NDLI: Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2

Content Provider	Journal of Biological Chemistry (JBC)
Author	Huang, Hsien-bin Horiuchi, Atsuko Watanabe, Takuo Shih, Su-Ru Tsay, Huey-Jen Li, Heng-Chun Greengard, Paul Nairn, Angus C.
Abstract	Phospho-DARPP-32 (where DARPP-32 is dopamine- and cAMP-regulated phosphoprotein, M r 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC50 ∼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 ∼800 nm), whereas further COOH-terminal deletion (I2-(1–84)) caused a moderate decrease in inhibitory potency (IC50∼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.
Related Links	http://www.jbc.org/content/274/12/7870.abstract
Ending Page	7878
Starting Page	7870
Page Count	9
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	12
Volume Number	274
DOI	10.1074/jbc.274.12.7870
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1999-03-19
Access Restriction	Open
Subject Keyword	Catalytic subunit of protein phosphatase-1 (PP1) Dopamine- and cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32) DARPP-32 (D32) Inhibitor-2 (I2) Myofibrillar PP1-binding protein (M110) Polyacrylamide gel electrophoresis (PAGE) PROTEIN CHEMISTRY AND STRUCTURE
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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