title: PMA-stimulated protein kinases and the regulation of transfected glucagon receptors
Creators: name: Tobias, Edward S.
Publisher: name: University of Glasgow
Year: 1997
Subjects: subject: Molecular biology, protein kinases, glucagon.

description: Previous observations had suggested that the reduction in glucagon-stimulated adenylyl cyclase activity that was elicited by challenge of hepatocytes with phorbol 12- myristate 13-acetate (PMA) may have been mediated by the action of protein kinase C. In order to gain further insight into the nature of the protein kinase species which conferred such an inhibitory effect in these cells, I used a model system consisting of COS-7 cells transiently transfected so as to overexpress glucagon receptors. In these cells glucagon elicited a profound, dose-dependent, increase in the intracellular cAMP concentration with an EC50 of 1.8+/-0.4 nM glucagon. This value was comparable to those reported previously for the response observed in intact hepatocytes. In the transfected cells, levels of cAMP accumulation were maximal after approximately 10 minutes of stimulation with glucagon and thereafter remained stably elevated. These studies were undertaken in the presence of the non-selective phosphodiesterase inhibitor, IBMX, which elicited an inhibition of &gt;96% of total cAMP phosphodiesterase (PDE) activity in these cells. Glucagon challenge of the transfected COS-7 cells failed to elicit a significant stimulation of IP3 production. However, in the presence of 0.3% (v/v) butan-1-ol, the hormone elicited an increase in the level of [3H]-PtdOH, although not of [3H]-PtdBut, suggesting that the hydrolysis of PtdCho by PLC, but not by PLD, was stimulated by glucagon in these cells. Intriguingly, in contrast to previous observations made using hepatocytes, treatment with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels in these transfected cells. Furthermore, PMA-induced inhibition of the response was not conferred by varying the quantity of transfected DNA or by treating with the potent protein phosphatase inhibitor, okadaic acid. Nor was it observed following the co-transfection of the cells with cDNAs encoding various protein kinase C isoforms (PKC-alpha, PKC-betaII and PKC-epsilon) or the PMA-activated G-protein receptor kinases, GRK2 and GRK3. A striking PMA-induced inhibition (51%) of the glucagon-stimulated cAMP accumulation was, however, observed in COS-7 cells which had been co-transfected with a cDNA encoding the novel diacyl glycerol/phorbol ester-stimulated protein kinase, protein kinase D (PKD). This PMA-induced inhibitory effect in these co-transfected COS-7 cells was dependent upon the catalytic activity of the kinase since PMA failed to elicit a reduction in glucagon-stimulated cAMP accumulation in COS-7 cells which had been co-transfected with the glucagon receptor and a kinase-inactive form of PKD. Moreover, the effect appeared to be directed at the level of cAMP synthesis rather than its degradation as studies of homogenate cAMP PDE activity showed no change in total PDE activity and IBMX was able to inhibit a similar fraction of the total activity. In the transfected cells, treatment with PMA did not inhibit either [125I]-glucagon binding or GTP-induced glucagon dissociation. Furthermore, the intracellular cAMP accumulation elicited by either cholera toxin or forskolin was not reduced by treatment with the phorbol ester. No statistically significant PMA-induced reduction in the isoprenaline-stimulated cAMP accumulation was detectable in COS-7 cells transfected with PKD and either the beta2AR or the beta3AR. This is consistent with the possibility that, in the co-transfected COS cells in which the inhibitory effect is observed, PKD phosphorylates the glucagon receptor itself. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS-7 cells, whilst transcripts for GRK3 were not found in either cell type. Immunoblotting studies indicated that PKC-alpha, PKC-betaII and PKC-epsilon were expressed both in hepatocytes and in COS-7 cells, although the level of PKC-betaII appeared to be lower in the latter cell type. Such studies also indicated that the levels of PKC-alpha, PKC-betaII, PKC-epsilon and PKC-zeta in hepatocytes isolated from streptozotocin diabetic rats were 1.4-2.0 fold higher than in hepatocytes obtained from healthy control animals. In contrast, the level of PKD-specific transcript in hepatocytes and adipose tissue appeared to be markedly reduced in diabetic rats, and was restored by insulin treatment. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase activity in vivo and that the loss, in the diabetic state, of such a regulatory mechanism might constitute an important factor in the pathogenesis of hyperglycaemia.