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

Abstract

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...

Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits

“…The KIQF sequence of I-1 and IKGI sequence of Inh2 are thought to function as the canonical VXF motif for PP1C binding. This concept makes interaction of PP1C with inhibitor proteins and targeting subunits mutually exclusive (4,39). Nevertheless, there are now several lines of evidence that indicate direct interaction of I-1 and Inh2 with PP1 holoenzymes, without dissociation (11)(12)(13).…”

Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5

“…Among the inhibitor proteins, the best characterized are inhibitor-1 (I-1; Endo et al, 1996), the I-1 homologue DARPP-32, and inhibitor-2 (I-2; Huang et al, 1999). Before purification and sequencing of their catalytic subunits, purified I-1 and I-2 were used to distinguish type 1 (i.e, PP1) from type 2 serine/threonine phosphatases (i.e., PP2A, PP2B [also called calcineurin], and PP2C).…”

“…Based on the crystal structure of the PP1-I-2 complex and other mostly in vitro biochemical studies, PP1 binds tightly to I-2 in a 1:1 stoichiometry, making multiple contacts with different parts of PP1, including an -helix of I-2 that covers the active site of PP1, inhibiting it (Huang et al, 1999;Hurley et al, 2007;Dancheck et al, 2011). PP1 is inactive within the in vitro PP1-I-2 complex, but can be quickly activated when I-2 is phosphorylated at threonine 72 (pT72) by GSK3 (Cohen, 1989), presumably removing the I-2 -helix away from the active site of PP1.…”

Wnt-5a-induced Phosphorylation of DARPP-32 Inhibits Breast Cancer Cell Migration in a CREB-dependent Manner

“…4). However, binding to T34A-DARPP-32 is not sufficient to inhibit PP1, since the phospho-Thr34 group is required to sterically block the catalytic site of PP1 (24). MCF-7 cells transfected with wt-DARPP-32 were left unstimulated, or stimulated with rWnt-5a (0.4 g/ml) or forskolin (1 M).…”