Protein kinase C phosphorylates the inositol 1,4,5-trisphosphate receptor type 2 and decreases the mobilization of Ca2+in pancreatoma AR4-2J cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor channel, which plays a major (IP3R) is an intracellular Ca2+ role in Ca2+ signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2 and IP3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP3R are poorly understood. AR4-2J cells, which express almost exclusively (~86%) the IP3R-2, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) influences IP3R-2-mediated Ca2+ release. Using an immunoprecipitation approach, we confirmed that AR4-2J cells express almost exclusively the IP3R-2 isoform. Using an in vitro phosphorylation assay, we showed that the immunopurified IP3R-2 was efficiently phosphorylated by exogenous PKC. In intact AR4-2J cells metabolically labelled with 32Pi, we showed that phorbol-12-myristate-13-acetate (PMA) and Ca2+ mobilizing agonists cause the phosphorylation of IP3R-2. In saponin-permeabilized AR4-2J cells, IP3-induced Ca2+ release was reduced after a pre-treatment with PMA or with exogenous PKC. PMA also reduced the Ca2+ response of intact AR4-2J cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. These results demonstrate that PKC decreases the Ca2+mobilizing activity of IP3R-2 and thus exerts a negative feedback on the agonists-induced Ca2+ response of AR4-2J cells.

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Mitogen stimulation of Na+-H+ exchange: differential involvement of protein kinase C

AJP Cell Physiology ◽

10.1152/ajpcell.1987.253.2.c219 ◽

1987 ◽

Vol 253 (2) ◽

pp. C219-C229 ◽

Cited By ~ 19

Author(s):

L. L. Muldoon ◽

G. A. Jamieson ◽

A. C. Kao ◽

H. C. Palfrey ◽

M. L. Villereal

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Types ◽

Intact Cells ◽

Kinase C ◽

Protein Kinase C Activity ◽

Human Fibroblast Strains ◽

Stimulation Of

The mitogen-induced activation of Na+-H+ exchange was investigated in two cultured human fibroblast strains (HSWP and WI-38 cells) that, based on previous studies, differed in their response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (L. M. Vincentini and M. L. Villereal, Proc. Natl. Acad. Sci. USA 82: 8053-8056, 1985). The role of protein kinase C in the activation of Na+-H+ exchange was investigated by comparing the effects of TPA on Na+ influx, in vitro phosphorylation, and in vivo phosphorylation in both cell types. Although both cell types have significant quantities of protein kinase C activity that can be activated by TPA in intact cells, the addition of TPA to intact cells stimulates Na+ influx in WI-38 cells but not in HSWP cells, indicating that in HSWP cells the stimulation of protein kinase C is not sufficient to activate the Na+-H+ exchanger. Cells were then depleted of protein kinase C activity by chronic treatment with high doses of TPA. Both HSWP and WI-38 cells were rendered protein kinase C deficient by this treatment as determined by in vitro and in vivo phosphorylation studies. Protein kinase C-deficient HSWP cells lose the ability for TPA to inhibit the serum-induced activation of Na+-H+ exchange, but there is no reduction in the stimulation of Na+ influx by serum, bradykinin, vasopressin, melittin, or vanadate, indicating that protein kinase C activity is not necessary for the mitogen-induced activation of Na+-H+ exchange in HSWP cells by agents known to stimulate phosphatidylinositol turnover (G. A. Jamieson and M. Villereal. Arch. Biochem. Biophys. 252: 478-486, 1987). In contrast, depletion of protein kinase C activity in WI-38 cells significantly reduces both the TPA- and the serum-induced activation of the Na+-H+ exchange system, suggesting that protein kinase C activity is necessary for at least a portion of the mitogen-induced activation of the Na+-H+ exchanger in WI-38 cells. These results indicate that the mechanisms for regulating Na+-H+ exchange can differ dramatically between different types of fibroblasts.

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Regulation of the phosphorylation of the inositol 1,4,5-trisphosphate receptor by protein kinase C

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.05.071 ◽

2004 ◽

Vol 319 (3) ◽

pp. 888-893 ◽

Cited By ~ 30

Author(s):

Elke Vermassen ◽

Rafael A Fissore ◽

Nael Nadif Kasri ◽

Veerle Vanderheyden ◽

Geert Callewaert ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Kinase C ◽

Inositol 1,4,5 Trisphosphate ◽

Trisphosphate Receptor

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Protein kinase C mediated activation and phosphorylation of Ca2+ pump in cardiac sarcolemma

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y92-171 ◽

1992 ◽

Vol 70 (9) ◽

pp. 1230-1235 ◽

Cited By ~ 11

Author(s):

Yi Qu ◽

Joseph Torchia ◽

Amar Kumar Sen

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Heart Tissue ◽

Cardiac Sarcolemma ◽

Pumping Rate ◽

Kinase C ◽

Calcium Affinity ◽

Intracellular Ca ◽

Sarcolemmal Vesicles

The effects of purified protein kinase C (PKC) on the Ca2+-pumping ATPase of cardiac sarcolemma were investigated. The addition of PKC to sarcolemmal vesicles resulted in a significant increase in ATP-dependent Ca2+ uptake, by increasing the calcium affinity by 2.8-fold (Km 0.14 vs. 0.4 μM for control) and by increasing Vmax from 5 to 6.8 nmol∙mg protein−1∙min−1. The addition of PKC also stimulated Ca2+ ATPase activity in sarcolemmal preparations. This activity was increased further upon the addition of calmodulin. These results suggest that PKC stimulates Ca2+ ATPase through a kinase-directed phosphorylation. The addition of PKC to a purified preparation of Ca2+ ATPase in the presence of [γ-32P]ATP resulted in a 100% increase in phosphorylation that was dependent on the presence of Ca2+, phosphatidylserine, and phorbol 12,13-dibutyrate. These results demonstrate that the Ca2+ ATPase of canine cardiac muscle can be phosphorylated by PKC in vitro, resulting in increased affinity of the Ca2+ ATPase for Ca2+ and increase in the Ca2+ pump pumping rate. The results suggest that the Ca2+-pumping ATPase in heart tissue can be stimulated by PKC, thereby regulating the intracellular Ca2+ levels in whole heart.Key words: protein kinase C, phosphorylation, plasma membrane, Ca2+ ATPase, heart.

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Staurosporine Mobilizes Ca2+ from Secretory Granules by Inhibiting Protein Kinase C in Rat Submandibular Acinar Cells

Journal of Dental Research ◽

10.1177/0810788 ◽

2002 ◽

Vol 81 (11) ◽

pp. 788-793 ◽

Cited By ~ 3

Author(s):

Y.J. Kim ◽

J.M. An ◽

D.M. Shin ◽

S.-I. Lee ◽

H. Sugiya ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Secretory Granules ◽

Acinar Cells ◽

Protein Kinase C Inhibitors ◽

Kinase C ◽

Inositol 1,4,5 Trisphosphate ◽

Intracellular Ca ◽

Inhibiting Protein

Staurosporine was previously shown to mobilize Ca2+ from the thapsigargin-insensitive Ca2+ store in rat submandibular acinar cells. However, the nature of the store is not yet known. Therefore, in the present study, the staurosporine-releasable intracellular Ca2+ store was characterized. Staurosporine increased the cytosolic Ca2+ concentration ([Ca2+]c) after the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store was depleted. Ionomycin caused only small increases in [Ca2+]c after the depletion of the IP3-sensitive Ca2+ store, whereas ionomycin+monensin caused large increases. However, ionomycin+monensin did not increase [Ca2+]c when added after [Ca2+]c was increased by staurosporine, indicating that the acidic Ca2+ store was the main source of Ca2+. The acidic Ca2+ store appeared to be associated with secretory granules, since ionomycin+monensin- and staurosporine-induced [Ca2+]c increases were significantly reduced when the acinar cells were degranulated. The effect of staurosporine on [Ca2+]c was mimicked by other protein kinase C inhibitors. Therefore, we conclude that staurosporine mobilizes Ca2+ from secretory granules, probably through the inhibition of protein kinase C in rat submandibular acinar cells.

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