Phospholipase D1 is located and activated by protein kinase Cα in the plasma membrane in 3Y1 fibroblast cell

The subcellular location of phospholipase D1 (PLD1) and its activation by protein kinase Cα (PKCα) were examined by subcellular fractionation and by microscopic observation of green fluorescent protein-fused PLD1 (GFP-PLD1) or PKCα (GFP-PKCα) in fibroblastic 3Y1 cells. Major PLD1 immunoreactivity and PKCα-stimulated PLD activity segregated with a plasma membrane marker, even though a significant amount was co-fractionated with markers for endoplasmic reticulum (ER) and Golgi. Upon treatment with phorbol myristate acetate (PMA), PKCα translocated from the cytosolic fraction to the membrane fraction to which PLD1 also localized. GFP-PLD1 was found in the plasma membrane as well as a in a perinuclear compartment consistent with ER and Golgi and in other dispersed vesicular structures in the cytoplasm. However, most of GFP-PKCα was translocated from the cytosol to the plasma membrane after treatment with PMA. From these results, we concluded that the plasma membrane is the major site of PLD1 activation by PKCα in 3Y1 cells.