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. 1986 Jul 1;237(1):53–61. doi: 10.1042/bj2370053

Protein kinase C and an endogenous substrate associated with adenohypophyseal secretory granules.

J L Turgeon, R H Cooper
PMCID: PMC1146947  PMID: 3026314

Abstract

Secretory granules isolated from anterior pituitary glands were examined for Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity as well as the occurrence of granule-associated substrate proteins. Sheep adenohypophyses were fractionated by differential and sucrose-density-gradient centrifugation to yield a granule fraction enriched for luteinizing-hormone (lutropin)-containing secretory granules. Marker-enzyme analysis showed no detectable cytosolic contamination, although there were small amounts of plasma membranes (2-4%) and lysosomes (4-6%) associated with the preparation. As determined by histone-H1 phosphorylation after DEAE-cellulose DE-52 chromatography, protein kinase C activity with a marked dependence on Ca2+ and lipid (4-fold increase in their presence) was evident in the secretory-granule fraction. Phosphorylation in vitro of the secretory-granule fraction by endogenous and exogenous protein kinase C revealed a protein of Mr 36,000, which by two-dimensional SDS/polyacrylamide-gel electrophoresis showed multiple sites of phosphorylation. The Mr-36,000 protein was not found in cytosolic or plasma-membrane fractions and was not phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Several secretory-granule proteins served as substrates for the catalytic subunit, the most prominent of which were of Mr 63,000, 23,000 and 21,000. From these data, we suggest that phosphorylation of secretory-granule-associated proteins by protein kinase C and by cyclic AMP-dependent protein kinase may be important in secretion regulation in the anterior pituitary gland.

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Selected References

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

  1. Beavo J. A., Bechtel P. J., Krebs E. G. Preparation of homogeneous cyclic AMP-dependent protein kinase(s) and its subunits from rabbit skeletal muscle. Methods Enzymol. 1974;38:299–308. doi: 10.1016/0076-6879(74)38046-9. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Brocklehurst K. W., Hutton J. C. Involvement of protein kinase C in the phosphorylation of an insulin-granule membrane protein. Biochem J. 1984 May 15;220(1):283–290. doi: 10.1042/bj2200283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cochet C., Gill G. N., Meisenhelder J., Cooper J. A., Hunter T. C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Feb 25;259(4):2553–2558. [PubMed] [Google Scholar]
  5. Corbin J. D., Reimann E. M. Assay of cyclic AMP-dependent protein kinases. Methods Enzymol. 1974;38:287–290. doi: 10.1016/0076-6879(74)38044-5. [DOI] [PubMed] [Google Scholar]
  6. Creutz C. E., Dowling L. G., Sando J. J., Villar-Palasi C., Whipple J. H., Zaks W. J. Characterization of the chromobindins. Soluble proteins that bind to the chromaffin granule membrane in the presence of Ca2+. J Biol Chem. 1983 Dec 10;258(23):14664–14674. [PubMed] [Google Scholar]
  7. Creutz C. E., Harrison J. R. Clathrin light chains and secretory vesicle binding proteins are distinct. Nature. 1984 Mar 8;308(5955):208–210. doi: 10.1038/308208a0. [DOI] [PubMed] [Google Scholar]
  8. Drust D. S., Martin T. F. Protein kinase C translocates from cytosol to membrane upon hormone activation: effects of thyrotropin-releasing hormone in GH3 cells. Biochem Biophys Res Commun. 1985 Apr 30;128(2):531–537. doi: 10.1016/0006-291x(85)90079-8. [DOI] [PubMed] [Google Scholar]
  9. Garrison J. C., Wagner J. D. Glucagon and the Ca2+-linked hormones angiotensin II, norepinephrine, and vasopressin stimulate the phosphorylation of distinct substrates in intact hepatocytes. J Biol Chem. 1982 Nov 10;257(21):13135–13143. [PubMed] [Google Scholar]
  10. Hatada Y., Munemura M., Fukunaga K., Yamamoto H., Maeyama M., Miyamoto E. Calmodulin and Ca2+- and calmodulin-dependent protein kinase in rat anterior pituitary gland. J Neurochem. 1983 Apr;40(4):1082–1089. doi: 10.1111/j.1471-4159.1983.tb08096.x. [DOI] [PubMed] [Google Scholar]
  11. Hirota K., Hirota T., Aguilera G., Catt K. J. Hormone-induced redistribution of calcium-activated phospholipid-dependent protein kinase in pituitary gonadotrophs. J Biol Chem. 1985 Mar 25;260(6):3243–3246. [PubMed] [Google Scholar]
  12. Ipata P. L. A coupled optical enzyme assay for 5'-nucleotidase. Anal Biochem. 1967 Jul;20(1):30–36. doi: 10.1016/0003-2697(67)90261-8. [DOI] [PubMed] [Google Scholar]
  13. Kikkawa U., Minakuchi R., Takai Y., Nishizuka Y. Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) from rat brain. Methods Enzymol. 1983;99:288–298. doi: 10.1016/0076-6879(83)99064-x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kraft A. S., Anderson W. B. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature. 1983 Feb 17;301(5901):621–623. doi: 10.1038/301621a0. [DOI] [PubMed] [Google Scholar]
  16. Labrie F., Lemaire S., Poirier G., Pelletier G., Boucher R. Adenohypophyseal secretory granules. I. Their phosphorylation and association with protein kinase. J Biol Chem. 1971 Dec 10;246(23):7311–7317. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lemaire S., Pelletier G., Labrie F. Adenosine 3',5'-monophosphate-dependent protein kinase from bovine anterior pituitary gland. II. Subcellular distribution. J Biol Chem. 1971 Dec 10;246(23):7303–7310. [PubMed] [Google Scholar]
  19. Lewin M. J., Reyl-Desmars F., Ling N. Somatocrinin receptor coupled with cAMP-dependent protein kinase on anterior pituitary granules. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6538–6541. doi: 10.1073/pnas.80.21.6538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Loh Y. P., Gainer H. Characterization of pro-opiocortin-converting activity in purified secretory granules from rat pituitary neurointermediate lobe. Proc Natl Acad Sci U S A. 1982 Jan;79(1):108–112. doi: 10.1073/pnas.79.1.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Melloni E., Pontremoli S., Michetti M., Sacco O., Sparatore B., Salamino F., Horecker B. L. Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring proteinase. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6435–6439. doi: 10.1073/pnas.82.19.6435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Naka M., Nishikawa M., Adelstein R. S., Hidaka H. Phorbol ester-induced activation of human platelets is associated with protein kinase C phosphorylation of myosin light chains. Nature. 1983 Dec 1;306(5942):490–492. doi: 10.1038/306490a0. [DOI] [PubMed] [Google Scholar]
  23. Nansel D. D., Gudelsky G. A., Porter J. C. Subcellular localization of dopamine in the anterior pituitary gland of the rat: apparent association of dopamine with prolactin secretory granules. Endocrinology. 1979 Nov;105(5):1073–1077. doi: 10.1210/endo-105-5-1073. [DOI] [PubMed] [Google Scholar]
  24. Nishizuka Y. Turnover of inositol phospholipids and signal transduction. Science. 1984 Sep 21;225(4668):1365–1370. doi: 10.1126/science.6147898. [DOI] [PubMed] [Google Scholar]
  25. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  26. Russell J. T. The isolation of purified neurosecretory vesicles from bovine neurohypophysis using isoosmolar density gradients. Anal Biochem. 1981 May 15;113(2):229–238. doi: 10.1016/0003-2697(81)90071-3. [DOI] [PubMed] [Google Scholar]
  27. Summers T. A., Creutz C. E. Phosphorylation of a chromaffin granule-binding protein by protein kinase C. J Biol Chem. 1985 Feb 25;260(4):2437–2443. [PubMed] [Google Scholar]
  28. Südhof T. C., Ebbecke M., Walker J. H., Fritsche U., Boustead C. Isolation of mammalian calelectrins: a new class of ubiquitous Ca2+-regulated proteins. Biochemistry. 1984 Mar 13;23(6):1103–1109. doi: 10.1021/bi00301a010. [DOI] [PubMed] [Google Scholar]
  29. Thams P., Capito K., Hedeskov C. J. Endogenous substrate proteins for Ca2+-calmodulin-dependent, Ca2+-phospholipid-dependent and cyclic AMP-dependent protein kinases in mouse pancreatic islets. Biochem J. 1984 Jul 1;221(1):247–253. doi: 10.1042/bj2210247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Turgeon J. L., Ashcroft S. J., Waring D. W., Milewski M. A., Walsh D. A. Characteristics of the adenohypophyseal Ca2+-phospholipid-dependent protein kinase. Mol Cell Endocrinol. 1984 Feb;34(2):107–112. doi: 10.1016/0303-7207(84)90061-3. [DOI] [PubMed] [Google Scholar]
  31. Turgeon J. L., Waring D. W. cAMP augmentation of secretagogue-induced luteinizing hormone secretion. Am J Physiol. 1986 Jan;250(1 Pt 1):E62–E68. doi: 10.1152/ajpendo.1986.250.1.E62. [DOI] [PubMed] [Google Scholar]
  32. Uchida T., Filburn C. R. Affinity chromatography of protein kinase C-phorbol ester receptor on polyacrylamide-immobilized phosphatidylserine. J Biol Chem. 1984 Oct 25;259(20):12311–12314. [PubMed] [Google Scholar]
  33. Werth D. K., Niedel J. E., Pastan I. Vinculin, a cytoskeletal substrate of protein kinase C. J Biol Chem. 1983 Oct 10;258(19):11423–11426. [PubMed] [Google Scholar]
  34. Witters L. A., Vater C. A., Lienhard G. E. Phosphorylation of the glucose transporter in vitro and in vivo by protein kinase C. 1985 Jun 27-Jul 3Nature. 315(6022):777–778. doi: 10.1038/315777a0. [DOI] [PubMed] [Google Scholar]
  35. Wolf M., LeVine H., 3rd, May W. S., Jr, Cuatrecasas P., Sahyoun N. A model for intracellular translocation of protein kinase C involving synergism between Ca2+ and phorbol esters. Nature. 1985 Oct 10;317(6037):546–549. doi: 10.1038/317546a0. [DOI] [PubMed] [Google Scholar]

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