title: Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells.
abstract: Stimulation of secretion from rat alveolar epithelial type II cells by the beta-adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion.
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
authors.forename: U J
authors.initials: UJ
authors.lastname: Zimmerman
authors.forename: M
authors.initials: M
authors.lastname: Wang
authors.forename: J B
authors.initials: JB
authors.lastname: Nelson
authors.forename: F S
authors.initials: FS
authors.lastname: Ekwunife
authors.forename: L
authors.initials: L
authors.lastname: Liu
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002469:Cell Separation; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D013726:Terbutaline / Q000494:pharmacology*
chemical_list: D000318:Adrenergic beta-Agonists; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; C069861:calpain inhibitor 2; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D002154:Calpain; D013726:Terbutaline