The Journal of Membrane Biology

, Volume 49, Issue 2, pp 123–138 | Cite as

Phosphorylation of casein by human erythrocyte membrane-bound protein kinase: competition of casein with endogenous substrates

  • J. D. Vickers
  • J. Brierley
  • M. P. Rathbone


The possibility that spectrin and band-3 protein are phosphorylated by the same membrane-bound protein kinase was investigated by adding casein to unsealed erythrocyte ghosts and examining competition of the three proteins for phosphorylation. The extent of spectrin and band-3 protein phosphorylation was reduced by up to approximately 55%. This indicated that casein was competing with these endogenous substrates for phosphorylation and was most probably phosphorylated by the same protein kinase(s). Furthermore, the extent of inhibition of the phosphorylation of the two endogenous substrates was indistinguishable over the range of casein concentrations tested (0.1 to 5mg/ml). This indicates that spectrin and band-3 protein may be phosphorylated by the same protein kinase. In contrast, casein was found to have no effect on the cAMP-dependent phosphorylation of band 4.5. This result indicates that casein only competes with the endogenous proteins phosphorylated by the cAMP-independent protein kinase(s).

The extent of reduction of endogenous substrate phosphorylation in the presence of casein was found to be constant over incubation periods of 1 to 15 min, indicating that this reduction was not due to consumption of ATP.

Since the spectrin and band-3 protein phosphorylations were specifically and identically reduced by casein and these reductions were not due to the ATP consumption or to a general alteration of the membrane, we conclude that the two substrates are likely phosphorylated by one kinase which also phosphorylates casein.


Protein Kinase Human Physiology Incubation Period Protein Phosphorylation Human Erythrocyte 
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  1. 1.
    Appel, S.H., Roses, A.D. 1976. Membrane biochemical studies in myotonic muscular dystrophy.In: Membranes and Disease L. Bolis, J.F. Hoffman and A. Leaf, editors. pp. 183–195. Raven Press, New YorkGoogle Scholar
  2. 2.
    Aster, R.H., Jandl, J.H. 1964. Platelet sequestration in man. I. Methods.J. Clin. Invest. 43:843PubMedGoogle Scholar
  3. 3.
    Avruch, J., Fairbanks, G. 1974. Phosphorylation of endogenous substrates by erythrocyte membrane protein kinases. I. A monovalent cation-stimulated reaction.Biochemistry 13:5507PubMedGoogle Scholar
  4. 4.
    Avruch, J., Fairbanks, G., Crapo, L.W. 1976. Regulation of plasma membrane protein phosphorylation in two mammalian cell types.J. Cell Physiol. 89:815PubMedGoogle Scholar
  5. 5.
    Bruni, A., Van Dijck, P.W.M., DeGier, J. 1975. The role of phospholipid acyl chains in the activation of mitochondrial ATPase complex.Biochim. Biophys. Acta 406:315PubMedGoogle Scholar
  6. 6.
    Buckley, T.B. 1977. Properties of human erythrocyte phosphatidylinositol kinase and inihibition by adenosine, ADP and related compounds.Biochim. Biophys. Acta 498:1PubMedGoogle Scholar
  7. 7.
    Coleman, R. 1973. Membrane-bound enzymes and membrane ultrastructure.Biochim. Biophys. Acta 300:1PubMedGoogle Scholar
  8. 8.
    Fairbanks, G., Avruch, J. 1974. Phosphorylation of endogenous substrates by erythrocyte membrane protein kinases. II. Cyclic adenosine monophosphate-stimulated reactions.Biochemistry 13:5514PubMedGoogle Scholar
  9. 9.
    Fairbanks, G., Steck, T.L., Wallach, D.F.H. 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membraneBiochemistry 10:2606PubMedGoogle Scholar
  10. 10.
    Garrett, R.J.B., Redman, C.M. 1975. Localization of enzymes involved in polyphosphoinositide metabolism on the cytoplasmic surface of the human erythrocyte membrane.Biochim. Biophys. Acta 382:58PubMedGoogle Scholar
  11. 11.
    Hosey, M.M., Tao, M. 1977. Selective phosphorylation of erythrocyte membrane proteins by the solubilized membrane protein kinases.Biochemistry 16:4578PubMedGoogle Scholar
  12. 12.
    Hosey, M.M., Tao, M. 1977. Protein kinases of rabbit and human erythrocyte membranes. Solubilization and characterization.Biochim. Biophys. Acta 482:348PubMedGoogle Scholar
  13. 13.
    Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurements with the Folin phenol reagent.J. Biol. Chem. 193:265PubMedGoogle Scholar
  14. 14.
    Missirlis, Y.F., Kohn, I., Vickers, J.D., Rathbone, M.P., Chui, D.H., McComas, A.J., Brain, M.C. 1977.In: Erythrocyte Membranes: Recent Clinical and Experimental Advances. W.C. Kruckeberg, J.W. Eaton, and G.J. Brewer, editors. pp. 189–200. Alan R. Liss, New YorkGoogle Scholar
  15. 15.
    Roelofsen, B., Schatzmann, H.J. 1977. The lipid requirement of the (Ca2++Mg2+)-ATPase in the human erythrocyte membrane, as studied by various highly purified phospholipases.Biochim. Biophys. Acta 464:17PubMedGoogle Scholar
  16. 16.
    Roses, A.D., Appel, S.H. 1973. Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy.Proc. Nat. Acad. Sci. USA 70:1855PubMedGoogle Scholar
  17. 17.
    Roses, A.D., Herbstreith, M.H., Appel, S.H. 1975. Membrane protein kinase alteration in Duchenne muscular dystrophy.Nature (London) 254:350Google Scholar
  18. 18.
    Semenuk, M., Vickers, J., Rathbone, M.P., Brain, M.C. 1977. The effect of cholesterolloading of red blood cells (RBCs) on membrane-bound protein kinase (PKase).Clin. Res. 25:695AGoogle Scholar
  19. 19.
    Sha'afi, R.I., Rodan, S.B., Hintz, R.L., Fernandez, S.M., Rodan, G.A. 1975. Abnormalities in membrane microviscosity and ion transport in genetic muscular dystrophy.Nature (London) 254:525Google Scholar
  20. 20.
    Steck, T.L. 1974. Preparation of impermeable inside-out and right-side-out vesicles from erythrocyte membranes.In: Methods in Membrane Biology. E. Korn, editor. Vol. 2, pp. 245–281. Plenum, New YorkGoogle Scholar
  21. 21.
    Vickers, J.D., McComas, A.J., Rathbone, M.P. 1977. Altered activity and temperature response of membrane-bound protein kinases in erythrocytes of patients with myotonic muscular dystrophy.Neurosci. Abstr. 3:224Google Scholar
  22. 22.
    Vickers, J.D., McComas, A.J., Rathbone, M.P. 1978. Alterations of membrane phosphorylation in erythrocyte membranes from patients with Duchenne muscular dystrophy.Can. J. Neurol. Sci. 5:437PubMedGoogle Scholar
  23. 23.
    Vickers, J.D., McComas, A.J., Rathbone, M.P. 1979. Myotonic muscular dystrophy: Abnormal temperature response of membrane phosphorylation in erythrocyte membranes.Neurology 29:791PubMedGoogle Scholar
  24. 24.
    Vickers, J.D., Rathbone, M.P. 1977. Effect of erythrocyte membrane cholesterol on a membrane protein kinase activity.Proc. Can. Fed. Biol. Soc. 20:65Google Scholar
  25. 25.
    Warren, G.B., Housley, M.D., Metcalf, J.C., Birdsall, N.J.M. 1975. Cholesterol is excluded from the phospholipid annulus surrounding an active calcium transport protein.Nature (London) 255:684Google Scholar

Copyright information

© Springer-Verlag New York Inc 1979

Authors and Affiliations

  • J. D. Vickers
    • 1
  • J. Brierley
    • 1
  • M. P. Rathbone
    • 1
  1. 1.MRC Group in Developmental Neurobiology, Department of NeurosciencesMcMaster University Medical CentreHamiltonCanada

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