Phosphorylation of the ribosomal protein S6 during agonist-induced exocytosis in exocrine glands is catalyzed by calcium-phospholipid-dependent protein kinase (protein kinase C). Experiments with guinea pig parotid glands.

The ribosomal protein S6 in exocrine cells is phosphorylated during stimulation of exocytosis by cAMP-dependent or calcium-dependent agonists. Under both conditions the same tryptic S6 phosphopeptides (termed A, B, and C) were found [Padel, Kruppa, Jahn & Söling (1983) FEBS Lett. 159, 112-118]. Studies have now been made of the phosphorylation pattern of protein S6 from purified guinea pig parotid ribosomes following in vitro phosphorylation with calmodulin-dependent, phospholipid-dependent, and cAMP-dependent protein kinases. Only the phospholipid-dependent enzyme led to the phosphorylation of peptides A, B, and C, while the cAMP-dependent enzyme phosphorylated only peptides A and C, and the calmodulin-dependent enzyme did not phosphorylate any of the phosphopeptides found in S6 from unstimulated or stimulated intact cells. Guinea pig parotid microsomes contain substantial phospholipid-dependent protein kinase activity. Stimulation of intact parotid glands with tetradecanoylphorbol acetate led to a significant phosphorylation of S6 and a similar tryptic S6 phosphopeptide pattern as seen with carbamoylcholine. It is concluded that activation of phospholipid-dependent protein kinase is responsible for the phosphorylation of protein S6 during stimulation with calcium-dependent and cAMP-dependent secretagogues.

Regulation of protein kinases in exocrine secretory cells during agonist-induced exocytosis.

Stimulation of exocytosis in exocrine glands is associated with an increased phosphorylation of several particulate proteins. Irrespective of the type of secretagogue (cAMP-dependent agonists, calcium-dependent agonists, calcium ionophores, phorbol esters) exocytosis is always accompanied by an enhanced phosphorylation of the ribosomal protein S6. It is shown by an analysis of the phosphopeptide pattern of the in vivo and the in vitro phosphorylated S6 protein that the protein kinase responsible for phosphorylation of the S6 protein during enhanced exocytosis is protein kinase C. This is so irrespective of whether the agonist uses cAMP or calcium as second messenger. Experiments with isolated guinea pig parotid gland lobules reveal that not only the acetylcholine analog carbamoylcholine, but also the beta-agonist isoproterenol lead within seconds to an increased formation of diacylglycerol. As diacylglycerol increases the affinity of protein kinase C for calcium this finding would explain why the phosphorylation pattern of the S6 protein reflects activation of protein kinase C also under conditions where (as in the case of stimulation with beta-agonists) cAMP is the primary second messenger. It would further explain why the changes of the phosphorylation of individual histones observed during agonist-induced exocytosis in the parotid gland are quite similar for isoproterenol on one hand and carbamoylcholine on the other. A 22 K protein which becomes phosphorylated only when cAMP serves as second messenger is located in the membrane of the endoplasmic reticulum. A possible relationship of this protein with the calcium transport ATPase of the endoplasmic reticulum is under investigation.

Phosphopeptide patterns of the ribosomal protein S6 following stimulation of guinea pig parotid glands by secretagogues involving either cAMP or calcium as second messenger.

Stimulation of secretion in exocrine cells is associated with the incorporation of up to 3 to 4 phosphates into the ribosomal protein S6. This occurs with secretagogues involving either cAMP or free calcium as second messenger. An analysis of the phosphorylation pattern of S6 from stimulated guinea pig parotid glands reveals 3 phosphopeptides (termed A,B,C). The phosphopeptide pattern was identical for cAMP- or calcium-mediated stimulation, whereas phosphorylation of the S6 protein in vitro with catalytic subunit of cAMP-dependent protein kinase resulted only in the formation of phosphopeptides A and C. Therefore, secretagogue-mediated phosphorylation is not or not exclusively catalyzed by cAMP-dependent protein kinase even when cAMP is the second messenger.

Absence of a direct role of phospholipid methylation in stimulus-secretion coupling and control of adenylate cyclase in guinea-pig and rat parotid gland.

The present study was undertaken to investigate a possible involvement of phospholipid methyltransferases in the coupling of receptor-mediated stimulation to secretion. Phospholipid methyltransferases were assayed in isolated parotid acini in the presence of carbamoylcholine or isoprenaline. Carbamoylcholine reduced the incorporation of methyl groups into phospholipids, whereas isoprenaline showed no effect. Amylase secretion stimulated either by carbamoylcholine or by isoprenaline could not be affected by inhibitors of methyltransferases (3-deaza-adenosine alone or plus homocysteine thiolactone) under conditions where phospholipid methylation was strongly inhibited. The activity of adenylate cyclase in isolated parotid microsomal membranes was not inhibited or stimulated by S-adenosyl-homocysteine or -methionine respectively. These results indicate that phospholipid methylation does not play an essential role in stimulus-secretion coupling in the parotid gland.