title: Recovery from acute glucagon challenge in isolated rat hepatocytes: is protein dephosphorylation synchronous or asynchronous?
abstract: One-dimensional SDS-PAGE of cytosolic phosphopeptides confirms that glucagon promotes the phosphorylation of 11 phosphopeptides in isolated rat hepatocytes pre-equilibrated with 32PO4(3-). Nine of these phosphopeptides are tentatively identified, whereas two phosphopeptides (48 kDa and 46 kDa) remain unidentified. Transfer of the glucagon-challenged hepatocytes to medium free of 32PO4(3-) and glucagon led to the rapid net dephosphorylation of the phosphopeptides and to a rapid decline in the specific radioactivity of the [32P]ATP pool. There were profound differences between the post-glucagon rates of net dephosphorylation of the different hepatic phosphopeptides, consistent with net dephosphorylation being asynchronous during the recovery phase from acute glucagon challenge. On the basis of descending rates of dephosphorylation, four major groups of phosphopeptides were delineated. Okadaic acid, a potent inhibitor of protein phosphatase 2A and to a lesser extent protein phosphatase 1, inhibited the dephosphorylation of all of the phosphopeptides. A role for protein phosphatase 2A in protein dephosphorylation may be indicated by the observation that spermine, a specific activator of protein phosphatase 2A, stimulates the dephosphorylation of some, but not all, of the glucagon-stimulated phosphopeptides. Although phosphorylation during the recovery phase from glucagon challenge may be a complicating factor, the results suggest that post-glucagon dephosphorylation is a complex asynchronous process. The physiological consequences of this asynchrony may be that the suppression of glycogenolysis and gluconeogenesis and the activation of glycolysis are early events in the recovery process.
authors.affiliation: Department of Biochemistry, University of Western Australia, Nedlands.
authors.forename: S R
authors.initials: SR
authors.lastname: Aggarwal
authors.forename: T N
authors.initials: TN
authors.lastname: Palmer
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004988:Ethers, Cyclic / Q000494:pharmacology; D005934:Glucagon / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D019319:Okadaic Acid; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D013096:Spermine / Q000494:pharmacology
chemical_list: D004988:Ethers, Cyclic; D010750:Phosphoproteins; D019319:Okadaic Acid; D013096:Spermine; D005934:Glucagon