Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells

We investigated the functional roles of ceramide, an intracellular lipid me diator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP ) in HeLa living cells. C-2-ceramide but not C-2-dihydroceramide induced tr anslocation of delta PKC-GFP to the Golgi complex, while alpha PKC- and zet a PKC-GFP did not respond to ceramide. The Golgi-associated delta PKC-GFP i nduced by ceramide was further translocated to the plasma membrane by phorb ol ester treatment. Ceramide itself accumulated to the Golgi complex where delta PKC was translocated by ceramide. Gamma interferon also induced the d elta PKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg2+-dependent neutral sphingomyelinase . Photobleaching studies showed that ceramide does not evoke tight binding of delta PKC-GFP to the Golgi complex but induces the continuous associatio n and dissociation of delta PKC with the Golgi complex. Ceramide inhibited the kinase activity of delta PKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of delta PKC-GFP immunoprec ipitated from ceramide-treated cells was increased. The immunoprecipitated delta PKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosin e kinase inhibitor abolished the ceramide-induced activation and tyrosine p hosphorylation of delta PKC-GFP. These results suggested that gamma interfe ron stimulation followed by ceramide generation through Mg2+-dependent sphi ngomyelinase induced delta PKC-specific translocation to the Golgi complex and that translocation results in delta PKC activation through tyrosine pho sphorylation of the enzyme.