Kim Y, Kim J E, Lee S D, Lee T G, Kim J H, Park J B, Han J M, Jang S K, Suh P G, Ryu S H
Department of Life Science, Pohang University of Science and Technology, South Korea.
Biochim Biophys Acta. 1999 Jan 4;1436(3):319-30. doi: 10.1016/s0005-2760(98)00120-9.
The subcellular location of phospholipase D1 (PLD1) and its activation by protein kinase C alpha (PKC alpha) were examined by subcellular fractionation and by microscopic observation of green fluorescent protein-fused PLD1 (GFP-PLD1) or PKC alpha (GFP-PKC alpha) in fibroblastic 3Y1 cells. Major PLD1 immunoreactivity and PKC alpha-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), PKC alpha 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-PKC alpha 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 PKC alpha in 3Y1 cells.
通过亚细胞分级分离以及在成纤维细胞3Y1中对绿色荧光蛋白融合的磷脂酶D1（GFP-PLD1）或蛋白激酶Cα（GFP-PKCα）进行显微镜观察，研究了磷脂酶D1（PLD1）的亚细胞定位及其被蛋白激酶Cα（PKCα）激活的情况。主要的PLD1免疫反应性和PKCα刺激的PLD活性与质膜标志物一起分离，尽管有相当数量的PLD1与内质网（ER）和高尔基体的标志物共分级分离。在用佛波酯肉豆蔻酸酯乙酸酯（PMA）处理后，PKCα从胞质部分转移到PLD1也定位的膜部分。GFP-PLD1存在于质膜以及与内质网和高尔基体一致的核周区室以及细胞质中的其他分散的囊泡结构中。然而，在用PMA处理后，大多数GFP-PKCα从胞质溶胶转移到质膜。从这些结果中，我们得出结论，质膜是3Y1细胞中PKCα激活PLD1的主要部位。
