Skip to main content
SpringerLink
Log in

Cyclic nucleotide- and calcium-independent phosphorylation of proteins in rat brain polyribosome: Effects of ACTH, spermine, and hemin

  • Original Articles
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

The incorporation of [γ-32P]ATP into proteins of rat brain polyribosomes was studied in vitro. The effects of cyclic nucleotides, calcium, hemin, ACTH, GTP, and spermine were examined. The incorporation of phosphate into proteins increased with time and phosphatase activity was very low; thus, the extent of phosphorylation was predominantly a reflection of protein kinase activity. Phosphorylation of proteins was not sensitive to Ca2+ in the presence or absence of either calmodulin or phosphatidylserine. Phosphorylation was also unaffected by cyclic nucleotides in the absence of exogenous enzymes. However, addition of a cMAP-dependent protein kinase together with cAMP resulted in a stimulation of the incorporation of phosphate into 4 phosphoproteins (pp70, pp58, pp43, and pp32); phosphorylation of pp32 was completely dependent on the addition of the kinase. ACTH (1–24), (11–24), and spermine inhibited the endogenous phosphorylation of one protein band (pp30). The phosphorylation of this 30 kD band was also selectively increased by hemin (5 μM). Higher concentrations of hemin exerted an inhibitory effect on the majority of the phosphoproteins. Protein phosphatase activity was not influenced by ACTH or spermine. The specific inhibition of pp30 phosphorylation by ACTH or spermine is most probably explained by an interaction with a cyclic nucleotide- and Ca2+-independent protein kinase.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Van Dijk, A. M. A., Benitez-King, G., Schotman, P., andGispen, W. H. 1981. Phosphorylation of proteins in a post-mitochondrial supernantant from rat brain stem affected by ACTH1–24 and cyclic nucleotides. Neurochem. Res. 6:847–860.

    Google Scholar 

  2. Schrama, L. H., Edwards, P. M., andSchotman, P. 1984. Modulation of protein synthesis in a cell-free system derived from rat brain by corticotrophin (ACTH), magnesium and spermine. J. Neurosci. Res. 11:67–77.

    Google Scholar 

  3. Schotman, P., Frankena, H., Schrama, L. H., andEdwards, P. M. 1982. Phosphorylation in relation to the modulation of brain protein synthesis by ACTH-like neuropeptides. Progr. Brain Res. 56:213–235.

    Google Scholar 

  4. Schotman, P., Van Heuven-Nolsen, D., andGispen, W. H. 1980. Protein synthesis in a cell-free system from rat brain sensitive to ACTH-like peptides. J. Neurochem. 34:1661–1670.

    Google Scholar 

  5. Floyd, G. A., andTraugh, J. A. 1980. Heme deficiency and phosphorylation of ribosome-associated proteins. Eur. J. Biochem. 106:269–277.

    Google Scholar 

  6. Ramsey, J. C., andSteele, W. J. 1977. Quantitative isolation and properties of nearly homogeneous populations of undegraded free and bound polysomes from rat brain. J. Neurochem. 28:517–527.

    Google Scholar 

  7. Zwiers, H., Veldhuis, H. D., Schotman, P., andGispen, W. H. 1976. ACTH, cyclic nucleotides, and brain protein phosphorylation in vitro. Neurochem. Res. 1:669–677.

    Google Scholar 

  8. Delgrande, R. W., andTraugh, J. A. 1982. Phosphorylation of 40S ribosomal subunits by cAMP-dependent, cGMP-dependent and protease-activated protein kinases. Eur. J. Biochem. 123:421–428.

    Google Scholar 

  9. Mumby, M., andTraugh, J. A. 1979. Dephosphorylation of translational components by phosphoprotein phosphatases from reticulocytes. Methods in Enzymol. 60:522–534.

    Google Scholar 

  10. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., andRandall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.

    Google Scholar 

  11. Munro, H. N., andFleck, A. 1966. The determination of nucleic acids. Meth. Biochem. Anal. 14:113–176.

    Google Scholar 

  12. Hunt, T., Vanderhoff, G., andLondon, I. M. 1972. Control of globin synthesis: the role of hemin. J. Mol. Biol. 66:471–481.

    Google Scholar 

  13. Ashby, C. D., andRoberts, S. 1975. Phosphorylation of ribosomal proteins in rat cerebral cortex in vitro. J. Biol. Chem. 250:2546–2555.

    Google Scholar 

  14. Roberts, S., andAshby, C. D. 1978. Ribosomal protein phosphorylation in rat cerebral cortex in vitro. J. Biol. Chem. 253:288–296.

    Google Scholar 

  15. Floyd, G. A., Merrick, W. C., andTraugh, J. A. 1979. Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis. Eur. J. Biochem. 96:277–286.

    Google Scholar 

  16. Roberts, S., andMorelos, B. S. 1979. Phosphorylation of multiple proteins of both ribosomal subunits in rat cerebral cortex in vivo. Biochem. J. 184:233–244.

    Google Scholar 

  17. Francis, T. A., andRoberts, S. 1982. Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome-protein kinase systems from rat cerebral cortex. Biochem. J. 208:289–300.

    Google Scholar 

  18. Wettenhall, R. E. H., andCohen, P. 1982. Isolation and characterization of cAMP-dependent phosphorylation sites from rat liver ribosomal protein S6. FEBS Lett. 140:263–269.

    Google Scholar 

  19. Wettenhall, R. E. H., Chesterman, C. N., Walker, T., andMorgan, F. J. 1983. Phosphorylation sites for S6 protein kinases in mouse 3T3 fibroblasts stimulated by PDGF. FEBS Lett. 162:171–177.

    Google Scholar 

  20. Traugh, J. A. 1981. Regulation of protein synthesis by phosphorylation. Biochem. Act. Horm. 8:167–208.

    Google Scholar 

  21. Weller, M. 1979. Protein phosphorylation. The nature, function and metabolism of proteins, which contain covalently bound phosphorus. PION, London, pp. 1–557.

    Google Scholar 

  22. England, P. J. 1980. Regulation by phosphorylation and dephosphorylation. Pages 292–344,in Freedman, R. B., andHawkins, R. C. (eds.), The enzymology of posttranslational modification of proteins, Vol. 1, Academic Press, New York.

    Google Scholar 

  23. Aloyo, V. J., Zwiers, H., andGispen, W. H. 1983. Phosphorylation of B-50 protein by calcium activated, phospholipid-dependent protein kinase and B-50 protein kinase. J. Neurochem. 41:649–653.

    Google Scholar 

  24. Hirsch, J. D., andMartelo, O. J. 1976. Inhibition of rabbit reticulocyte protein kinases by hemin. Biochem. Biophys. Res. Comm. 71:926–932.

    Google Scholar 

  25. Datta, A., De Haro, C., andOchoa, S. 1978. Translational control by hemin is due to binding to cyclic AMP dependent protein kinase. Proc. Natl. Acad. Sci. USA 75:1148–1152.

    Google Scholar 

  26. Kodiček, M., Hrkal, Z., andVodrážka, Z. 1983. Kinetics of haem binding to human albumin and haemopexin. Non-specific interactions of haem with proteins. Int. J. Biol. Macromol. 5:194–199.

    Google Scholar 

  27. Cenatiempo, Y., Cozzone, A. J., Genot, A., andReboud, J. P. 1977. In vitro phosphorylation of proteins from free and membrand bound rat liver polysomes. FEBS Lett. 79:165–169.

    Google Scholar 

  28. Witter, A. 1980. On the presence of receptors for ACTH neuropeptides in the brain. Pages 407–414,in Pepeu, G. C., Kuhar, M., andEnna, L. (eds.), Receptors for neurotransmitters and peptide hormones, Raven Press, New York.

    Google Scholar 

  29. Rasmussen, H. 1982. Hormone action: 1981. Pages 127–143,in Corradino, R. A. (ed.), Functional regulation at the cellular and molecular levels, Elsevier North Holland, Amsterdam.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Molecular Neurobiology Institute of Molecular Biology Rudolf Magnus Institute for Pharmacology, State University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands

    L. H. Schrama, H. Frankena, P. M. Edwards &  P. Schotman

  2. Laboratory of Physiological Chemistry, State University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands

    L. H. Schrama, H. Frankena, P. M. Edwards &  P. Schotman

Authors
  1. L. H. Schrama
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. Frankena
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P. M. Edwards
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P. Schotman
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schrama, L.H., Frankena, H., Edwards, P.M. et al. Cyclic nucleotide- and calcium-independent phosphorylation of proteins in rat brain polyribosome: Effects of ACTH, spermine, and hemin. Neurochem Res 9, 1267–1281 (1984). https://doi.org/10.1007/BF00973039

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00973039

Keywords

Search

Navigation