Autophosphorylation suppresses whereas kinase inhibition augments the translocation of protein kinase Cα in response to diacylglycerol

Abstract

We have seen that protein kinase Cα (PKCα) is transiently translocated to the plasma membrane by carbachol stimulation of neuroblastoma cells. This is induced by the Ca2+ increase, and PKCα does not respond to diacylglycerol (DAG). The unresponsiveness is dependent on structures in the catalytic domain of PKCα. This study was designed to investigate if and how the kinase activity and autophosphorylation are involved in regulating the translocation. PKCα enhanced green fluorescent protein translocation was studied in living neuroblastoma cells by confocal microscopy. Carbachol stimulation induced a transient translocation of PKCα to the plasma membrane and a sustained translocation of kinase-dead PKCα. In cells treated with the PKC inhibitor GF109203X, wild-type PKCα also showed a sustained translocation. The same effects were seen with PKCβ1, PKCβII, and PKCδ. Only Kinase-dead and not wild-type PKCα translocated in response to 1,2-dioctanoylglycerol. To examine whether autophosphorylation regulates relocation to the cytosol, the autophosphorylation sites in PKCα were mutated to glutamate, to mimic phosphorylation, or alanine, to mimic the non-phosphorylated protein. After stimulation with carbachol, glutamate mutants behaved like wild-type PKCα, whereas alanine mutants behaved like kinase-dead PKCα. When the alanine mutants were treated with 1,2-dioctanoylglycerol, all cells showed a sustained translocation of the protein. However, neither carbachol nor GF109203X had any major effects on the level of autophosphorylation, and GF109203X potentiated the translocation of the glutamate mutants. We, therefore, hypothesize that 1) autophosphorylation of PKCα limits its sensitivity to DAG and 2) that kinase inhibitors augment the DAG sensitivity of PKCα, perhaps by destabilizing the closed conformation.