Hemmings H C, Nairn A C, Elliott J I, Greengard P
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021-6399.
J Biol Chem. 1990 Nov 25;265(33):20369-76.
Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1.
基于苏氨酸磷酸化位点以及多巴胺和环磷酸腺苷调节的磷蛋白（DARPP - 32，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定其分子量为32,000）的推测抑制位点，制备了合成肽，并将其作为环磷酸腺苷依赖性蛋白激酶、蛋白磷酸酶 - 1c、 - 2Ac（分别为蛋白磷酸酶 - 1和2A的催化亚基）以及 - 2B的底物进行分析，同时作为蛋白磷酸酶 - 1c的抑制剂进行研究。对环磷酸腺苷依赖性蛋白激酶催化肽磷酸化的动力学研究表明，可磷酸化苏氨酰残基羧基末端区域在促进磷酸化过程中起重要作用。对磷酸化肽去磷酸化的研究表明，它们能被蛋白磷酸酶 - 2A和 - 2B有效去磷酸化，而被蛋白磷酸酶 - 1去磷酸化的程度较差。通过测定合成磷酸肽对蛋白磷酸酶 - 1c活性的影响，分析了磷酸化DARPP - 32的活性抑制区域。磷酸化D32 - (8 - 48)和磷酸化D32 - (8 - 38)对蛋白磷酸酶 - 1c的抑制作用的IC50值分别为2×10⁻⁸和4×10⁻⁸ M，而完整的磷酸化DARPP - 32的IC50值为8×10⁻⁹ M。磷酸化D-32 - (9 - 38)与磷酸化D32 - (8 - 38)具有同等效力；然而，进一步的氨基末端缺失导致IC50值显著降低。一种含有磷酸丝氨酰残基而非磷酸苏氨酰残基的活性DARPP - 32磷酸肽类似物的IC50值也大幅降低。这些数据确定了磷酸化DARPP - 32的必需抑制区域为9 - 38位残基，其中包含磷酸化位点（Thr34）。该区域与蛋白磷酸酶 - 1的一种独特抑制剂磷酸酶抑制剂 - 1具有广泛的氨基酸序列同源性。对强效抑制剂磷酸化D32 - (9 - 38)以及弱效抑制剂磷酸化D32 - (10 - 38)抑制蛋白磷酸酶 - 1c的动力学研究表明，其抑制机制为混合竞争性/非竞争性，这与之前对完整磷酸化DARPP - 32和完整磷酸化抑制剂 - 1的研究结果一致。这些发现支持了这样一种假说，即磷酸化DARPP - 32氨基末端区域的一个30个氨基酸的结构域足以抑制蛋白磷酸酶 - 1。
