Wang Y, MacDonald J I, Kent C
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
J Biol Chem. 1993 Mar 15;268(8):5512-8.
The mechanism by which oleate induced the translocation of CTP:phosphocholine cytidylyltransferase from the soluble to particulate fraction was studied in HeLa cells. Addition of 0.5 mM oleate caused a 10-fold stimulation of [methyl-3H]choline incorporation into phosphatidylcholine. The stimulation of phosphatidylcholine synthesis was correlated with a decrease in phosphocholine and an increase in CDP-choline, indicating that cytidylyltransferase is regulatory under these conditions. The stimulation of cytidylyltransferase activity upon oleate treatment was concomitant with the translocation of this enzyme from the soluble to particulate fraction. Immunoblot analysis revealed the soluble form as multiple slowly migrating bands, and the particulate form as a faster migrating, single band. The slowly migrating form could be converted to the fast migrating form by phosphatase treatment, suggesting that the mobility difference was due to phosphorylation. In vivo 32P labeling showed that the soluble cytidylyltransferase was highly phosphorylated and the particulate form was much less phosphorylated. The kinetics of the translocation showed that the conversion of the majority of cytidylyltransferase from the soluble to the particulate form occurred within about 15 min following addition of oleate and that the translocation showed a good correlation with dephosphorylation. In contrast, the relocation of cytidylyltransferase from the particulate to the soluble fraction after the removal of oleate occurred within 1 min, and extensive phosphorylation was not required for cytidylyltransferase to leave the membrane. The relocated, soluble cytidylyltransferase was phosphorylated at a much slower rate than that at which the enzyme left the membrane. Immunolocalization showed that the cytidylyltransferase translocated to the nuclear envelope instead of the endoplasmic reticulum. These results indicate that activation of cytidylyltransferase by dephosphorylation and translocation to the nuclear envelope may be a general mechanism occurring in many cell types. Moreover, in HeLa cells, multiple mechanism exist for regulating cytidylyltransferase activity.
在HeLa细胞中研究了油酸诱导CTP：磷酸胆碱胞苷转移酶从可溶性部分转位至颗粒部分的机制。添加0.5 mM油酸可使[甲基 - 3H]胆碱掺入磷脂酰胆碱的量增加10倍。磷脂酰胆碱合成的刺激与磷酸胆碱的减少和CDP - 胆碱的增加相关，表明胞苷转移酶在这些条件下具有调节作用。油酸处理后胞苷转移酶活性的刺激与该酶从可溶性部分转位至颗粒部分同时发生。免疫印迹分析显示可溶性形式为多个迁移缓慢的条带，颗粒形式为迁移较快的单个条带。通过磷酸酶处理，迁移缓慢的形式可转化为迁移快速的形式，表明迁移率差异是由于磷酸化所致。体内32P标记显示可溶性胞苷转移酶高度磷酸化，颗粒形式的磷酸化程度则低得多。转位动力学表明，在添加油酸后约15分钟内，大多数胞苷转移酶从可溶性形式转化为颗粒形式，并且转位与去磷酸化具有良好的相关性。相反，去除油酸后，胞苷转移酶从颗粒部分重新定位至可溶性部分在1分钟内发生，并且胞苷转移酶离开膜不需要广泛的磷酸化。重新定位的可溶性胞苷转移酶磷酸化的速率比该酶离开膜的速率慢得多。免疫定位显示胞苷转移酶转位至核膜而非内质网。这些结果表明，通过去磷酸化激活胞苷转移酶并转位至核膜可能是许多细胞类型中普遍存在的机制。此外，在HeLa细胞中，存在多种调节胞苷转移酶活性的机制。
