Okadaic acid identifies a phosphorylation/dephosphorylation cycle controlling the inhibitory guanine-nucleotide-binding regulatory protein Gi2.
Recently, the alpha-subunit of the inhibitory guanine-nucleotide-binding protein Gi2 (alpha-Gi2) has been shown to be a substrate for phosphorylation both by protein kinase C and also by other unidentified kinase(s) which are activated as a result of elevated cyclic AMP levels in intact rat hepatocytes [Bushfield, Murphy, Lavan, Parker, Hruby, Milligan & Houslay (1990) Biochem. J. 268, 449-457]. Here we show that the incorporation of [32P]Pi into alpha-Gi2 was enhanced 3-fold by incubation of intact hepatocytes with the tumour promoter and protein phosphatase (1 and 2A) inhibitor, okadaic acid. This action was both time- and concentration-dependent and was accompanied by a loss of guanine-nucleotide-induced inhibition of adenylate cyclase. The increased labelling of alpha-Gi2 induced by okadaic acid was partially additive with that elicited by 8-bromo cyclic AMP, but not with that elicited by the protein kinase C activator phorbol 12-myristate 13-acetate. We suggest that, in the absence of hormones, the activity of alpha-Gi2 is under the control of a dynamic phosphorylation/dephosphorylation system involving protein kinase C and protein phosphatases 1 and/or 2A. This highlights the regulation of kinases and phosphatases as both providing potentially important mechanisms for causing 'cross-talk' between different signalling systems, in this instance controlling cellular responsiveness through regulation of alpha-Gi2 phosphorylation.
Bushfield
M
M
Department of Biochemistry, University of Glasgow, U.K.
Lavan
B E
BE
Houslay
M D
MD
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004988:Ethers, Cyclic / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D005934:Glucagon / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D019319:Okadaic Acid; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
D004988:Ethers, Cyclic; D007476:Ionophores; D046911:Macromolecular Substances; D014667:Vasopressins; D019319:Okadaic Acid; D015124:8-Bromo Cyclic Adenosine Monophosphate; D005934:Glucagon; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate