Agrawal H C, Sprinkle T J, Agrawal D
Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110.
Neurochem Res. 1994 Jun;19(6):721-8. doi: 10.1007/BF00967712.
2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [gamma-32]ATP with cAMP (5 microM) and cAMP (5 microM)+catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that it is likely a PKA site.
2',3'-环核苷酸3'-磷酸水解酶（CNP）在体内、脑片和无细胞体系中均发生了磷酸化。对体内和脑片中标记的免疫沉淀CNP进行磷酸氨基酸分析，结果显示磷酸丝氨酸（94%）和磷酸苏氨酸（5%）残基发生了磷酸化。用福斯高林孵育脑片后，CNP的磷酸化增加了3倍。同样，将分离的髓磷脂与[γ-32]ATP、5μM的环磷酸腺苷（cAMP）以及5μM的cAMP加环磷酸腺苷依赖性蛋白激酶催化亚基一起孵育，分别使CNP2的磷酸化显著增加了4倍和6倍。CNP2主要在其氨基末端肽的丝氨酸和/或苏氨酸残基上发生磷酸化，且这种磷酸化由蛋白激酶A催化，这是可行的。用佛波酯孵育脑片，CNP1和CNP2的磷酸化增加了2倍。福斯高林和佛波酯增加了单一但不同的CNP肽段的磷酸化。我们首次提供了生化证据，表明以蛋白质量为基础，CNP2的磷酸化程度远高于CNP1，这表明CNP2上的磷酸化位点比CNP1更多，且至少有一个位点位于CNP2的20个氨基酸末端，并且很可能是一个蛋白激酶A位点。
