The Endoplasmic Reticulum, A Novel Organelle for PKCδ Translocation

Protein Kinase C delta (PKCδ), a member of the novel PKC family, is ubiquitously expressed and involved in many intracellular signal pathways. Compared to conventional PKCs, the translocation behavior of the nPKCs is unclear, although they also contain C1 and C2 domains. We found that, when co-expressing fluorescent protein fusion proteins of PKCα and PKCδ in the same cell, following “physiological” stimulation of the cells with ATP (100μM), PKCα translocated to the plasma membrane and PKCδ to intracellular membrane structures that we identified as the ER. In order to investigate whether C1 or C2 domains of the PKC were responsible for such a different re-distribution patterns, we designed several chimera PKCs by exchanging C1 and C2 domains between PKCα and PKCδ and analysing subcellular translocation with various stimuli. Chimera II and III with their α-C2 domain not only displayed rapid and Ca2+ dependent plasma membrane translocation but also retained the slower ER translocation of wt-PKCδ. In contrast, chimeras I and IV did not display ATP or Ca2+ dependent translocation while their PMA-induced behavior was still intact. These data demonstrated the independent translocation capabilities of C1 and C2 domains in PKCs. But we were still puzzled to why the C1 domains of PKCs cause different recruitment schemes. To further enlighten this, we designed constructs in which we swapped C1a and C1b domains of PKCα and PKCδ. Expression and translocation experiments unequivocally showed that the C1b rather than C1a domain of PKCδ determined ER targeting, which was further confirmed by single amino acid mutation in C1b. To elucidate the signalling cascade contributing to PKCδ translocation, several specific chemical compounds and genetic inhibitors were applied. The results strongly indicated that the Gαs-cAMP-EPAC signal pathway played an essential role in the ER targeting PKCδ translocation.