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

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