Watkins J D, Wang Y L, Kent C
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Arch Biochem Biophys. 1992 Feb 1;292(2):360-7. doi: 10.1016/0003-9861(92)90003-f.
Immunoprecipitation of 32P-labeled CTP:phosphocholine cytidylyltransferase from freshly isolated rat hepatocytes followed by trypsin digestion and two-dimensional peptide mapping revealed multiple phosphorylation sites. Treatment of the hepatocytes with 0.5 mM of the cAMP analog, 8-(4-chlorophenylthio)-adenosine 3':5'-monophosphate or elevation of intracellular cAMP levels by cholera toxin activated the cAMP-dependent protein kinase activity in intact cells. Despite the activation of cAMP-dependent protein kinase no change in the rate of [3H]choline incorporation into phosphatidylcholine was detected. In addition, the activity of cytidylyltransferase in total cell homogenates and its distribution between soluble and particulate fractions remained unchanged. Comparison of peptide maps of 32P-labeled cytidylyltransferase obtained from control and cholera-toxin-treated hepatocytes did not reveal any differences in the phosphorylation state of cytidylyltransferase. Furthermore, only [32P]phosphoserine residues were detected following phosphoamino acid analysis. We conclude that cytidylyltransferase activity is not altered solely by the activation of the cAMP-dependent kinase in fresh hepatocytes.
对新鲜分离的大鼠肝细胞中经32P标记的CTP：磷酸胆碱胞苷转移酶进行免疫沉淀，随后进行胰蛋白酶消化和二维肽图分析，发现了多个磷酸化位点。用0.5 mM的cAMP类似物8-(4-氯苯基硫代)-腺苷3':5'-单磷酸处理肝细胞，或通过霍乱毒素提高细胞内cAMP水平，均可激活完整细胞中依赖cAMP的蛋白激酶活性。尽管依赖cAMP的蛋白激酶被激活，但未检测到[3H]胆碱掺入磷脂酰胆碱的速率有变化。此外，全细胞匀浆中胞苷转移酶的活性及其在可溶性和颗粒性组分之间的分布保持不变。比较从对照和霍乱毒素处理的肝细胞中获得的32P标记的胞苷转移酶的肽图，未发现胞苷转移酶磷酸化状态有任何差异。此外，磷酸氨基酸分析后仅检测到[32P]磷酸丝氨酸残基。我们得出结论，在新鲜肝细胞中，胞苷转移酶活性不会仅因依赖cAMP的激酶的激活而改变。
