Abstract
Protein kinase C-delta (PKC-delta) is involved in growth, differentiation, tumor suppression, and regulation of other cellular processes. PKC-delta 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-delta and explored the mechanisms. In rat pancreatic acini under basal conditions, PKC-delta is almost exclusively located in cytosol. CCK and TPA stimulated a rapid PKC-delta translocation to membrane and nuclear fractions, which was transient with CCK. CCK stimulated rapid tyrosine phosphorylation of PKC-delta and increased kinase activity. Using tyrosine kinase (B44) and a tyrosine phosphatase inhibitor (orthovanadate), changes in both CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation were shown to correlate with changes in its kinase activity but not translocation. Both PKC-delta 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-delta tyrosine phosphorylation and activation. In contrast, PP2 also had a lesser effect on CCK- but not TPA-stimulated PKC-delta translocation. CCK stimulated the association of Src kinases with PKC-delta, demonstrated by co-immunoprecipitation. These results demonstrate that CCKA receptor activation results in rapid translocation, tyrosine phosphorylation, and activation of PKC-delta. Stimulation of PKC-delta 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.
Highlights
Protein kinase C-␦ (PKC-␦) is involved in growth, dif- nases [1,2,3,4,5]
We found that CCK-8 and TPA caused a translocation of protein kinase C (PKC)-␦ (Figs. 1–3) as well as activation of PKC-␦ (Figs. 4 and 5) and stimulated its tyrosine phosphorylation (Figs. 6 – 8)
Pretreatment of pancreatic acini with B44 did not affect basal PKC-␦ kinase activity (Fig. 5, top panel). This pretreatment inhibited almost completely PKC-␦ activation induced by CCK-8 or TPA (Fig. 5). These results demonstrate that inhibition of PKC-␦ tyrosine phosphorylation resulted in inhibition of its activation, suggesting a relationship exists between tyrosine phosphorylation and kinase activity in pancreatic acinar cells for PKC-␦
Summary
Protein kinase C-␦ (PKC-␦) is involved in growth, dif- nases [1,2,3,4,5]. This family is divided, based on their structure and ferentiation, tumor suppression, and regulation of other allosteric requirements, into three general subgroups including cellular processes. 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 (␦, ⑀, , ), and atypical PKCs (, /, and ) [2,3,4]. CCK stimulated the association of Src kinases with PKC-␦, demonstrated by co-immunoprecipitation These results demonstrate that CCKA receptor activation results in rapid translocation, tyrosine phosphorylation, and activation of PKC-␦. With the G protein-coupled receptors (GPCRs) and to a varying degree with the other stimuli, the relationship between these three processes (translocation, tyrosine phosphorylation, and serine-threonine kinase activation) upon PKC-␦ stimulation as well as the cellular mechanisms mediating these changes, remains unclear. Recent studies demonstrate that activation of the CCKA receptor, a heptahelical GPCR that mediates the action of
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