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Okadaic acid identifies a phosphorylation/dephosphorylation cycle controlling the inhibitory guanine-nucleotide-binding regulatory protein Gi2.

ABSTRACT: 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.

SUBMITTER: Bushfield M

PROVIDER: S-EPMC1150139 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

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Project description:A phosphorylated form of alpha-Gi-2 (the alpha-subunit of Gi-2), immunoprecipitated from hepatocytes under basal conditions, migrated as a single species of pI approximately 5.7, the labelling of which increased approximately 2-fold in cells challenged with either vasopressin or phorbol 12-myristate 13-acetate (PMA); agents which activate protein kinase C. In contrast, treatment of hepatocytes with 8-bromo-cyclic AMP produced a more acidic species of phosphorylated alpha-Gi-2 having a pI of approximately 5.4 and whose labelling was increased approximately 3-fold. Trypsin digestion of labelled alpha-Gi-2 isolated from hepatocytes under basal conditions identified, on two-dimensional peptide analyses, three positively charged phosphoserine-containing peptides (C1, C2 and C3), with only peptides C1 and C2 being evident upon less extensive digestion with trypsin. These are suggested to reflect a single site of phosphorylation, with proteolysis by trypsin being incomplete, and where C2 is larger than C1, which is larger than C3. An identical pattern of tryptic phosphopeptides was seen in hepatocytes treated with either vasopressin or PMA, although labelling of this group of peptides was increased by approximately 2-fold compared with the basal state. In contrast, treatment of hepatocytes with glucagon, 8-bromo-cyclic AMP or forskolin not only resulted in increased labelling of the 'basal' sites approximately 3-fold, but identified a novel positively charged tryptic phosphoserine-containing peptide (AN). All four tryptic peptides were susceptible to proteolysis by V8 protease. Treatment of labelled alpha-Gi-2 from basal and PMA-treated cells produced a pattern of peptides which was identical with those found when the tryptic phosphopeptide was treated with V8 protease. We tentatively suggest that, on alpha-Gi-2, Ser144 is phosphorylated through the action of protein kinase C and Ser207 is phosphorylated upon elevation of the intracellular concentrations of cyclic AMP.

Project description:We have previously shown that the stimulatory effects of guanine nucleotides, N-ethylcarboxamide-adenosine and other agonists on adenylate cyclase activity were diminished in aorta and heart sarcolemma of spontaneously hypertensive rats (SHR) [Anand-Srivastava (1988) Biochem. Pharmacol. 37, 3017-3022]. In the present studies, we have examined whether the decreased response of these agonists is due to the defective GTP-binding proteins (G-proteins) which couple the receptors to adenylate cyclase, and have therefore measured the levels of G-proteins in aorta and heart from SHR and their respective Wistar-Kyoto (WKY) controls by using pertussis toxin (PT)- and cholera toxin (CT)-catalysed ADP-ribosylations and immunoblotting techniques using specific antibodies against G-proteins. The labelling with [32P]NAD+ and PT identified a 40/41 kDa protein in heart and aorta from WKY and SHR and was significantly increased in the hearts (approximately 100%) and aorta (approximately 30-40%), from SHR as compared with WKY. Immunoblotting revealed an increase in the levels of the G-protein alpha-subunits Gi alpha-2 and Gi alpha-3 in heart and Gi alpha-2 in aorta, whereas no change in Go alpha was observed in heart from SHR and WKY. On the other hand, no differences were observed in CT labelling or immunoblotting of stimulatory G-protein (Gs) in heart and aorta from WKY and SHR. In addition, CT stimulated the adenylate cyclase activity in heart sarcolemma from WKY and SHR to a similar extent. These results were correlated with adenylate cyclase inhibition and stimulation by various hormones. Angiotensin II (AII), atrial natriuretic factor (ANF) and oxotremorine-mediated inhibition was found to be greater in SHR as compared with WKY, whereas the stimulatory effects of adrenaline, isoprenaline, dopamine and forskolin were diminished in SHR aorta as compared to WKY. These results indicate that regulatory protein G(i) is more expressed in SHR, which may be associated with the decreased responsiveness of stimulatory hormones and increased sensitivity of inhibitory hormones to stimulate/inhibit adenylate cyclase activity. It may thus be suggested that the enhanced G(i) activity may be one of the mechanisms responsible for the diminished vascular tone and impaired myocardial functions in hypertension.

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Okadaic acid identifies a phosphorylation/dephosphorylation cycle controlling the inhibitory guanine-nucleotide-binding regulatory protein Gi2.

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