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. 1977 Sep 1;165(3):469–477. doi: 10.1042/bj1650469

Purification and properties of a nuclear protein kinase from rat mammary gland

Gopal C Majumder 1,*
PMCID: PMC1164929  PMID: 921760

Abstract

A nuclear protein kinase that shows a high degree of substrate specificity for the phosphorylation of the acidic proteins casein, phosvitin and non-histone chromatin proteins, rather than the basic proteins histones and protamine, was partially purified from lactatingrat mammary gland. The enzyme is associated with the acidic protein fraction of chromatin. Nuclear kinase requires Co2+ for activity, and other bivalent cations such as Mg2+ and Mn2+ can substitute partially for Co2+. The kinase is further activates (2–3-fold) by various salts, their concentration for maximum stimulation being: NaCl, 150mm; KCl, 200mm; sodium acetate, 300mm. The sedimentation coefficient of the nuclear kinase is 8.9S and its mol.wt. is approx. 300000 by gel-exclusion chromatography. The enzyme is not activated by cyclic AMP or cyclic GMP and is inhibited neither by the regulatory subunit of mammary cyclic AMP-dependent protein kinase nor by the heat-stable protein kinase inhibitor from ox heart. Analysis of 32P-labelled protein products reveals that the kinase transfers the terminal phosphate of ATP to serine and threonine residues of proteins. The enzyme, however, has specificity for the phosphorylation of threonine in casein and serine in phosvitin. Molecular size and enzymic characteristics of the nuclear protein kinase are clearly different from those of the cytosol enzyme previously characterized.

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Selected References

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

  1. Ashby C. D., Walsh D. A. Characterization of the interaction of a protein inhibitor with adenosine 3',5'-monophosphate-dependent protein kinases. I. Interaction with the catalytic subunit of the protein kinase. J Biol Chem. 1972 Oct 25;247(20):6637–6642. [PubMed] [Google Scholar]
  2. CHAUVEAU J., MOULE Y., ROUILLER C. Isolation of pure and unaltered liver nuclei morphology and biochemical composition. Exp Cell Res. 1956 Aug;11(2):317–321. doi: 10.1016/0014-4827(56)90107-0. [DOI] [PubMed] [Google Scholar]
  3. Desjardins P. R., Lue P. F., Liew C. C., Gornall A. G. Purification and properties of rat liver nuclear protein kinases. Can J Biochem. 1972 Dec;50(12):1249–1259. doi: 10.1139/o72-170. [DOI] [PubMed] [Google Scholar]
  4. Erlichman J., Hirsch A. H., Rosen O. M. Interconversion of cyclic nucleotide-activated and cyclic nucleotide-independent forms of a protein kinase from beef heart. Proc Natl Acad Sci U S A. 1971 Apr;68(4):731–735. doi: 10.1073/pnas.68.4.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gill G. N., Garren L. D. Role of the receptor in the mechanism of action of adenosine 3':5'-cyclic monophosphate. Proc Natl Acad Sci U S A. 1971 Apr;68(4):786–790. doi: 10.1073/pnas.68.4.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Higashino H., Takeda M. Changes in adenosine 3',5'-monophosphate level and protein kinase activity by glucagon in rat liver nuclei. J Biochem. 1974 Jan;75(1):189–191. doi: 10.1093/oxfordjournals.jbchem.a130374. [DOI] [PubMed] [Google Scholar]
  7. Kadohama N., Turkington R. W. Changes in acidic chromatin proteins during mammary cell differentiation. Can J Biochem. 1973 Aug;51(8):1167–1176. doi: 10.1139/o73-153. [DOI] [PubMed] [Google Scholar]
  8. Kamiyama M., Dastugue B., Defer N., Kruh J. Liver chromatin non-histone proteins. Partial fractionation and mechanism of action on RNA synthesis. Biochim Biophys Acta. 1972 Sep 14;277(3):576–583. doi: 10.1016/0005-2787(72)90101-3. [DOI] [PubMed] [Google Scholar]
  9. Kish V. M., Kleinsmith L. J. Nuclear protein kinases. Evidence for their heterogeneity, tissue specificity, substrate specificities, and differential responses to cyclic adenosine 3':5'-monophosphate. J Biol Chem. 1974 Feb 10;249(3):750–760. [PubMed] [Google Scholar]
  10. Kleinsmith L. J., Allfrey V. G. Nuclear phosphoproteins. I. Isolation and characterization of a phosphoprotein fraction from calf thymus nuclei. Biochim Biophys Acta. 1969 Feb 4;175(1):123–135. [PubMed] [Google Scholar]
  11. Kleinsmith L. J., Heidema J., Carroll A. Specific binding of rat liver nuclear proteins to DNA. Nature. 1970 Jun 13;226(5250):1025–1026. doi: 10.1038/2261025a0. [DOI] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Langan T. A. Protein kinases and protein kinase substrates. Adv Cyclic Nucleotide Res. 1973;3:99–153. [PubMed] [Google Scholar]
  14. Majumder G. C., Turkington R. W. Adenosine 3',5'-monophosphate-dependent and -independent protein phosphokinase isoenzymes from mammary gland. J Biol Chem. 1971 Apr 25;246(8):2650–2657. [PubMed] [Google Scholar]
  15. Majumder G. C., Turkington R. W. Hormonal regulation of protein kinases and adenosine 3',5'-monophosphate-binding protein in developing mammary gland. J Biol Chem. 1971 Sep 25;246(18):5545–5554. [PubMed] [Google Scholar]
  16. Majumder G. C., Turkington R. W. Hormone-dependent phosphorylation of ribosomal and plasma membrane proteins in mouse mammary gland in vitro. J Biol Chem. 1972 Nov 25;247(22):7207–7217. [PubMed] [Google Scholar]
  17. McCarty K. S., Stafford D., Brown O. Resolution and fractionation of macromolecules by isokinetic sucrose density gradient sedimentation. Anal Biochem. 1968 Aug;24(2):314–329. doi: 10.1016/0003-2697(68)90185-1. [DOI] [PubMed] [Google Scholar]
  18. Miyamoto E., Petzold G. L., Harris J. S., Greengard P. Dissociation and concomitant activation of adenosine 3',5'-monophosphate-dependent protein kinase by histone. Biochem Biophys Res Commun. 1971 Jul 16;44(2):305–312. doi: 10.1016/0006-291x(71)90600-0. [DOI] [PubMed] [Google Scholar]
  19. Reimann E. M., Walsh D. A., Krebs E. G. Purification and properties of rabbit skeletal muscle adenosine 3',5'-monophosphate-dependent protein kinases. J Biol Chem. 1971 Apr 10;246(7):1986–1995. [PubMed] [Google Scholar]
  20. Ruddon R. W., Anderson S. L. Presence of multiple protein kinase activities in rat liver nuclei. Biochem Biophys Res Commun. 1972 Feb 25;46(4):1499–1508. doi: 10.1016/0006-291x(72)90777-2. [DOI] [PubMed] [Google Scholar]
  21. Shea M., Kleinsmith L. J. Template-specific stimulation of RNA synthesis by phosphorylated non-histone chromatin proteins. Biochem Biophys Res Commun. 1973 Jan 23;50(2):473–477. doi: 10.1016/0006-291x(73)90864-4. [DOI] [PubMed] [Google Scholar]
  22. Spelsberg T. C., Hnilica L. S. Deoxyribonucleoproteins and the tissue-specific restriction of the deoxyribonucleic acid in chromatin. Biochem J. 1970 Nov;120(2):435–437. doi: 10.1042/bj1200435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Takeda M., Yamamura H., Oga Y. Phosphoprotein kinases associated with rat liver chromatin. Biochem Biophys Res Commun. 1971 Jan 8;42(1):103–110. doi: 10.1016/0006-291x(71)90368-8. [DOI] [PubMed] [Google Scholar]
  24. Tao M., Salas M. L., Lipmann F. Mechanism of activation by adenosine 3':5'-cyclic monophosphate of a protein phosphokinase from rabbit reticulocytes. Proc Natl Acad Sci U S A. 1970 Sep;67(1):408–414. doi: 10.1073/pnas.67.1.408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Teng C. S., Teng C. T., Allfrey V. G. Studies of nuclear acidic proteins. Evidence for their phosphorylation, tissue specificity, selective binding to deoxyribonucleic acid, and stimulation effects on transcription. J Biol Chem. 1971 Jun 10;246(11):3597–3609. [PubMed] [Google Scholar]
  26. Turkington R. W., Majumder G. C., Kadoama N., MacIndoe J. H., Frantz W. L. Hormonal regulation of gene expression in mammary cells. Recent Prog Horm Res. 1973;29:417–455. doi: 10.1016/b978-0-12-571129-6.50015-4. [DOI] [PubMed] [Google Scholar]
  27. Turkington R. W., Riddle M. Hormone-dependent formation of polysomes in mammary cells in vitro. J Biol Chem. 1970 Oct 10;245(19):5145–5152. [PubMed] [Google Scholar]
  28. Ueda K., Reeder R. H., Honjo T., Nishizuka Y., Hayaishi O. Poly adenosine diphosphate ribose synthesis associated with chromatin. Biochem Biophys Res Commun. 1968 May 10;31(3):379–385. doi: 10.1016/0006-291x(68)90486-5. [DOI] [PubMed] [Google Scholar]

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