Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

Abstract

Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840-G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 muM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 muM) and cholecystokinin-(26-33)-peptide amide (CCK8, 0.1 muM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK8-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-alpha and, to a lesser extent, PKC-epsilon. In addition, TPA-induced inhibition of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-alpha. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca2+]i. Taken together, the present findings are in agreement with the idea that PKC-alpha-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca2+]i oscillations and eventually to inhibition of high-affinity Ca2+ signalling through the native CCK receptor in pancreatic acinar cells.

PKC‐mediated inhibitory feedback of the cholecystokinin 1 receptor controls the shape of oscillatory Ca2+ signals

“…Full recovery from TPA inhibition occurred within 1 h and was paralleled by the disappearance of PKCa, suggesting that of the three PKC isoforms present (a, e and f) this conventional PKC isoform, which is activated by the combined action of increased Ca 2+ and 1,2-DAG, mediates the inhibitory effect of TPA. Of note, the same conclusion was reached for pancreatic acinar cells [22]. Here we used CCKR1-mt cells to investigate the role of PKC phosphorylation in the transduction of extracellular hormonal signals into [Ca 2+ ] c oscillations at the experimental and theoretical level.…”

“…CCK-8-induced phosphorylation of CCK1R was not observed in cells in which PKC was downregulated by long-term exposure to TPA, indicat-ing that PKC mediates the effect of receptor activation on receptor phosphorylation. PKC downregulation increased the duration of the agonist-induced [Ca 2+ ] c oscillations while decreasing their frequency [22]. On the other hand, acute addition of TPA to cells displaying ongoing agonist-induced [Ca 2+ ] c oscillations decreased the frequency of these oscillations at lower TPA concentrations and immediately blocked these oscillations at higher TPA concentrations [23].…”

The novel protein kinase C isoforms -δ and -ε modulate caerulein-induced zymogen activation in pancreatic acinar cells

“…PKC has been shown to phosphorylate the CCK receptor (9,31). To determine whether the PKC effects we observed were downstream of the receptor, we examined the effects of kinase isoform inhibition in a cell-free reconstitution system.…”

Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCKA receptor

“…At first sight, this observation seems to exclude any important role for a staurosporine‐insensitive receptor kinase (GRK) in the attenuation of CCK‐8‐induced cyclic AMP formation in otherwise untreated CHO‐CCK A WT cells. However, in a previous study we provided evidence that PKC phosphorylation of the CCK A receptor is required for the action of a GRK (Smeets et al , 1998b).…”

“…The observation that TPA potently inhibits cell activation by physiological concentrations of CCK is in agreement with the idea that receptor phosphorylation leads to desensitization (for a review, see Willems et al , 1997). In a recent study, using freshly isolated pancreatic acinar cells in which PKC activity was down‐regulated by the prolonged action of TPA, we provided evidence that a basal level of PKC‐mediated receptor phosphorylation is required for the action of a putative receptor kinase (Smeets et al , 1998b). Together with the finding that CCK receptors engineered to have mutations in two (S260 and S264) of the four consensus sites for PKC action were not phosphorylated in response to CCK (Rao et al , 1997), this led us to speculate that CCK A receptors are phosphorylated in a hierarchical way, with PKC sites phosphorylated initially, making potential phosphorylation sites for a putative receptor kinase more accessible.…”