TPA induces translocation but not down-regulation of new PKC isoform η in macrophages, MDCK cells and astrocytes

New type protein kinase C (PKC) η was found to be expressed in RAW 264.7 and J774A.1 macrophages, Madin-Darby canine kidney (MDCK) cells and astrocytes by Western blot analysis. Both cytosol and membrane in macrophages and astrocytes express this isoform, however, the expression in the membrane is more abundant than that in the cytosol. On the other hand, only membrane PKCη was detected in MDCK cells. Exposure of the cells to 1 μM TPA for 10 min resulted in the translocation of PKCη from the cytosolic to the membrane fraction. This translocation maintained at a constant level after 1.5, 3, 6 and 24 h TPA treatment. However, another new type PKCδ which expressed in the macrophages and astrocytes was down-regulated after long-term (6 and 24 h) TPA treatment. The immunoreactive band of PKCη in J774A.1 macrophages was blocked by the control PKCη antigenic peptide. Incubation of RAW 264.7 macrophages with UTP (1, 10 and 100 μM) resulted in the accumulation of inositol phosphates, indicating the presence of P2 receptor-coupled PLC pathway in these cells. This natural activator UTP also induced translocation of PKCη from cytosol to the membrane in RAW 264.7 macrophages after 1, 5 or 10 min treatment. Immunofluorescence microscopy revealed that in RAW 264.7 cells, PKCη is located in the cytoplasm organelle, plasma membrane and nuclear envelope. Stimulation of the cells with TPA resulted in translocation to the plasma membrane. This translocation of PKCη was still apparent after 24 h treatment with TPA.