Huang H B, Horiuchi A, Watanabe T, Shih S R, Tsay H J, Li H C, Greengard P, Nairn A C
Institute of Biochemistry, Tzu Chi College of Medicine and Humanities, Hualien 970, Taiwan.
J Biol Chem. 1999 Mar 19;274(12):7870-8. doi: 10.1074/jbc.274.12.7870.
Phospho-DARPP-32 (where DARPP-32 is dopamine- and cAMP-regulated phosphoprotein, Mr 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC50 approximately 1 nM) of the catalytic subunit of protein phosphatase-1 (PP1). Our previous studies have indicated that a region encompassing residues 6-11 (RKKIQF) and phospho-Thr-34, of phospho-DARPP-32, interacts with PP1. However, little is known about specific regions of inhibitor-2 that interact with PP1. We have now characterized in detail the interaction of phospho-DARPP-32 and inhibitor-2 with PP1. Mutagenesis studies indicate that within DARPP-32 Phe-11 and Ile-9 play critical roles, with Lys-7 playing a lesser role in inhibition of PP1. Pro-33 and Pro-35 are also important, as is the number of amino acids between residues 7 and 11 and phospho-Thr-34. For inhibitor-2, deletion of amino acids 1-8 (I2-(9-204)) or 100-204 (I2-(1-99)) had little effect on the ability of the mutant proteins to inhibit PP1. Further deletion of residues 9-13 (I2-(14-204)) resulted in a large decrease in inhibitory potency (IC50 approximately 800 nM), whereas further COOH-terminal deletion (I2-(1-84)) caused a moderate decrease in inhibitory potency (IC50 approximately 10 nM). Within residues 9-13 (PIKGI), mutagenesis indicated that Ile-10, Lys-11, and Ile-13 play critical roles. The peptide I2-(6-20) antagonized the inhibition of PP-1 by inhibitor-2 but had no effect on inhibition by phospho-DARPP-32. In contrast, the peptide D32-(6-38) antagonized the inhibition of PP1 by phospho-DARPP-32, inhibitor-2, and I2-(1-120) but not I2-(85-204). These results indicate that distinct amino acid motifs contained within the NH2 termini of phospho-DARPP-32 (KKIQF, where italics indicate important residues) and inhibitor-2 (IKGI) are critical for inhibition of PP1. Moreover, residues 14-84 of inhibitor-2 and residues 6-38 of phospho-DARPP-32 share elements that are important for interaction with PP1.
磷酸化多巴胺和环磷腺苷调节的磷蛋白32（DARPP - 32，分子量32,000）、其同系物磷酸化抑制蛋白1以及抑制蛋白2是蛋白磷酸酶1（PP1）催化亚基的强效抑制剂（半数抑制浓度约为1 nM）。我们之前的研究表明，磷酸化DARPP - 32中包含第6至11位氨基酸残基（RKKIQF）和磷酸化苏氨酸34的区域与PP1相互作用。然而，对于抑制蛋白2与PP1相互作用的特定区域知之甚少。我们现在详细研究了磷酸化DARPP - 32和抑制蛋白2与PP1的相互作用。诱变研究表明，在DARPP - 32中，苯丙氨酸11和异亮氨酸9起关键作用，赖氨酸7在抑制PP1方面作用较小。脯氨酸33和脯氨酸35也很重要，残基7至11与磷酸化苏氨酸34之间的氨基酸数量同样重要。对于抑制蛋白2，缺失氨基酸1至8（I2 - (9 - 204)）或100至204（I2 - (1 - 99)）对突变蛋白抑制PP1的能力影响不大。进一步缺失残基9至13（I2 - (14 - 204)）导致抑制效力大幅下降（半数抑制浓度约为800 nM），而进一步的羧基末端缺失（I2 - (1 - 84)）导致抑制效力适度下降（半数抑制浓度约为10 nM）。在残基9至13（PIKGI）范围内，诱变表明异亮氨酸10、赖氨酸11和异亮氨酸13起关键作用。肽I2 - (6 - 20)拮抗抑制蛋白2对PP - 1的抑制作用，但对磷酸化DARPP - 32的抑制作用无影响。相反，肽D32 - (6 - 38)拮抗磷酸化DARPP - 32、抑制蛋白2和I2 - (1 - 120)对PP1的抑制作用，但对I(85 - 204)无影响。这些结果表明，磷酸化DARPP - 32（KKIQF，斜体表示重要残基）和抑制蛋白2（IKGI）的氨基末端包含的不同氨基酸基序对于抑制PP1至关重要。此外，抑制蛋白2的残基14至84和磷酸化DARPP - 32的残基6至38具有对与PP1相互作用很重要的共同元件。
