Subtype-Specific Translocation of the δ Subtype of Protein Kinase C and Its Activation by Tyrosine Phosphorylation Induced by Ceramide in HeLa Cells

Abstract

We investigated the functional roles of ceramide, an intracellular lipid mediator, 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. C₂-ceramide but not C₂-dihydroceramide induced translocation of δPKC-GFP to the Golgi complex, while αPKC- and ζPKC-GFP did not respond to ceramide. The Golgi-associated δPKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where δPKC was translocated by ceramide. Gamma interferon also induced the δPKC-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 δPKC-GFP to the Golgi complex but induces the continuous association and dissociation of δPKC with the Golgi complex. Ceramide inhibited the kinase activity of δPKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of δPKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated δPKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of δPKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg²⁺-dependent sphingomyelinase induced δPKC-specific translocation to the Golgi complex and that translocation results in δPKC activation through tyrosine phosphorylation of the enzyme.

ACKNOWLEDGMENT

This work was supported by grants from the Ministry of Education, Science, Sports, and Culture in Japan (09NP0601, 11780448, 12680754, 12210107), the Sankyo Foundation of Life Science, the Uehara Memorial Foundation, and the Hyogo Science and Technology Association.