Han Jung Min, Kim Jae Ho, Lee Byoung Dae, Lee Sang Do, Kim Yong, Jung Yon Woo, Lee Sukmook, Cho Wonhwa, Ohba Motoi, Kuroki Toshio, Suh Pann-Ghill, Ryu Sung Ho
Department of Life Science and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, South Korea.
J Biol Chem. 2002 Mar 8;277(10):8290-7. doi: 10.1074/jbc.M108343200. Epub 2001 Dec 13.
Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.
许多研究表明，蛋白激酶C（PKC）是磷脂酶D（PLD）的重要生理调节因子。然而，PKC在激动剂诱导的PLD激活中的作用主要是围绕PLD1进行研究的，PLD1是迄今为止克隆的两种PLD同工酶（PLD1和PLD2）之一。由于PLD2的表达显著增强了佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA）或缓激肽诱导的大鼠嗜铬细胞瘤PC12细胞中的PLD活性，我们研究了PC12细胞中PLD2的调节机制。两种不同的PKC抑制剂，GF109203X和Ro - 31 - 8220，完全阻断了PMA诱导的PLD2激活。此外，rottlerin对PKCδ的特异性抑制阻止了PMA刺激的PC12细胞中PLD2的激活。在PC12细胞用PMA或缓激肽处理后，伴随着PLD2的激活，PLD2发生了磷酸化。此外，rottlerin阻断了PC12细胞中PMA或缓激肽诱导的PLD2磷酸化。使用腺病毒介导的基因转移表达PKCδ的激酶缺陷型突变体抑制了PMA诱导的PC12细胞中PLD2的磷酸化和激活，这表明在PC12细胞中PKCδ激酶活性介导了PLD2的磷酸化依赖性调节。PKCδ与PC12细胞提取物中的PLD2共免疫沉淀，并在体外以PMA依赖性方式与PLD2结合。对从PMA处理的PC12细胞中免疫沉淀的磷酸化PLD2和在体外被PKCδ磷酸化的PLD2进行二维磷酸肽图谱分析并比较。至少有七个磷酸肽迁移位置相同，表明细胞内PKCδ直接对PLD2进行了磷酸化。PC12细胞的免疫细胞化学研究表明，用PMA处理后，PKCδ从细胞质转移到了主要定位有PLD2的质膜。这些结果表明，PKCδ依赖性直接磷酸化在PC12细胞中PLD2活性的调节中起重要作用。
