Girault J A, Hemmings H C, Williams K R, Nairn A C, Greengard P
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
J Biol Chem. 1989 Dec 25;264(36):21748-59.
DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.
多巴胺和cAMP调节的磷蛋白（DARPP - 32，十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定其分子量为32,000）是蛋白磷酸酶 - 1的抑制剂，在具有D1多巴胺受体的多巴胺感受神经元中含量丰富。纯化的牛DARPP - 32在体外被酪蛋白激酶II磷酸化，磷酸化化学计量比大于2摩尔磷酸盐/摩尔蛋白质，而两种结构和功能相关的蛋白质，蛋白磷酸酶抑制剂 - 1和G底物，是该酶的不良底物。对酪蛋白激酶II磷酸化的牛DARPP - 32的胰凝乳蛋白酶和嗜热菌蛋白酶磷酸肽进行测序表明，主要的磷酸化残基是Ser45和Ser102。对于大鼠DARPP - 32，通过手动Edman降解证实了这些磷酸化位点的鉴定。磷酸化残基位于酸性氨基酸残基的氨基末端，这是酪蛋白激酶II磷酸化位点的一个特征。酪蛋白激酶II磷酸化DARPP - 32的表观Km值为3.4 microM，kcat值为0.32 s-1。Ser102磷酸化的kcat值比Ser45的大5 - 6倍。使用包含每个磷酸化位点的合成肽进行的研究证实了两个位点磷酸化kcat值之间的这种差异。在用[32P]磷酸盐预标记的大鼠尾状核 - 壳核切片中，DARPP - 32在基础条件下在丝氨酸残基上被磷酸化。通过嗜热菌蛋白酶磷酸肽图谱的比较和手动Edman降解对磷酸化残基的测定确定完整细胞中的主要磷酸化位点为Ser102。在体外，酪蛋白激酶II磷酸化的DARPP - 32被蛋白磷酸酶 - 1和 - 2A去磷酸化。酪蛋白激酶II的磷酸化不影响DARPP - 32作为蛋白磷酸酶 - 1抑制剂的效力，其效力仅取决于cAMP依赖性蛋白激酶对Thr34的磷酸化。然而，酪蛋白激酶II对DARPP - 32的磷酸化促进了cAMP依赖性蛋白激酶对Thr34的磷酸化，Vmax增加2.2倍，表观Km增加1.4倍。因此，完整细胞中酪蛋白激酶II对DARPP - 32的磷酸化可能会响应cAMP水平的升高而调节其磷酸化。
