Abstract
Protein kinase C epsilon (PKCε) contributes to multiple signaling pathways affecting human disease. The function of PKCε requires it to undergo changes in subcellular distribution in response to signaling events. While the mechanisms underlying this translocation are incompletely understood, it involves the receptor for activated C kinase protein (RACK2/β'-COP). This receptor also functions as a vesicle coat protein in the secretory pathway where it is regulated by the small GTP-binding protein ADP-ribosylation factor, ARF1. We inhibited ARF1 activation to test the requirement for RACK2/β'-COP in PKCε localization in NIH3T3 fibroblasts. We found that steady-state localization of PKCε at the Golgi complex requires ARF1-regulated RACK2/β'-COP function. By contrast, we did not observe any defects in phorbol ester-induced translocation when ARF1 was inhibited. We also found that PKCε bound to isolated membranes through two distinct mechanisms. One mechanism was dependent upon RACK2/β'-COP while a second was RACK2/β'-COP-independent and stimulated by phorbol esters. Finally, we show that RACK2/β'-COP affects the subcellular distribution of a constitutively active form of PKCε, in a manner similar to what we observed for wild-type PKCε. Together, our data support a role for RACK2/β'-COP in the steady-state localization of PKCε at the Golgi apparatus, which may be independent of its role during PKCε translocation to the cell surface.