Cholecystokinin-stimulated Protein Kinase C-δ Kinase Activation, Tyrosine Phosphorylation, and Translocation Are Mediated by Src Tyrosine Kinases in Pancreatic Acinar Cells

Abstract

Protein kinase C-␦ (PKC-␦) is involved in growth, differentiation, tumor suppression, and regulation of other cellular processes. PKC-␦ activation causes translocation, tyrosine phosphorylation, and serine-threonine kinase activity. However, little is known about the ability of G protein-coupled receptors to activate these processes or the mediators involved. In the present study, we explored the ability of the neurotransmitter/hormone, CCK, to stimulate these changes in PKC-␦ and explored the mechanisms. In rat pancreatic acini under basal conditions, PKC-␦ is almost exclusively located in cytosol. CCK and TPA stimulated a rapid PKC-␦ translocation to membrane and nuclear fractions, which was transient with CCK. CCK stimulated rapid tyrosine phosphorylation of PKC-␦ and increased kinase activity. Using tyrosine kinase (B44) and a tyrosine phosphatase inhibitor (orthovanadate), changes in both CCK-and TPA-stimulated PKC-␦ tyrosine phosphorylation were shown to correlate with changes in its kinase activity but not translocation. Both PKC-␦ tyrosine phosphorylation and activation occur exclusively in particulate fractions. The Src kinase inhibitors, SU6656 and PP2, but not the inactive related compound, PP3, inhibited CCK-and TPA-stimulated PKC-␦ tyrosine phosphorylation and activation. In contrast, PP2 also had a lesser effect on CCK-but not TPA-stimulated PKC-␦ translocation. CCK stimulated the association of Src kinases with PKC-␦, demonstrated by co-immunoprecipitation. These results demonstrate that CCK A receptor activation results in rapid translocation, tyrosine phosphorylation, and activation of PKC-␦. Stimulation of PKC-␦ translocation precedes tyrosine phosphorylation, which is essential for activation to occur. Activation of Src kinases is essential for the tyrosine phosphorylation and kinase activation to occur and plays a partial role in translocation.

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

“…1, B and D, hGH induced a robust tyrosine phosphorylation of c-Src. This effect was abolished by 10 M PP2, a specific inhibitor of Src kinases (20,48,57,61), which has been shown to be without effect on JAK2 activation induced by hGH (20,61 Fig. 2A), consistent with previous findings (60).…”

“…Means Ϯ SE derived from 3 separate experiments for each are shown (*P Ͻ 0.05 for a chance difference by ANOVA). [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50], were cultured in RPMI 1640 tissue culture medium containing 10% (vol/vol) FBS, 100 IU/ml penicillin, and 100 g/ml streptomycin. The BRIN-BD11 ␤-cell line was established by electrofusion of RINm5F insulinoma cells with New England Deaconess Hospital (NEDH) rat pancreatic islet cells and shares the major features of normal pancreatic ␤-cells (35,36).…”

Down-regulation of Caspase-2 by Rottlerin via Protein Kinase C-δ–Independent Pathway

“…However, several studies reported that rottlerin acts on additional targets besides PKCy (40)(41)(42)(43)(44)(45)(46). For example, rottlerin acts as a mitochondrial uncoupler independent of its effects on PKCy (40,43,46).…”

Regulated Binding of Importin-α to Protein Kinase Cδ in Response to Apoptotic Signals Facilitates Nuclear Import

“…Our laboratory and others have shown that tyrosine phosphorylation of PKC␦ in response to various stimuli regulates its subcellular localization as well as other aspects of its function (9,27,(33)(34)(35)(36)(37)(38)(39)(40). To explore a potential role for tyrosine phosphorylation in regulating binding of importin-␣ to PKC␦ in response to H 2 O 2 , we first examined the kinetics of tyrosine phosphorylation of PKC␦ in response to H 2 O 2 using a pan phospho-tyrosine antibody (4G10).…”