scholarly journals Multi-site phosphorylation of the inhibitory guanine nucleotide regulatory protein Gi-2 occurs in intact rat hepatocytes

1994 ◽  
Vol 301 (3) ◽  
pp. 693-702 ◽  
Author(s):  
N J Morris ◽  
M Bushfield ◽  
B E Lavan ◽  
M D Houslay
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Single Species ◽  
Kinase C ◽  
Protease Treatment ◽  
Guanine Nucleotide Regulatory Protein ◽  
Positively Charged

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.

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

1991 ◽  
Vol 274 (2) ◽  
pp. 317-321 ◽  
Author(s):  
M Bushfield ◽  
B E Lavan ◽  
M D Houslay
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Okadaic Acid ◽  
Regulatory Protein ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Kinase C ◽  
Guanine Nucleotide Binding

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.

scholarly journals Diabetes-induced alterations in the expression, functioning and phosphorylation state of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes

1990 ◽  
Vol 271 (2) ◽  
pp. 365-372 ◽  
Author(s):  
M Bushfield ◽  
S L Griffiths ◽  
G J Murphy ◽  
N J Pyne ◽  
J T Knowler ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylate Cyclase ◽  
G Protein ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Immunoblot Analysis ◽  
Kinase C ◽  
Liver Plasma Membranes ◽  
Guanine Nucleotide Regulatory Protein

Levels of the G-protein alpha-subunits alpha-Gi-2, alpha-Gi-3 and the 42 kDa, form of alpha-Gs were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in alpha-Gi subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of Gs were increased. G-protein beta subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of Gi-3, caused an increase in Gs transcripts and decreased transcript number for Gi-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, alpha-Gi-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than alpha-Gi-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of alpha-Gi-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of alpha-Gi-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5′-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.

Inhibition of epithelial Na+ transport by atriopeptin, protein kinase c, and pertussis toxin

1987 ◽  
Vol 253 (2) ◽  
pp. F372-F376 ◽  
Author(s):  
M. Mohrmann ◽  
H. F. Cantiello ◽  
D. A. Ausiello
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Regulatory Protein ◽  
Guanine Nucleotide ◽  
Kinase C ◽  
Guanine Nucleotide Regulatory Protein ◽  
Na Uptake ◽  
Atrial Natriuretic

We have recently shown the selective inhibition of an amiloride-sensitive, conductive pathway for Na+ by atrial natriuretic peptide and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) in the renal epithelial cell line, LLC-PK1. Using 22Na+ fluxes, we further investigated the modulation of Na+ transport by atrial natriuretic peptide and by agents that increase cGMP production, activate protein kinase c, or modulate guanine nucleotide regulatory protein function. Sodium nitroprusside increases intracellular cGMP concentrations without affecting cAMP concentrations and completely inhibits amiloride-sensitive Na+ uptake in a time- and concentration-dependent manner. In contrast, 8-BrcAMP is without effect on Na+ uptake through the Na+ channel. 1-Oleoyl 2-acetylglycerol (10 micrograms/ml) and phorbol 12-myristate 13-acetate (100 nM), activators of protein kinase c, inhibit Na+ uptake by 93 +/- 13 and 51 +/- 10%, respectively. Prolonged incubation with phorbol ester results in the downregulation of protein kinase c activity and reduces the inhibitory effect of atrial natriuretic peptide, suggesting that the action of this peptide involves stimulation of protein kinase c. Pertussis toxin, which induces the ADP-ribosylation of a 41-kDa guanine nucleotide regulatory protein in LLC-PK1 cells, inhibits 22Na+ influx to the same extent as amiloride. Thus, increasing cGMP, activating protein kinase c, and ADP-ribosylating a guanine nucleotide regulatory protein all inhibit Na+ uptake. These events may be sequentially involved in the action of atrial natriuretic peptide.

Phorbol esters inhibit adenylate cyclase activity in cultured collecting tubular cells

1988 ◽  
Vol 254 (1) ◽  
pp. C183-C191 ◽  
Author(s):  
B. S. Dixon ◽  
R. Breckon ◽  
C. Burke ◽  
R. J. Anderson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Regulatory Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Guanine Nucleotide ◽  
Tubular Cells ◽  
Kinase C ◽  
Guanine Nucleotide Regulatory Protein

Activators of protein kinase C, a calcium- and phospholipid-dependent protein kinase, inhibit vasopressin-stimulated water flow in toad bladder. To determine the biochemical mechanisms of this inhibition, we examined the effects of activators of protein kinase C on arginine vasopressin (AVP)-stimulated adenylate cyclase activity in cultured rabbit cortical collecting tubular cells. The phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), the diacylglycerol, 1-oleyl-2-acetyl glycerol (OAG), and the diacylglycerol kinase inhibitor, R59022, all rapidly activate protein kinase C in collecting tubular cells. Pretreatment with PMA produces a delayed inhibition (greater than or equal to 4 h) of AVP-stimulated adenylate cyclase activity. The 4-h time lag suggests that the effects of protein kinase C are mediated indirectly, possibly as a consequence of stimulating cell proliferation. PMA does not inhibit cholera toxin- or forskolin-stimulated adenylate cyclase activity, suggesting an effect on the vasopressin receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. Neither prostaglandins nor the inhibitory guanine nucleotide regulatory protein appear to mediate this effect. In contrast, treatment with either OAG or R59022 produces a rapid inhibition of both AVP- and forskolin-stimulated adenylate cyclase activity suggesting a prominent distal site of action, presumably at the catalytic subunit of adenylate cyclase. The results demonstrate that different activators of protein kinase C inhibit AVP-stimulated adenylate cyclase activity by distinctly different mechanisms possibly by altering the substrate specificity or activating multiple forms of the kinase. These results have important implications when using different activators to study the biological effects of protein kinase C.

Regulation of oxytocin secretion by the ovine corpus luteum: effect of activators of protein kinase C

1990 ◽  
Vol 124 (2) ◽  
pp. 225-232 ◽  
Author(s):  
J. J. Hirst ◽  
G. E. Rice ◽  
G. Jenkin ◽  
G. D. Thorburn
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Corpus Luteum ◽  
Phospholipase C ◽  
Tissue Slices ◽  
Kinase C ◽  
Luteal Tissue ◽  
Dose Dependent ◽  
Stimulation Of

ABSTRACT The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234·4 ± 32·8 pmol/g per h (n = 24) during 60-min incubations.Activators of protein kinase C: phorbol 12,13-dibutyrate (n = 8), phorbol 12-myristate,13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0·2 μmol/l). Phospholipase C (PLC; 50–250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4). These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum. Journal of Endocrinology (1990) 124, 225–232

scholarly journals Ca2+-induced insulin secretion from electrically permeabilized islets. Loss of the Ca2+-induced secretory response is accompanied by loss of Ca2+-induced protein phosphorylation

1992 ◽  
Vol 285 (3) ◽  
pp. 973-978 ◽  
Author(s):  
P M Jones ◽  
S J Persaud ◽  
S L Howell
Keyword(s):  
Protein Kinase C ◽  
Insulin Secretion ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Prolonged Exposure ◽  
Kinase C ◽  
Dependent Protein ◽  
32P Incorporation ◽  
Rat Islets Of Langerhans

Increasing the cytosolic Ca2+ concentration of electrically permeabilized rat islets of Langerhans caused rapid increases in insulin secretion and in 32P incorporation into islet proteins. However, the secretory responsiveness of permeabilized islets was relatively transient, with insulin secretion approaching basal levels within 20-30 min despite the continued presence of stimulatory concentrations of Ca2+. The loss of Ca2(+)-induced insulin secretion was accompanied by a marked reduction in Ca2(+)-dependent protein phosphorylation, but not in cyclic AMP-dependent protein phosphorylation. Similarly, permeabilized islets which were no longer responsive to Ca2+ were able to mount appropriate secretory responses to cyclic AMP and to a protein kinase C-activating phorbol ester. These results suggest that prolonged exposure to elevated cytosolic Ca2+ concentrations results in a specific desensitization of the secretory mechanism to Ca2+, perhaps as a result of a decrease in Ca2(+)-dependent kinase activity. Furthermore, these studies suggest that secretory responses of B-cells to cyclic AMP and activators of protein kinase C are not dependent upon the responsiveness of the cells to changes in cytosolic Ca2+.

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