]
  9. Icely P. L., Gros P., Bergeron J. J., Devault A., Afar D. E., Bell J. C. TIK, a novel serine/threonine kinase, is recognized by antibodies directed against phosphotyrosine. J Biol Chem. 1991 Aug 25;266(24):16073–16077. [PubMed] [Google Scholar]
  10. Kennedy M. J., Prestigiacomo L. J., Tyler G., May W. S., Davidson N. E. Differential effects of bryostatin 1 and phorbol ester on human breast cancer cell lines. Cancer Res. 1992 Mar 1;52(5):1278–1283. [PubMed] [Google Scholar]
  11. Koromilas A. E., Roy S., Barber G. N., Katze M. G., Sonenberg N. Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science. 1992 Sep 18;257(5077):1685–1689. doi: 10.1126/science.1382315. [DOI] [PubMed] [Google Scholar]
  12. Krust B., Galabru J., Hovanessian A. G. Further characterization of the protein kinase activity mediated by interferon in mouse and human cells. J Biol Chem. 1984 Jul 10;259(13):8494–8498. [PubMed] [Google Scholar]
  13. Langland J. O., Jacobs B. L. Cytosolic double-stranded RNA-dependent protein kinase is likely a dimer of partially phosphorylated Mr = 66,000 subunits. J Biol Chem. 1992 May 25;267(15):10729–10736. [PubMed] [Google Scholar]
  14. Lengyel P. Tumor-suppressor genes: news about the interferon connection. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5893–5895. doi: 10.1073/pnas.90.13.5893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Levin D. H., Petryshyn R., London I. M. Characterization of double-stranded-RNA-activated kinase that phosphorylates alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) in reticulocyte lysates. Proc Natl Acad Sci U S A. 1980 Feb;77(2):832–836. doi: 10.1073/pnas.77.2.832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maurides P. A., Akkaraju G. R., Jagus R. Evaluation of protein phosphorylation state by a combination of vertical slab gel isoelectric focusing and immunoblotting. Anal Biochem. 1989 Nov 15;183(1):144–151. doi: 10.1016/0003-2697(89)90182-6. [DOI] [PubMed] [Google Scholar]
  17. Merrick W. C. Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev. 1992 Jun;56(2):291–315. doi: 10.1128/mr.56.2.291-315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Meurs E. F., Galabru J., Barber G. N., Katze M. G., Hovanessian A. G. Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):232–236. doi: 10.1073/pnas.90.1.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Montine K. S., Henshaw E. C. Serum growth factors cause rapid stimulation of protein synthesis and dephosphorylation of eIF-2 in serum deprived Ehrlich cells. Biochim Biophys Acta. 1989 Dec 14;1014(3):282–288. doi: 10.1016/0167-4889(89)90224-3. [DOI] [PubMed] [Google Scholar]
  20. Mundschau L. J., Faller D. V. Oncogenic ras induces an inhibitor of double-stranded RNA-dependent eukaryotic initiation factor 2 alpha-kinase activation. J Biol Chem. 1992 Nov 15;267(32):23092–23098. [PubMed] [Google Scholar]
  21. Rhoads R. E. Regulation of eukaryotic protein synthesis by initiation factors. J Biol Chem. 1993 Feb 15;268(5):3017–3020. [PubMed] [Google Scholar]
  22. Safer B. 2B or not 2B: regulation of the catalytic utilization of eIF-2. Cell. 1983 May;33(1):7–8. doi: 10.1016/0092-8674(83)90326-4. [DOI] [PubMed] [Google Scholar]
  23. Safer B., Adams S. L., Anderson W. F., Merrick W. C. Binding of MET-TRNAf and GTP to homogeneous initiation factor MP. J Biol Chem. 1975 Dec 10;250(23):9076–9082. [PubMed] [Google Scholar]
  24. Samuel C. E. The eIF-2 alpha protein kinases, regulators of translation in eukaryotes from yeasts to humans. J Biol Chem. 1993 Apr 15;268(11):7603–7606. [PubMed] [Google Scholar]
  25. Sen G. C., Lengyel P. The interferon system. A bird's eye view of its biochemistry. J Biol Chem. 1992 Mar 15;267(8):5017–5020. [PubMed] [Google Scholar]
  26. SenGupta D. N., Silverman R. H. Activation of interferon-regulated, dsRNA-dependent enzymes by human immunodeficiency virus-1 leader RNA. Nucleic Acids Res. 1989 Feb 11;17(3):969–978. doi: 10.1093/nar/17.3.969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  28. Szyszka R., Kudlicki W., Kramer G., Hardesty B., Galabru J., Hovanessian A. A type 1 phosphoprotein phosphatase active with phosphorylated Mr = 68,000 initiation factor 2 kinase. J Biol Chem. 1989 Mar 5;264(7):3827–3831. [PubMed] [Google Scholar]
  29. Taga T., Kishimoto T. Cytokine receptors and signal transduction. FASEB J. 1992 Dec;6(15):3387–3396. doi: 10.1096/fasebj.6.15.1334470. [DOI] [PubMed] [Google Scholar]
  30. Thomis D. C., Samuel C. E. Mechanism of interferon action: evidence for intermolecular autophosphorylation and autoactivation of the interferon-induced, RNA-dependent protein kinase PKR. J Virol. 1993 Dec;67(12):7695–7700. doi: 10.1128/jvi.67.12.7695-7700.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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