Decoding of short-lived Ca2+ influx signals into long term substrate phosphorylation through activation of two distinct classes of protein kinase C

Abstract

In electrically excitable cells, membrane depolarization opens voltage-dependent Ca2+ channels eliciting Ca2+ influx, which plays an important role for the activation of protein kinase C (PKC). However, we do not know whether Ca2+ influx alone can activate PKC. The present study was conducted to investigate the Ca2+ influx-induced activation mechanisms for two classes of PKC, conventional PKC (cPKC; PKCα) and novel PKC (nPKC; PKCθ), in insulin-secreting cells. We have demonstrated simultaneous translocation of both DsRed-tagged PKCα to the plasma membrane and green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate to the cytosol as a dual marker of PKC activity in response to depolarization-evoked Ca2+ influx in the DsRed-tagged PKCα and GFP-tagged myristoylated alanine-rich C kinase substrate co-expressing cells. The result indicates that Ca2+ influx can generate diacylglycerol (DAG), because cPKC is activated by Ca2+ and DAG. We showed this in three different ways by demonstrating: 1) Ca2+ influx-induced translocation of GFP-tagged C1 domain of PKCγ, 2) Ca2+ influx-induced translocation of GFP-tagged pleckstrin homology domain, and 3) Ca2+ influx-induced translocation of GFP-tagged PKCθ, as a marker of DAG production and/or nPKC activity. Thus, Ca2+ influx alone via voltage-dependent Ca2+ channels can generate DAG, thereby activating cPKC and nPKC, whose activation is structurally independent of Ca2+.