The crystal structure of mammalian protein phosphatase-1, complexed with the toxin microcystin and determined at 2.1 A resolution, reveals that it is a metalloenzyme unrelated in architecture to the tyrosine phosphatases. Two metal ions are positioned by a central beta-alpha-beta-alpha-beta scaffold at the active site, from which emanate three surface grooves that are potential binding sites for substrates and inhibitors. The carboxy terminus is positioned at the end of one of the grooves such that regulatory sequences following the domain might modulate function. The fold of the catalytic domain is expected to be closely preserved in protein phosphatases 2A and 2B (calcineurin).

Phospho-DARPP-32 (where DARPP-32 is dopamineand cAMP-regulated phosphoprotein, M r 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC 50 ϳ1 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 (IC 50 ϳ800 nM), whereas further COOH-terminal deletion (I2-(1-84)) caused a moderate decrease in inhibitory potency (IC 50 ϳ10 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 NH 2 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.Protein phosphatase-1 (PP1) 1 is a major eukaryotic protein serine/threonine phosphatase that regulates diverse cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription, and neuronal signaling (1-4). The catalytic subunit of PP1 is regulated by the heat-stable protein inhibitors, inhibitor-1, its homolog DARPP-32 (dopamine-and cAMP-regulated phosphoprotein, M r 32,000), and inhibitor-2 (1, 2). Phosphorylation of inhibitor-1 at Thr-35 or of DARPP-32 at Thr-34 by cAMP-dependent protein kinase converts either protein into a potent inhibitor of PP1. In contrast, unphosphorylated inhibitor-2 interacts with the catalytic subunit of PP1 leading first to inhibition of enzyme activity and subsequently to an inactive complex, termed Mg-ATP-dependent PP1 (1, 5). The Mg-ATPdependent form of PP1 can then be re-activated following phosphorylation of Thr-72 of inhibitor-2 by glycogen synthase kinase-3 (GSK-3). PP1 is also regulated by its interaction with a variety of protein subunits that act in a manner distinct from the inhibitor proteins and t...

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