Protein kinase C and an endogenous substrate associated with adenohypophyseal secretory granules.
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.
D015720:5'-Nucleotidase; D000818:Animals; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005966:Glucuronidase / Q000378:metabolism; D007986:Luteinizing Hormone / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism; D010766:Phosphorylation; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D011493:Protein Kinase C / Q000378:metabolism*; D012756:Sheep; D013347:Subcellular Fractions / Q000378:metabolism
D007986:Luteinizing Hormone; D011493:Protein Kinase C; D009708:Nucleotidases; D015720:5'-Nucleotidase; D005966:Glucuronidase