Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 Mar 1;218(2):285–294. doi: 10.1042/bj2180285

Characterization of the protein kinase activities of human platelet supernatant and particulate fractions.

S E Salama, R J Haslam
PMCID: PMC1153340  PMID: 6324754

Abstract

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.

Full text

PDF
285

Images in this article

289Fig. 1.
on p.289
290Fig. 2.
on p.290
291Fig. 3.
on p.291

Selected References

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

  1. Bishop G. A., Rozenberg M. C. The effect of ADP, calcium and some inhibitors of platelet aggregation on protein phosphokinases from human blood platelets. Biochim Biophys Acta. 1975 Mar 28;384(1):112–119. doi: 10.1016/0005-2744(75)90100-x. [DOI] [PubMed] [Google Scholar]
  2. Booyse F. M., Marr J., Yang D. C., Guiliani D., Rafelson M. E., Jr Adenosine cyclic 3',5'-monophosphate-dependent protein kinase from human platelets. Biochim Biophys Acta. 1976 Jan 23;422(1):60–72. doi: 10.1016/0005-2744(76)90008-5. [DOI] [PubMed] [Google Scholar]
  3. Chambers D. A., Nachman R. L., Evarts J., Kinoshita T. Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling. Biochim Biophys Acta. 1982 Nov 24;719(2):208–214. doi: 10.1016/0304-4165(82)90090-3. [DOI] [PubMed] [Google Scholar]
  4. Corbin J. D., Keely S. L., Park C. R. The distribution and dissociation of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues. J Biol Chem. 1975 Jan 10;250(1):218–225. [PubMed] [Google Scholar]
  5. Corbin J. D., Sugden P. H., Lincoln T. M., Keely S. L. Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue. J Biol Chem. 1977 Jun 10;252(11):3854–3861. [PubMed] [Google Scholar]
  6. Dreyfuss G., Schwartz K. J., Blout E. R. Compartmentalization of cyclic AMP-dependent protein kinases in human erythrocytes. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5926–5930. doi: 10.1073/pnas.75.12.5926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Flockhart D. A., Corbin J. D. Regulatory mechanisms in the control of protein kinases. CRC Crit Rev Biochem. 1982 Feb;12(2):133–186. doi: 10.3109/10409238209108705. [DOI] [PubMed] [Google Scholar]
  8. Fox J. E., Say A. K., Haslam R. J. Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23187 or to prostaglandin E1. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport. Biochem J. 1979 Dec 15;184(3):651–661. doi: 10.1042/bj1840651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haslam R. J., Lynham J. A. Activation and inhibition of blood platelet adenylate cyclase by adenosine or by 2-chloroadenosine. Life Sci II. 1972 Dec 8;11(23):1143–1154. doi: 10.1016/0024-3205(72)90269-x. [DOI] [PubMed] [Google Scholar]
  10. Haslam R. J., Lynham J. A., Fox J. E. Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets. Biochem J. 1979 Feb 15;178(2):397–406. doi: 10.1042/bj1780397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hathaway D. R., Eaton C. R., Adelstein R. S. Regulation of human platelet myosin light chain kinase by the catalytic subunit of cyclic AMP-dependent protein kinase. Nature. 1981 May 21;291(5812):252–256. doi: 10.1038/291252a0. [DOI] [PubMed] [Google Scholar]
  12. Hofmann F., Beavo J. A., Bechtel P. J., Krebs E. G. Comparison of adenosine 3':5'-monophosphate-dependent protein kinases from rabbit skeletal and bovine heart muscle. J Biol Chem. 1975 Oct 10;250(19):7795–7801. [PubMed] [Google Scholar]
  13. Inoue M., Kishimoto A., Takai Y., Nishizuka Y. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. II. Proenzyme and its activation by calcium-dependent protease from rat brain. J Biol Chem. 1977 Nov 10;252(21):7610–7616. [PubMed] [Google Scholar]
  14. Juhl H., Esmann V. Purification and properies of cAMP dependent and independent histone kinases from human leukocytes. Mol Cell Biochem. 1979 Jul 15;26(1):3–18. doi: 10.1007/BF00226816. [DOI] [PubMed] [Google Scholar]
  15. Kaulen H. D., Gross R. Purification and properties of a soluble cyclic AMP-dependent protein kinase from human platelets. Hoppe Seylers Z Physiol Chem. 1974 Apr;355(4):471–480. doi: 10.1515/bchm2.1974.355.1.471. [DOI] [PubMed] [Google Scholar]
  16. Kawahara Y., Takai Y., Minakuchi R., Sano K., Nishizuka Y. Phospholipid turnover as a possible transmembrane signal for protein phosphorylation during human platelet activation by thrombin. Biochem Biophys Res Commun. 1980 Nov 17;97(1):309–317. doi: 10.1016/s0006-291x(80)80169-0. [DOI] [PubMed] [Google Scholar]
  17. Keely S. L., Jr, Corbin J. D., Park C. R. On the question of translocation of heart cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1501–1504. doi: 10.1073/pnas.72.4.1501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kikkawa U., Takai Y., Minakuchi R., Inohara S., Nishizuka Y. Calcium-activated, phospholipid-dependent protein kinase from rat brain. Subcellular distribution, purification, and properties. J Biol Chem. 1982 Nov 25;257(22):13341–13348. [PubMed] [Google Scholar]
  19. Kishimoto A., Kajikawa N., Shiota M., Nishizuka Y. Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease. J Biol Chem. 1983 Jan 25;258(2):1156–1164. [PubMed] [Google Scholar]
  20. Kishimoto A., Takai Y., Mori T., Kikkawa U., Nishizuka Y. Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J Biol Chem. 1980 Mar 25;255(6):2273–2276. [PubMed] [Google Scholar]
  21. Käser-Glanzmann R., Gerber E., Lüscher E. F. Regulation of the intracellular calcium level in human blood platelets: cyclic adenosine 3',5'-monophosphate dependent phosphorylation of a 22,000 dalton component in isolated Ca2+-accumulating vesicles. Biochim Biophys Acta. 1979 Dec 12;558(3):344–347. doi: 10.1016/0005-2736(79)90271-2. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lohmann S. M., Walter U., Greengard P. Identification of endogenous substrate proteins for cAMP-dependent protein kinase in bovine brain. J Biol Chem. 1980 Oct 25;255(20):9985–9992. [PubMed] [Google Scholar]
  24. Lyons R. M. Compartmentalization of adenosine 3':5'-monophosphate-binding proteins in human platelets. Thromb Res. 1980 Aug 1;19(3):317–332. doi: 10.1016/0049-3848(80)90260-1. [DOI] [PubMed] [Google Scholar]
  25. Lyons R. M., Stanford N., Majerus P. W. Thrombin-induced protein phosphorylation in human platelets. J Clin Invest. 1975 Oct;56(4):924–936. doi: 10.1172/JCI108172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rubin C. S. Characterization and comparison of membrane-associated and cytosolic cAMP-dependent protein kinases. Studies on human erythrocyte protein kinases. J Biol Chem. 1979 Dec 25;254(24):12439–12449. [PubMed] [Google Scholar]
  27. Salama S. E., Haslam R. J. Subcellular distribution of cyclic AMP-dependent protein kinase activity and of cyclic AMP-binding proteins in human platelets. Modification by Ca2+-dependent proteolysis. FEBS Lett. 1981 Aug 3;130(2):230–234. doi: 10.1016/0014-5793(81)81127-1. [DOI] [PubMed] [Google Scholar]
  28. Salzman E. W., Weisenberger H. Role of cyclic AMP in platelet function. Adv Cyclic Nucleotide Res. 1972;1:231–247. [PubMed] [Google Scholar]
  29. Steiner M. Endogenous phosphorylation of platelet membrane proteins. Arch Biochem Biophys. 1975 Nov;171(1):245–254. doi: 10.1016/0003-9861(75)90029-6. [DOI] [PubMed] [Google Scholar]
  30. Sugden P. H., Corbin J. D. Adenosine 3':5'-cyclic monophosphate-binding proteins in bovine and rat tissues. Biochem J. 1976 Nov;159(2):423–437. doi: 10.1042/bj1590423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Takai Y., Kishimoto A., Inoue M., Nishizuka Y. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J Biol Chem. 1977 Nov 10;252(21):7603–7609. [PubMed] [Google Scholar]
  32. Takai Y., Kishimoto A., Iwasa Y., Kawahara Y., Mori T., Nishizuka Y. Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. J Biol Chem. 1979 May 25;254(10):3692–3695. [PubMed] [Google Scholar]
  33. Takai Y., Minakuchi R., Kikkawa U., Sano K., Kaibuchi K., Yu B., Matsubara T., Nishizuka Y. Membrane phospholipid turnover, receptor function and protein phosphorylation. Prog Brain Res. 1982;56:287–301. doi: 10.1016/S0079-6123(08)63780-2. [DOI] [PubMed] [Google Scholar]
  34. Uno I., Ueda T., Greenglad P. Differences in properties of cytosol and membrane-derived protein kinases. J Biol Chem. 1976 Apr 10;251(7):2192–2195. [PubMed] [Google Scholar]
  35. Walter U., Kanof P., Schulman H., Greengard P. Adenosine 3':5'-monophosphate receptor proteins in mammalian brain. J Biol Chem. 1978 Sep 10;253(17):6275–6280. [PubMed] [Google Scholar]
  36. Yamaki T., Nishikawa M., Hidaka H. Calmodulin and acidic compounds alter basic protein phosphorylation by the protein kinase from human platelets. Biochim Biophys Acta. 1982 Feb 2;714(2):257–264. doi: 10.1016/0304-4165(82)90332-4. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES