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J. Biol. Chem., Vol. 275, Issue 30, 22635-22644, July 28, 2000

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Interaction of Inhibitor-2 with the Catalytic Subunit of Type 1 Protein Phosphatase

IDENTIFICATION OF A SEQUENCE ANALOGOUS TO THE CONSENSUS TYPE 1 PROTEIN PHOSPHATASE-BINDING MOTIF^*

Jie Yang, Thomas D. Hurley, and Anna A. DePaoli-Roach

From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122

Received for publication, April 11, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibitor-2 (I-2) is the regulatory subunit of a cytosolic type 1 Ser/Thr protein phosphatase (PP1) and potently inhibits the activity of the free catalytic subunit (CS1). Previous work from the laboratory had proposed that the interaction of I-2 with CS1 involved multiple sites (Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269, 28919-28928). The present study refines the earlier analysis and arrives at a more detailed model for the interaction between I-2 and CS1. Although the NH₂-terminal I-2 regions containing residues 1-35 and 1-64 have no inhibitory activity on their own, they increase the IC₅₀ for I-2 by ~30-fold, indicating the presence of a CS1-interacting site. Based on several experimental approaches, we have also identified the sequence Lys¹⁴⁴-Leu-His-Tyr¹⁴⁷ as a second site of interaction that corresponds to the RVXF motif present in many CS1-binding proteins. The peptide I-2(135-151) significantly increases the IC₅₀ for I-2 and attenuates CS1 inhibition. Replacement of Leu and Tyr with Ala abolishes the ability to counteract inhibition by I-2. The I-2(135-151) peptide, but not I-2(1-35), also antagonizes inhibition of CS1 by DARPP-32 in a pattern similar to that of I-2. Furthermore, a peptide derived from the glycogen-binding subunit, R_GL/G_M(61-80), which contains a consensus CS1-binding motif, completely counteracts CS1 inhibition by I-2 and DARPP-32. The NH₂-terminal 35 residues of I-2 bind to CS1 at a site that is specific for I-2, whereas the KLHY sequence interacts with CS1 at a site shared with other interacting proteins. Other results suggest the presence of yet more sites of interaction. A model is presented in which multiple "anchoring interactions" serve to position a segment of I-2 such that it sterically occludes the catalytic pocket but need not make high affinity contacts itself.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Type 1 protein phosphatases (PP1)¹ constitute a major proportion of the cellular Ser/Thr phosphatases and play important roles in the regulation of many cellular functions including metabolism, hormone receptor activation, muscle contraction, cell growth and division, and gene expression (1-4). The enzymes are oligomers that consist of one of four highly homologous catalytic subunits (CS1) and different regulatory/targeting subunits (5). These latter subunits target the holoenzyme to distinct subcellular compartments in proximity to physiological substrates, confer substrate specificity, and may be involved in the regulation of enzyme activity (6-8). About two dozen CS1-binding proteins have been identified. They include the glycogen-binding subunits, R_GL/G_M (9, 10) G_L (11), PTG/R5/U5 (12-14), and R6 (15), which target the phosphatase to glycogen, the myosin-associating subunits, M₁₁₀ (16), NIPP-1 (17), p99/PNUTS (18, 19), and Sds22 (20), which may direct the phosphatase to the nucleus. In addition, there are cytosolic protein inhibitors, inhibitor-1 (I-1) and its brain homologue DARPP-32, and inhibitor-2 (I-2) (21, 22). Other inhibitor proteins recently identified include CPI-17 (23), HCGV/I-3 (24), and PHI-1 (25). Only one CS1-binding protein is found in any given holoenzyme, suggesting that the interaction with regulatory subunits is mutually exclusive.

I-2 associates with CS1 to form the ATP-Mg²⁺-dependent protein phosphatase whose activity is regulated by the phosphorylation of I-2 (3, 4, 26, 27, 28). I-2 has two key properties, it inhibits free CS1 and it controls the cyclic inactivation/activation of CS1 in the ATP-Mg²⁺-dependent phosphatase complex. Inhibition occurs rapidly and leaves the CS1 in its "active" conformation even though its activity is blocked. Inactivation is slower and involves conversion of CS1 to an inactive conformation. The inactive CS1·I-2 complex can be reactivated through phosphorylation of I-2 at Thr⁷² by glycogen synthase kinase-3 (27, 28) or the extracellular-regulated kinase 2 (29). I-2 can also be phosphorylated on Ser⁸⁶ by casein kinase II. This phosphorylation alone does not activate the complex but enhances the effect of glycogen synthase kinase-3 (27, 30, 31). Studies in our laboratory have indicated that different domains of I-2 are involved in inhibition, inactivation, and reactivation of the phosphatase (30). Deletion of the NH₂-terminal 35 residues of I-2 increased the IC₅₀ by two orders of magnitude, suggesting a role in inhibition. Within this domain, residues 10-13 (IKGI) were recently implicated in inhibition (32). The region surrounding Thr⁷² was shown to be important for the inactivation of CS1, and the COOH-terminal region of I-2 may be required for reactivation of the inactive CS1·I-2 complex (30). Mutation of these domains individually was not sufficient to disrupt complex formation.

In contrast to I-2, I-1 and DARPP-32 require phosphorylation at a threonine residue by the cAMP-dependent protein kinase for inhibitory activity (21, 22, 33). CS1 is also inhibited by a number of naturally occurring toxins, including okadaic acid, microcystin, calyculin A, and tautomycin (34). The crystal structure of CS1 complexed with microcystin has provided a basis to understand the mechanism by which the toxin interacts at the active site to block access of the substrates (35). Genetic and mutagenesis studies have implicated the 12-13 loop of CS1 in inhibition by both toxins and protein inhibitors (36, 37). Thus, although structurally different, the natural toxins bind to CS1 at a site that is shared or overlaps with that of the protein inhibitors, explaining their mutual competition (38, 39).

Although there is no extensive sequence homology among the different CS1 regulatory subunits, biochemical and crystallographic studies and peptide library screening have identified a consensus (K/R)(V/I)X(F/W) motif that is present in a number of CS1-binding proteins (40-42), including the glycogen-, myosin-, and nuclear-targeting subunits, I-1 and DARPP-32. The crystal structure of CS1 complexed with a 13-amino acid peptide containing this motif showed that the residues RRVSFA occupy a hydrophobic groove, opposite to the catalytic site, flanked by a negatively charged region that accommodates the NH₂-terminal basic residues in the peptide (41). DARPP-32 and I-1 contain the sequence KIQF, similar to RVXF, and the interaction of the KIQF motif in I-1 and DARPP-32 with CS1 is essential for inhibition (43, 44). Multiple sites of interaction have recently been shown also for NIPP-1, in which the RVXF and a flanking basic inhibitory sequence appear to be required to stabilize the association with CS1 (45). Thus, similar to what was originally proposed for I-2 (30), the notion has evolved that the association of inhibitory proteins with CS1 involves multiple contacts.

No sequence analogous to the consensus RVXF motif has been reported for I-2. However, the fact that I-2 and other regulatory subunits are mutually exclusive for CS1 binding (46, 47) and that a DARPP-32 peptide containing the KIQF could antagonize CS1 inhibition by I-2 (44) suggested that I-2 may contain a similar sequence. In this study we provide evidence that residues Lys¹⁴⁴-Leu-His-Tyr¹⁴⁷, serve this role in I-2. Comparison of the effects of various peptides on CS1 inhibition by I-2 and DARPP-32 indicates that the NH₂ terminus of I-2 interacts with CS1 at a site that is unique for I-2, whereas the KLHY sequence interacts with CS1 at a site that is common to R_GL and DARPP-32 and most likely other CS1-binding proteins. Evidence is also presented for the existence of additional sites in I-2 that interact with CS1. The implication is that interaction of the various binding subunits involves shared as well as unique sites that may be important for the diverse mechanisms by which different regulatory proteins control enzyme activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Other Materials and Methods-- Glycogen phosphorylase, phosphorylase kinase, and CS1 were purified from rabbit skeletal muscle as described previously (27, 29). The C catalytic subunit of the cyclic AMP-dependent protein kinase was a generous gift from Dr. Michael Uhler (University of Michigan, Ann Arbor, MI). The polyclonal anti-CS1 antibody raised in chicken against the CS1 peptide was provided by Dr. John Lawrence (University of Virginia, Charlottesville, VA). Horseradish peroxidase-conjugated rabbit anti-chicken antibodies were purchased from Sigma. Recombinant human DARPP-32 was from Chemicon. Restriction enzymes were obtained from New England Biolabs. The fast flow Q-Sepharose and reagents for enhanced chemiluminescence were purchased from Amersham Pharmacia Biotech. Toyopearl AF-Heparin-650 M was from TosoHass. Microcystin-LR was from Life Technologies, Inc., and okadaic acid was from Roche Molecular Biochemicals. The following peptides, R_GL(61-80) peptide, SGGRRVSFADNFGFNLVSVK; I-2(1-35), AASTASHRPIKGILKNKTSSTSSRVASAEQPRGSV; I-2(135-151), KKRQFEMKRKLHYNEGL and I-2(70-93), PSTPYHSMIGDDDDAYSDTETTEA, were synthesized on an Applied Biosystems 430A synthesizer, by the Biochemistry Biotechnology Facility, Indiana University School of Medicine. Two mutant I-2 peptides, I-2(135-151)L145A/Y147A, in which both Leu¹⁴⁵ and Tyr¹⁴⁷ were replaced by Ala, and I-2(135-151)F139A, where Phe¹³⁹ was substituted by Ala, were synthesized by Macromolecular Resources, Colorado State University. Radionuclides were from NEN Life Science Products. General chemicals were from Life Technologies, Inc., Sigma, Roche Molecular Biochemicals, and Bio-Rad. The coordinates of the CS1 and R_GL peptide complex were kindly provided by Dr. David Barford (Institute of Cancer Research, London, United Kingdom).

Preparation of Recombinant CS1 and -- CS1 and  cDNAs were obtained by reverse transcription-polymerase chain reaction. CS1 cDNA was synthesized from rabbit skeletal muscle total RNA, using SUPERSCRIPT^TM II RNase H reverse transcriptase (Life Technologies, Inc.). Oligonucleotides 5'-GCCCATATGTCCGACAGCGAGAA-3' and 5'-CCCGTCGACTATTTCTTGGCTTTG-3' were used as the 5'-primer and 3'-primer, respectively. CS1 cDNA was amplified from rat liver total cDNA (CLONTECH), using oligonucleotides 5'-ACGCATATGGCGGATATCGATAAA-3' as the 5'-primer and 5'-AGTGTCGACTATTTCTTTGCTTGC-3' as the 3'-primer. An NdeI site was engineered at the ATG start codon in the 5'-primers, and a SalI site was introduced after the TAG stop codon in the 3'-primers (sites are underlined). The polymerase chain reaction fragments were subcloned into pCR II vector (Invitrogen) and sequenced. The CS1 and  cDNA fragments were then excised and inserted into the pTacTac vector (48) at the NdeI and SalI sites. Protein expression and purification were performed by modification of published procedures (49). The proteins were purified to apparent homogeneity, greater than 95% as judged by SDS-PAGE, with a specific activity for both CS1 and  of 13,000-18,000 units/mg.

Preparation of I-2 Mutants-- The wild type I-2, the COOH-terminally truncated mutant, I-2(1-145), and the NH₂-terminally truncated I-2, I-2(36-204), were prepared as described previously (30). The cDNAs for the I-2 NH₂-terminal polypeptides I-2(1-64), I-2(1-75), and I-2(1-114) were obtained by polymerase chain reaction, using I-2·pET8d as template (30). Amplification utilized a common 5'-oligonucleotide primer and different 3'-primers. The 5'-primer was 5'-TACATATGGGCTCCATGGCGGCCTCGACGG-3' (NcoI site underlined); the three 3'-oligonucleotides for the different fragments were 5'-TCGCTCAGCTATAAACCATAGTCTTTGTCTGC-3' for I-2(1-64), 5'-CCGCTCAGCTAATGGTAAGGAGTGCTTGGTT-3' for I-2(1-75), and 5'-TGGCTCAGCTACCGATACTTTGGCTCTGAG-3' for I-2(1-114) (Bpu1102 I site underlined). The amplified DNAs were subcloned into the pCRII vector and sequenced. The NcoI-Bpu1102 I fragments were then cloned into pET8c vector. The I-2 mutants, I-2(F139A), in which Phe¹³⁹ was mutated to Ala, and I-2(L145A/Y147A), in which both Leu¹⁴⁵ and Tyr¹⁴⁷ were replaced by Ala, were generated by overlap extension polymerase chain reaction (50) using I-2·pET8d as template. The set of primers for mutating Phe¹³⁹ to Ala were the 5'-primer 5'-GCGACAAGCTGAAATGAAAAGG-3' and the 3'-primer 5'-CATTTCAGCTTGTCGCTTTTTTTC-3'. The primers for replacing both Leu¹⁴⁵ and Tyr¹⁴⁷ with Ala were the 5'-primer 5'-GGAAGGCTCACGCCAATGAAGGACTA-3' and the 3'-primer 5'-CATTGGCGTGAGCCTTCCTTTTCATT-3'. The altered codons are underlined. The full-length I-2 mutant cDNAs were cloned into the pET21d vector (Novagene), and the mutations were verified by DNA sequencing.

Expression in Escherichia coli was carried out as described (30) except that 0.1 mM isopropyl-1-thio--D-galactopyranoside was used for induction of I-2(F139A) and I-2(L145A/Y147A). Proteins were purified to homogeneity by the previously reported procedure (30) as modified by Dr. Tania Barshevsky at New England Biolabs. Briefly, the heat-treated cell lysate was centrifuged at 11,000 × g for 20 min at 4 °C, and the cleared extract was loaded onto a Q-Sepharose fast flow column. After extensive washing with buffer A (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 50 µg/ml N-p-tosyl-L-lysine chloromethyl ketone, 25 mM -mercaptoethanol) plus 0.05 M NaCl, the column was developed with a linear gradient of 0.05-0.45 M NaCl in buffer A (200 ml). Fractions containing the I-2 protein were combined, dialyzed against buffer A, and then applied onto a Toyopearl AF-Heparin-650 M column. The column was washed with buffer A plus 0.04 M NaCl, and the bound proteins were eluted with a 0.04-0.4 M NaCl linear gradient in buffer A (200 ml). I-2(1-64) and I-2(1-75) were purified directly by AF-Heparin-650M chromatography, using conditions similar to those described above. Fractions containing I-2 proteins were stored at 80 °C. All I-2 polypeptides were purified to near homogeneity as judged by SDS-PAGE (Fig. 1, B and C).

Phosphorylation of DARPP-32-- DARPP-32 was phosphorylated in 0.1 ml of 10 mM Tris-HCl, pH 7.5, containing 16 µM of DARPP-32, 2 µM of C catalytic subunit of the cyclic AMP-dependent protein kinase, 1 mM ATP, and 10 mM MgCl₂ at 30 °C for 3 h. The reaction was terminated by inactivation of the enzyme at 100 °C for 10 min. The sample was then diluted with 0.1 ml of 10 mM Tris-HCl, pH 7.5, 10% glycerol and extensively dialyzed against the same buffer to remove the unreacted ATP. The phosphorylation level was determined in a parallel reaction containing [-³²P]ATP. The stoichiometry of phosphorylation was ~1 mol/mol.

Phosphatase Activity Assay-- Phosphatase activity was measured as described previously (30) in the presence or absence of either I-2 or DARPP-32 and in combination with various peptides. CS1 purified from rabbit skeletal muscle (29) was used in most of the studies, because, unlike recombinant CS1, it does not require Mn²⁺ for activity, which could interfere with the inhibition assays. Furthermore, the native but not the recombinant CS1 is inhibited effectively by I-1 (51). To determine the effect of peptides on the I-2 IC₅₀, fixed concentrations of the peptide and varied amounts of I-2 were preincubated with CS1 at 30 °C for 5 min. The phosphatase reaction was then initiated by the addition of [³²P]phosphorylase a (300-4000 cpm/pmol) to a final concentration of 1 mg/ml, and the incubation was continued for an additional 10 min. The reaction was terminated by the addition of 17% trichloroacetic acid. To evaluate the antagonistic effect of the peptides, fixed amounts of I-2 (4 nM) or phosphorylated DARPP-32 (2 nM), which inhibit CS1 by ~70-75%, were preincubated with CS1 and various concentrations of peptides at 30 °C for 5 min. Phosphatase activity was then measured as described above. The effect of the peptides was expressed either as their ability to alter the IC₅₀ for I-2 or by their ability to release CS1 inhibition by I-2 or DARPP-32. One unit of phosphorylase phosphatase activity is defined as the amount of enzyme that releases 1 nmol of phosphate/min at 30 °C.

CS1·I-2 Complex Formation-- CS1·I-2 complexes were formed by incubating recombinant CS1 and I-2 proteins at a 1:1.3 ratio for 40 min at room temperature. Incubation was in the presence or absence of a ~100-fold molar excess of I-2(1-35), a ~200-fold molar excess of I-2-(135-151), or a combination of the two peptides. To detect the effect of microcystin-LR on complex formation, CS1 was preincubated with a 2-fold excess of I-2 for 40 min before the addition of microcystin at a concentration equimolar to I-2. All samples were analyzed by native PAGE performed similarly to Laemmli SDS-PAGE (52) except that SDS was omitted, and both stacking and separating gel buffer were at pH 8.8. Gels were run at low voltage (100 V) with cold buffer or at 4 °C to prevent heat denaturation of the proteins.

CS1 Overlay-- Different forms of I-2 were separated on 13% Tricine-SDS-PAGE (53) and transferred onto nitrocellulose membranes. The membranes were blocked in 5% nonfat dry milk in TBST (Tris-buffered saline with 0.05% Tween-20) for 2 h before overnight incubation with 4 µg/ml of purified recombinant CS1 at 4 °C. Membranes were then washed with TBST and incubated with anti-CS1 peptide antibody for 2 h at room temperature. After treatment with horseradish peroxidase-conjugated rabbit-anti-chicken secondary antibodies, signals were detected by enhanced chemiluminescence.

Molecular Modeling of the KLHY Sequence onto the CS1 Structure-- The structure of CS1 complexed with the R_GL peptide (41) was utilized as the starting point for modeling the I-2 KLHY tetrapeptide bound to CS1. The program O (54) was used to change the amino acid side chains on the R_GL peptide to those of the I-2 peptide. A library of standard amino acid side chain rotomers was used to find the best conformation of each mutated residue within the complex. Two amino acid side chains (Leu²⁴³ and Leu²⁸⁹) in the catalytic subunit were also rotated using the standard library to more commonly observed side chain rotomers to complement better the binding of the Leu side chain on the I-2 peptide. No atomic contacts with distances less than 2.6 Å are found in the modeled complex. The backbone atoms in both the catalytic subunit and the bound R_GL peptide were not altered during this modeling study. No energy minimization of the resulting CS1·I-2 complex was performed since the complex closely resembled that found for the R_GL complex and no unfavorable contacts were produced by the amino acid substitutions in the R_GL peptide.

Determination of Protein and Peptide Concentrations-- Concentrations of CS1 and , DARPP-32, and I-2 polypeptides were determined by the method of Bradford (55), using bovine serum albumin as standard. Peptide concentrations based on weight were confirmed by quantitative amino acid analysis performed in the Department of Biochemistry at Purdue University, West Lafayette, Indiana on a Beckman System Gold high pressure liquid chromatography system. For this determination, the peptide samples were hydrolyzed under vacuum in 6 N HCl vapor phase at 110 °C for 22 h. The hydrolysates were then derivatized with a Waters AccQ-Fluor Reagent Kit, and the amino acids were separated on a Waters AccQ-Tag column.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of the Inhibitory Properties of I-2 Deletion Mutants-- Previous work from our laboratory had indicated that I-2 interacts with CS1 at multiple sites, and that the NH₂-terminal 35 residues were required for high potency inhibition (30). An NH₂-terminally truncated I-2, I-2(36-204), exhibited an IC₅₀ for inhibition of CS1 that was ~200-fold higher than full-length I-2 (30). In the course of the present study, a second NH₂-terminally truncated protein, I-2(12-204), was fortuitously generated by proteolysis during purification of the recombinant full-length I-2 (Fig. 1B). Loss of the NH₂-terminal 11 residues increased the IC₅₀ to 7 nM, 5-fold higher than that of the intact protein (Fig. 1A), a finding consistent with a critical role for this region in inhibition. Our previous data had indicated that I-2 lacking the COOH-terminal 59 residues, I-2(1-145), had an inhibitory potency similar to that of the wild type I-2 (30) (Fig. 1A). Therefore, we generated four NH₂-terminal polypeptides, I-2(1-35), I-2(1-64), I-2(1-75), and I-2(1-114). All the polypeptides were purified to >95% homogeneity as estimated by Coomassie Blue staining of SDS-PAGE (Fig. 1C). The synthetic peptide comprising residues 1-35 had no inhibitory activity at concentrations up to 200 µM, and I-2(1-64) was not inhibitory at concentrations over 100 µM (56) (Fig. 1A). I-2(1-75) exhibited a weak inhibition, with an IC₅₀ of ~50 µM, whereas I-2(1-114) displayed high inhibitory activity with an IC₅₀ of 10 nM, only 6-fold greater than that of the wild type I-2, which under the same conditions was ~1.5 nM (Fig. 1A). Interestingly, the phosphatase activity was not completely inhibited by I-2(1-114) even at 100 µM concentration. This result is similar to that observed with I-2(1-145) (30) (Fig. 1A), supporting the proposal of additional CS1 interacting site(s) in the COOH-terminal 59 residues. The increased inhibitory potency of I-2(1-114) as compared with I-2(1-64) also implies that the region between 64-114 contributes significantly to the inhibitory properties of I-2.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Analysis of wild type and mutant I-2. A, inhibition of CS1 by wild type and mutant I-2. The indicated concentrations of various I-2 polypeptides were preincubated with 4-7 mU of rabbit skeletal muscle CS1 at 30 °C for 5 min prior to the determination of the phosphatase activity as described under "Experimental Procedures." Phosphatase activity is expressed as percent of the activity in the absence of I-2. The IC50 was extrapolated from the inhibition curve. Data are presented as mean ± S.D. from three to five experiments each carried out in duplicate. B, SDS-PAGE of purified wild type and mutant I-2 polypeptides. Lane 1, wild type I-2; lane 2, I-2(12-204); lane 3, I-2(36-204); lane 4, I-2(1-145); lane 5, I-2(F139A); lane 6, I-2(L145A/Y147A). Each lane contains 0.5 µg of purified protein. C, Tricine-SDS-PAGE of purified full-length and COOH-terminally truncated I-2. Lane 1, full-length I-2; lane 2, I-2(1-114); lane 3, I-2(1-75); lane 4, I-2(1-64). Each lane contains 1 µg of purified I-2.

Antagonism of CS1 Inhibition by NH₂-terminal Fragments of I-2-- The results described above indicated that the NH₂-terminal region of I-2 has no inhibitory activity by itself, even though analysis of the NH₂-terminally deleted I-2 mutants showed that it was important for inhibition (30). To further evaluate the effect of the NH₂-terminal residues on CS1 activity, we tested the ability of the I-2(1-35) and I-2(1-64) to affect CS1 inhibition by full-length I-2. I-2(1-35) at 25 µM or I-2(1-64) at 1 µM caused 35- or 16-fold increases in the I-2 IC₅₀, respectively (56) (Fig. 2A). Consistent with this result, I-2(1-35) also antagonized inhibition of enzyme activity, measured as release of CS1 inhibition by I-2 (Fig. 2B). At a concentration of 10 µM the I-2(1-35) peptide fully released I-2 inhibition. Half-maximal release was achieved at ~1 µM peptide. These results confirm that the NH₂-terminal 35 residues of I-2 contain a binding site for CS1 even though alone they are not sufficient for inhibition.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effect of I-2(1-35) and I-2(1-64) on CS1 inhibition by I-2 or DARPP-32. A, shift of I-2 IC50 by I-2(1-35) or I-2(1-64). Different amounts of I-2 were incubated with 4-6 mU of rabbit skeletal muscle CS1 in the absence (closed squares) or presence of 25 µM of I-2(1-35) peptide (open circles) or 1 µM of I-2(1-64) (open triangles) for 5 min at 30 °C prior to phosphatase activity determination. The data shown represent an average of two experiments, each carried out in duplicate. B, effect of I-2(1-35) on the release of CS1 inhibition by I-2 or DARPP-32. The indicated concentrations of I-2(1-35) peptide were incubated with CS1 in the absence (closed squares) or presence of 4 nM of I-2 (open squares) or 2 nM of phosphorylated DARPP-32 (open triangles) for 5 min at 30 °C prior to determination of the phosphatase activity. Phosphatase activity is expressed as the percent of the activity in the absence of inhibitors and peptides. Data are presented as mean ± S.D. from three independent experiments with duplicate measurements.

I-1 and I-2 have been reported to compete for CS1 inhibition (46), and a DARPP-32 peptide containing the KIQF sequence antagonized inhibition by I-2 (44). Interaction of I-1 and its homolog DARPP-32 with CS1 involves two subdomains, one surrounding the phosphorylated Thr³⁴/Thr³⁵ and the other containing the KIQF motif (43, 44). Both subdomains are located within the NH₂-terminal 38 residues. Therefore, we questioned whether the I-2(1-35) peptide could also antagonize inhibition by DARPP-32. Under the conditions used, phosphorylated DARPP-32 had an IC₅₀ of ~1 nM, a value similar to that reported by other laboratories (33, 44). As shown in Fig. 2B, I-2(1-35) was more than 100-fold less effective in releasing inhibition by DARPP-32 than by I-2 (56). As a control, an unrelated peptide, RRAAEELDSRAGPSPQL, based on the sequence of eukaryotic elongation factor 2, was also tested. At concentrations up to 200 µM, it did not affect CS1 activity, nor did it release CS1 inhibition either by I-2 or DARPP-32 (data not shown). These data indicated that amino acids within the NH₂-terminal 35 residue of I-2 interact with CS1 at a site that is specific for I-2 and that is not shared by DARPP-32. However, this interaction alone cannot account for the inhibitory activity of I-2.

Binding of Wild Type and Mutant I-2 Proteins to CS1-- To confirm that the mutant I-2 proteins physically associated with CS1, an overlay assay was performed. Binding of CS1 was observed with all the I-2 mutants analyzed (Fig. 3). The binding of CS1 was specific because no signal was detected with bovine serum albumin (Fig. 3, lane 5). Deletion of the NH₂-terminal 11 residues of I-2, which interrupted the IKGI motif after the Lys, had little effect on interaction with CS1 and caused only ~20% decrease in binding, as estimated by densitometry. Deletion of 35 residues reduced binding by 60%. Interaction of the NH₂-terminal polypeptides, I-2(1-145), I-2(1-114), I-2(1-75), and I-2(1-64) with CS1 was reduced but still significant (Fig. 3). Interestingly, in the overlay assay, I-2(1-145) showed weaker interaction, ~25%, with CS1 as compared with the wild type, even though its inhibitory potency was similar to that of the full-length I-2. An independent analysis of interactions in solution, monitoring the formation of the complexes either by native gel electrophoresis or isoelectric focusing, showed that all truncated I-2 forms associated with CS1 (data not shown). These data are consistent with those of the overlay assays. The results directly demonstrate that sites of interaction with CS1 are present in both the NH₂ and the COOH termini of I-2.

View larger version (104K): [in this window] [in a new window]   Fig. 3.   Binding of CS1 to wild type and mutant I-2 proteins by overlay assay. Purified wild type I-2 or I-2 truncation mutants were separated on 13% Tricine-SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk and then probed with 4 µg/ml of recombinant CS1, in the presence of 0.2 mM Mn2+. Binding of CS1 was detected by incubation of the membrane with CS1 antibody followed by enhanced chemiluminescence and autoradiography. Lanes 1-4, 0.1 µg of wild type I-2, I-2(12-204), I-2(36-204), or I-2(1-145), respectively; lane 5, 0.5 µg of bovine serum albumin and 0.5 µg each of I-2(1-114), I-2(1-75), and I-2(1-64); lane 6, 20 ng of CS1. A representative experiment is shown.

Effect of R_GL(61-80) and I-2(135-151) on the Inhibition of CS1 by I-2 or DARPP-32-- To determine whether I-2 interacts with CS1 at the same site as other PP1-binding proteins, we analyzed the ability of a R_GL peptide (61-80), which contains the prototypical RVSF sequence, to alter I-2 or DARPP-32 inhibition of CS1. As shown in Fig. 4A, the peptide antagonized inhibition by I-2 and DARPP-32 with the same potency. Full release of inhibition was achieved at 1 µM peptide, and 50% release was attained at 0.1 µM. At 1 µM, the peptide also caused a ~25-fold increase in I-2 IC₅₀ (data not shown). As observed by Johnson et al. (40) with the R_GL peptide containing residues 63-75, the 60-81 peptide alone caused a small but consistent increase in phosphatase activity (Fig. 4A). These results support the hypothesis that I-2 shares a similar or an overlapping binding site on CS1 with R_GL and DARPP-32.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effect of RGL-(60-81) peptide and wild type or mutant I-2(135-151) peptides on CS1 inhibition by I-2 or DARPP-32. The indicated concentrations of peptides were incubated with 4-7 mU of rabbit skeletal muscle CS1, in the absence (closed squares) or presence of 4 nM I-2 (open squares) or 2 nM of phosphorylated DARPP-32 (open triangles) for 5 min at 30 °C, prior to the determination of phosphatase activity. A, RGL (60-81) peptide; B, I-2(135-151) peptide; C, I-2(135-151)F139A mutant peptide; D, I-2(135-151)L145A/Y147A mutant peptide. Data are presented as mean ± S.D. from three experiments except for D, which shows the average from two experiments with duplicate measurements.

It has been speculated that residues Lys¹³⁵-Lys-Arg-Gln-Phe¹³⁹ in I-2 resemble the RKKIQF motif in DARPP-32 (44). However, close scrutiny of the I-2 sequence revealed another region, Lys¹⁴²-Arg-Lys-Leu-His-Tyr-Asn¹⁴⁸, that we believe shows closer similarity to the consensus motif. A synthetic peptide, I-2(135-151), containing both candidate sequences, released CS1 inhibition by either I-2 or DARPP-32 in a very similar pattern (56) (Fig. 4B). Maximal effect was observed at 25 µM and 50% release was attained at ~7 µM. Similarly to the R_GL peptide, I-2(135-151) caused a ~20% increase in phosphatase activity at concentrations up to 10 µM and became inhibitory at higher concentrations. This inhibitory effect may account for the apparent inability to completely relieve the inhibition by I-2 or DARPP-32. The I-2(135-151) peptide at 25 µM also increased the IC₅₀ for I-2 by ~5-fold (data not shown). These results suggest that the region between 135-151 in I-2 may interact with CS1 at a site that is shared with other CS1-binding proteins.

We have previously shown that mutations of Thr⁷² and Ser⁸⁶ had no effect on the inhibitory or inactivating properties of I-2, even though activation of the CS1·I-2 complex was altered. However, deletion of residues 76-85 or substitution of amino acids 77-81 to Pro resulted in incomplete inactivation of the complex, without significantly affecting inhibitory potency (30). To further address the role of this region of I-2, a synthetic peptide comprising residues 70-93 was tested for its ability to antagonize inhibition by I-2. The peptide either by itself or in combination with I-2 had no effect on CS1 activity (data not shown).

Identification of KLHY as a CS1 Binding Motif in I-2-- Either of the two sequences KRQF or KLHY in the I-2(135-151) peptide could account for the observed antagonism of I-2 inhibition. Three approaches were taken to identify the residues involved. Taking advantage of the presence of an intervening methionine (Met¹⁴²) in I-2(135-151), the peptide was cleaved with CNBr to generate two short peptide fragments. One, I-2(135-141), contained the KRQF sequence and the other, I-2(142-151), the KLHY sequence. The I-2(142-151) peptide but not I-2(135-141) retained the ability to release I-2 inhibition, albeit to a lesser extent as compared with the full-length peptide (data not shown).

The second approach utilized two synthetic mutant peptides: I-2(135-151)L145A/Y147A, where both Leu¹⁴⁵ and Tyr¹⁴⁷ were substituted by Ala, and I-2(135-151)F139A, in which Phe¹³⁹ was replaced by Ala. The F139A mutant peptide behaved similarly to the wild type peptide (Fig. 4C) and caused maximal release of CS1 inhibition by both I-2 and DARPP-32 at ~25 µM. The concentration required for half-maximal effect by the F139A or the wild type peptide was very similar, 8-10 µM. The F139A mutant peptide alone also caused a small but significant activation at concentrations below 10 µM, similar to what was observed with the wild type peptide (Fig. 4, B and C). In contrast, the L145A/Y147A mutant peptide did not antagonize CS1 inhibition by either I-2 or DARPP-32, and by itself it did not activate (Fig. 4D). These data support the proposal that Leu¹⁴⁵ and Tyr⁴⁷ are involved in the interaction with CS1.

Further support for the thesis that KLHY and not KRQF is a CS1-binding site was gained by introducing point mutations in full-length I-2. Consistent with the analysis of the I-2(135-151) peptide, mutation of Phe¹³⁹ to Ala did not alter inhibitory activity. I-2(F139A) had an IC₅₀ comparable to that of the wild type I-2 (Fig. 5). I-2(L145A/Y147A), in which both Leu and Tyr were replaced by Ala, actually created a more potent inhibitor, with an IC₅₀ ~4-fold lower than that of the wild type protein (Fig. 5). This increased potency could be explained if one considers that the wild type I-2(135-151) peptide has a small but significant activating effect (Fig. 4B), as did the R_GL peptide containing the RVXF motif (40). Regardless of the increased potency, the results are consistent with the hypothesis that KLHY is a CS1 interacting sequence.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Inhibitory activities of I-2(F139A) and I-2(L145A/Y147A). Different amounts of wild type I-2 (squares), I-2(F139A) (circles), or I-2(L145A/Y147A) (triangles) were incubated with ~4 mU of rabbit skeletal muscle CS1 at 30 °C for 5 min, prior the phosphatase activity determination. Data shown are the average from two experiments with duplicate measurements.

View larger version (67K): [in this window] [in a new window]   Fig. 6.   Molecular modeling of the KLHY interaction with CS1. KLHY was modeled onto the RVSF position in the crystal structure of CS1 and RGL RVSF peptide complex. Top, ribbon diagram of the CS1 and stick representation of the RGL peptide. The active site is indicated by the arrow. Bottom, space filling model showing the interaction of RVSF (right, dark) or KLHY (left, dark) with a portion of CS1 (gray).

Molecular Modeling of the KLHY Peptide Bound to CS1-- Compared with the consensus (R/K)(V/I)X(F/W) motif, KRQF lacks a Val or Ile, which contributes an important hydrophobic interaction (41). The KLHY sequence has a Leu instead of Val or Ile and a Tyr instead of Phe or Trp. From the crystal structure of the R_GL peptide bound to CS1, it was implied that Leu would not be favorable at position 2 due to its bulky side chain (41). However, modeling of a bound KLHY tetrapeptide molecule using the coordinates from the CS1 and R_GL peptide complex showed that the KLHY sequence made interactions with CS1 similar to the R_GL peptide (Fig. 6). The side chain of Lys forms an ion pair comparable to Arg. The side chain of His can form hydrogen bonds with the carbonyl oxygen of Thr²⁸⁸ on CS1 through a water molecule, as is seen for Ser⁶⁷ in the R_GL peptide. In contrast to the predictions (41), in our model, Leu makes favorable Van der Waal's contacts with the side chains of Leu²⁴³, Ile¹⁶⁹, and Leu²⁸⁹ in the CS1 molecule, which create the hydrophobic pocket. In fact, it appears that Leu fits more completely in this hydrophobic pocket and contacts more hydrophobic surface area than Val in the R_GL peptide. The aromatic ring of Tyr maintains the same hydrophobic contact with Phe²⁵⁷ in CS1, as is seen for Phe in the R_GL peptide. Furthermore, the hydroxyl group of Tyr is hydrogen-bonded (3.3 Å) to the carbonyl oxygen of Arg²⁶¹ in the CS1 molecule. Preceding the KLHY sequence are two basic residues, another feature of the consensus PP1-binding motif. These residues may further interact with the adjacent acidic channel and thus strengthen the interaction at this site, as proposed by Egloff et al. (41). On the other hand, the Arg in the KRQF is too big to fit into the Val position without disturbing the structure of the peptide or CS1. Also, replacing an aliphatic residue with Arg would obviously weaken the hydrophobic interaction.

Effect of Microcystin on CS1·I-2 Complex Formation-- Studies from several laboratories (36, 37, 39), including our own (38), have indicated that the protein inhibitors of PP1 including I-1, DARPP-32, and I-2 share a site of interaction on CS1 with small molecule toxins, such as microcystin and okadaic acid, located on the 12-13 loop. Utilizing native PAGE, we showed direct competition between microcystin and I-2 for binding to recombinant CS1 (Fig. 7A). Microcystin at a concentration equimolar with I-2 impaired the formation of the CS1·I-2 complex (Fig. 7A, lanes 2 and 3), and disrupted the pre-formed complex (Fig. 7A, lane 4). Note that, for reasons that are not understood, free CS1 does not enter the native gel. Neither decreasing the concentration of acrylamide or bisacrylamide nor inverting the polarity of the current allowed CS1 to enter the gel (data not shown). Incubation of CS1 with -octylglucoside or a chaotropic salt, such as lithium chloride, was also ineffective.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Competition between toxins and I-2 or I-2 peptides for interaction with CS1. A, competition between microcystin-LR and I-2 for CS1 binding. Recombinant CS1, 2.7 µM, was incubated with 5.5 µM I-2, 5.5 µM microcystin, or both. Proteins were separated on 7% native PAGE and stained with Coomassie Blue (upper panel), and the CS1·I-2 complex formed was quantitated densitometrically (lower panel). Lane 1, CS1 incubated with I-2 for 40 min at room temperature; lane 2, CS1 incubated with microcystin before addition of I-2 and incubation for another 20 min; lane 3, I-2 and microcystin were mixed together before incubation with CS1; lane 4, CS1 incubated with I-2 before addition of microcystin. The position of the CS1·I-2 complex and the I-2 bands are indicated. The density of the complex formed in the absence of microcystin (lane 1) was taken as 100%. B, effect of I-2 or RGL peptides on CS1 inhibition by okadaic acid. I-2 or RGL peptides were preincubated with ~5 mU of rabbit skeletal muscle CS1 in the presence or absence of 50 nM okadaic acid at 30 °C for 5 min, before measurement of phosphatase activity. P1, 25 µM I-2(135-151); P2, 25 µM I-2(1-35); P3, 100 µM I-2(70-93); P4, 10 µM RGL(60-81); OA, okadaic acid. Data are presented as mean ± S.D. from three experiments with duplicate measurements.

Effect of the R_GL and I-2 Peptides on the Inhibition of CS1 by Okadaic Acid-- The notion that okadaic acid and I-2 bind to CS1 at a shared site is supported by fluorescence anisotropy (38) as well as biochemical (39) and mutagenesis studies (36). To examine whether either the NH₂-terminal 35 residues or the 135-151 region of I-2 interacted with CS1 at the same site as okadaic acid, peptide competition assays were conducted. None of the peptides tested, I-2(1-35), I-2(135-151), I-2(70-93), or R_GL(60-81), antagonized inhibition by okadaic acid, even at concentrations significantly higher than those effective for competition with I-2 (Fig. 7B). These results suggest that a distinct region of I-2 may be involved in the competition with okadaic acid implying that I-2 interacts with CS1 at an additional site.

Competition of I-2(135-151) and R_GL(61-80) Peptides for CS1 Inhibition by I-2(1-114) and Complex Formation-- The observation that I-2(1-114) was an effective inhibitor (IC₅₀ of 10 nM) is intriguing, since the KLHY motif lies COOH-terminal to residue 114. Competition assays showed that I-2-(135-151) and R_GL (61-80) increased the IC₅₀ of I-2(1-114) by 4- and 30-fold, respectively (Fig. 8). These results and the significant difference observed between the inhibitory potency of I-2(1-64) and I-2(1-114) (Fig. 1A) support the hypothesis that there are additional sites in the region of residues 64-114 that interact with CS1 at site(s) close to where I-2(135-151) and the consensus motif bind. We then examined whether I-2(1-35), I-2(135-151), or their combination was able to disrupt the complex formed by I-2(1-114) and CS1. Analysis by native gel electrophoresis revealed that the I-2(1-35) peptide, at a concentration 100-fold over I-2, reduced the formation of the complex between CS1 and I-2(1-114) by 40% (Fig. 9A, lane 4). I-2(135-151) by itself was less effective (Fig. 9A, lane 3), but in combination with I-2(1-35), the complex formation was reduced by 60% (Fig. 9A, lane 5). However, neither I-2(1-35), I-2(135-151), nor the combination of both peptides disrupted the complex formed between CS1 and the full-length I-2 (Fig. 9B), consistent with the existence of additional interacting sites in the COOH-terminal 114-204 region of I-2.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Effect of I-2(135-151) or RGL(61-80) peptide on CS1 inhibition by I-2(1-114). The indicated concentrations of I-2(1-114) were preincubated with 5-6 mU of rabbit skeletal muscle CS1 in the absence (open squares) or presence of 25 µM of I2(135-151) (closed squares) or 1 µM of RGL(61-80) (closed circles) at 30 °C for 5 min; phosphatase activity was then determined. The values shown represent averages of two to three experiments with duplicate measurements.

View larger version (28K): [in this window] [in a new window]   Fig. 9.   Effect of I-2 peptides on CS1·I-2(1-114) or CS1·I-2 complex formation. A, effect of I-2 peptides on CS1·I-2(1-114) (A) or CS1·I-2 (B) complex formation. Four µM CS1 and 5.6 µM I-2(1-114) (A) or 2.6 µM CS1 and 3.2 µM I-2 (B) were incubated at room temperature for 1 h in the absence or presence of I-2(1-35), I-2(135-151), or both peptides and 0.4 mM MnCl2. Lane 1, I-2(1-114) or I-2; lane 2, CS1 incubated with I-2(1-114) or I-2 without peptide; lane 3, I-2(1-114) or I-2 and 200-fold molar excess of I-2(135-151) peptide were incubated with CS1; lane 4, I-2(1-114) or I-2 and 100-fold molar excess of I-2(1-35) incubated with CS1; lane 5, I-2(1-114) or I-2 and 200-fold excess of I-2(135-151) and 100-fold excess of I-2(1-35) peptides were incubated with CS1. Samples were separated on 7% native PAGE, stained with Coomassie Blue (left panel) and quantitated for complex formation (right panel) by densitometric analysis. The density of the complex formed in the absence of peptide (lane 1) was taken as 100%. A representative experiment is shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

I-2 was initially identified as one of two heat stable protein inhibitors of PP1, although it subsequently became clear that I-2 is a regulatory subunit of the ATP-Mg²⁺-dependent phosphatase, whose activity state is modulated by phosphorylation of I-2. Previous work from our laboratory had suggested the existence of multiple interactions between I-2 and CS1 (30). This study expands the earlier observations and identifies residues Lys¹⁴⁴-Leu-His-Tyr¹⁴⁷ as the I-2 counterpart of the RVXF motif, common to many CS1-binding proteins (41). Our results are consistent with a model in which the NH₂-terminal 35 residues of I-2 bind to CS1 at a site that is specific for I-2, whereas the KLHY sequence interacts with CS1 at a site shared by other CS1-binding proteins. Several lines of evidence also point to the presence of three or more other contacts between CS1 and I-2. One site may be located within residues 64-114, another in the COOH-terminal 59 amino acids of I-2, and an additional site may also be present around Trp⁴⁶, corresponding to Phe³³ in the Drosophila I-2 (57). Mutation of Trp⁴⁶ to Ala in rabbit I-2 resulted in a 10-fold increase in the IC₅₀ (data not shown).

Taking into account these and previous studies, we propose a model for the complex interaction between I-2 and CS1 as depicted in Fig. 10. The physical properties of I-2, including the low sedimentation coefficient (s_20,w = 1.75 S) and large Stokes radius (3.5 nm), stability to heat and acid treatment, and the low content of hydrophobic amino acids, suggest that it is not a globular protein (58). Circular dichroism studies² indicate that I-2 has ~50% random coil structure, which could correlate with an elongated shape. An extended conformation would provide a structural basis for a model in which I-2 wraps around CS1 to establish multiple contacts. The NH₂-terminal region, subdomain 1, contains the IKGI sequence (32) and binds to site A. The region comprised of residues 135-151, subdomain 3, contains the KLHY motif and binds to site C. Residues within positions 64-114, subdomain 2, interact with site B, and a region near the COOH terminus, subdomain 4, may bind to site D. Subdomain 5, which surrounds Trp⁴⁶ may interact at a distinct site, site E. Sites A and C are both located on CS1 opposite to the active site (41, 59). Site C, the site shared with other CS1-binding proteins, is characterized by a hydrophobic channel and adjacent acidic residues. Site A is unique for I-2 binding, is characterized by a cluster of charged amino acids (59), and may be the site where the first I-2 contact is established (30). Although we have no direct evidence, we speculate that site B may be the 12-13 loop. Based on our model, it is unlikely that the Trp⁴⁶ subdomain 5 binds to site B or C, because the I-2(1-64) is not inhibitory. The finding that the Drosophila I-2 F33A mutant (equivalent to the rabbit W46A mutant) is more sensitive to antagonism by peptides containing the canonical RVXF sequence, as compared with wild type I-2 (57), cannot be taken as proof that the Phe³³ region interacts at the common CS1 site. It is equally possible that the mutation affects another contact whose disruption weakens the interaction with CS1, so that binding of the peptide to site C more readily releases inhibition. More importantly, it has not been shown that peptides encompassing Phe³³/Trp⁴⁶ can antagonize other CS1-binding proteins that have the consensus motif. Thus, we favor positioning the Trp⁴⁶ subdomain 5 as interacting at a separate site, site E. 

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Model of interaction between I-2 and CS1. A schematic diagram of the regions of I-2 that interact with CS1 is shown in the upper part. The IKGI and the KLHY motifs are indicated as subdomains 1 and 3, respectively. Subdomains 5, 2, and 4 denote, respectively, the region of I-2 comprising Trp46, residues 64-114, and a site in the COOH terminus of I-2. The lower part shows a cartoon of I-2 (thick line) wrapped around the CS1. The interacting sites on CS1 are indicated by capital letters A, B, C, D, and E. Interaction of I-2 with CS1 positions the masking region in front of the active site (*), thus blocking the access of substrates. Sites A and C are located at the back of the active site and indicated the binding sites for IKGI and the KLHY, respectively. Site B may correspond to the 12-13 loop of CS1. Site D is a site that may interact with the COOH terminus of I-2, and site E indicates the Trp46 binding site. Weakening of any of the interactions renders the masking region more mobile thus allowing access of substrates to the active site.

We propose that interactions at sites A, B, and C serve as anchors to bring a "masking region" close to the active site, thus blocking access to substrates. This mechanism is reminiscent of the inhibition of calcineurin by the FKBP12·FK506 complex, which does not inhibit the phosphatase by direct interaction with the active site. Instead, the FKBP12·FK506 complex interacts with calcineurin such that access of the substrate to the catalytic pocket is physically hindered (60). An important feature of our model is that the anchoring sites on I-2 are important to achieve inhibition but the primary sequence in the masking region is relatively less important, since it would not contact the catalytic site directly. This explains why mutations in this region, between Thr⁷² and Ser⁸⁶, do not alter the inhibitory potency of I-2 (30, 31). The lack of inhibitory activity for the NH₂-terminal peptides, I-2(1-35) and I-2(1-64), can be explained by the absence of an anchor in their COOH termini. The reduced potency of the NH₂-terminally truncated I-2 polypeptides would result from the removal of the strong NH₂-terminal anchor, IKGI. Competition with peptides at any of the anchoring sites will destabilize the CS1·I-2 complex making it easier to dislodge the masking region and allow partial access to the catalytic pocket. The anchoring mechanism can also account for the ability of several truncated I-2 mutants still to interact with CS1, albeit with much lower affinity than wild-type I-2 (Fig. 3).

In the ATP-Mg²⁺-dependent protein phosphatase, activation of CS1 by I-2 involves the conversion of the inactive CS1 to an active state following phosphorylation of I-2 at Thr⁷² (61). This event induces conformational changes in I-2 (38) that may loosen one or more of the anchors such that the masking region moves and no longer occludes the catalytic site. However, since there is no dissociation of the complex, some anchoring interactions must persist after phosphorylation. As dephosphorylation of Thr⁷² is required for activity toward exogenous substrates (61), our model positions Thr⁷² in front of the active site, but not necessarily in direct contact with CS1. In fact, an I-2 peptide containing residues 70-93 was not inhibitory and did not antagonize inhibition by I-2, suggesting that this region does not directly contribute greatly to CS1 binding. Previous work had shown that I-2(1-145) had the same inhibitory potency as wild type I-2 and formed a complex with CS1 that was not activated by glycogen synthase kinase-3 (30). The retention of the basic and the Leu residues in subdomain 3 may be sufficient for some degree of interaction at site C, which is further strengthened by the anchors in subdomains 1 and 2. These contacts, by positioning the masking domain in front of the catalytic pocket, could account for the similar inhibitory potencies of I-2(1-145) and wild type I-2. The weaker binding to CS1, as detected in the overlay assay, may be due to faster on and off rates, caused by the absence of the binding site in the COOH-terminal domain. As to the failure of glycogen synthase kinase-3 to activate the CS1·I-2(1-145) complex, it is possible that the conformational changes required for activation do not occur without full interaction with the KLHY sequence and/or with subdomain 4. It should be noted, however, that the CS1 was in the inactive form, because treatment with trypsin alone did not result in activation (30).

The much greater inhibitory potency of I-2(1-114) as compared with I-2(1-64) implies that there are at least two anchors within residues 1-114 that effectively position the masking region. One is the NH₂-terminal IKGI sequence and the other is most likely located within residues 64-114. The ability of the I-2(135-151) and the R_GL(61-80) peptides to antagonize inhibition by I-2(1-114) (Fig. 8) suggested that one of the anchors in the 1-114 region interacts with CS1 at a site that is close to or overlapping with site C. Indeed, recent work (59) has shown that mutation of CS1 residues near site C decreases the sensitivity to inhibition by I-2, but not by I-1, indicating that the I-2-specific site A is close to site C. The proximity of sites A and C may allow for binding at one site to affect interaction at the other. This could explain why the I-2(135-151) peptide attenuates inhibition by I-2(1-114) and the weak antagonizing effect of the I-2(1-35) peptide on DARPP-32 inhibition (Fig. 2B). However, the presence of subdomain 1 alone cannot account for the inhibitory potency of I-2(1-114) because I-2(1-64), which contains the same region, is not inhibitory. An additional site must be present between residues 64 and 114.

The molecular basis for CS1 inhibition by I-2 is different from that by DARPP-32 or I-1, which do not form stable complexes with the catalytic subunit. However, although the three inhibitors do not share obvious primary structure homology, they are mutually exclusive for inhibition of CS1 indicating that they share common binding site(s). Inhibition by DARPP-32/I-1 involves the interaction of phosphothreonine-34/35 directly with the active site (43, 44), whereas inhibition by I-2 does not involve a similar interaction of the phosphorylated Thr⁷². Nevertheless, all these inhibitors make multiple contacts with CS1, and they share at least two binding sites, one where the KLHY sequence in I-2 and the KIQF sequence in DARPP-32/I-1 bind and the other on the 12-13 loop (residues 267-282), where the toxins also bind (35-37). The regions of the inhibitors that interact with the 12-13 loop have not been defined, as none of the peptides examined, based either on I-2 (Fig. 7B) or DARPP-32 (44), antagonized CS1 inhibition by okadaic acid.

Note that inhibitory activity and binding of I-2 to CS1 are not always correlated in the sense that binding can be independent of inhibition. In fact I-2(1-64) interacts with CS1 (Figs. 2A and 3) but by itself it does not inhibit enzyme activity (Fig. 1A). The binding of I-2(1-145) with CS1, as determined by the overlay assay, is much weaker as compared with the full-length I-2 (Fig. 3), but the IC₅₀ is similar to that of the wild type I-2 (Fig. 1A). Our model for interaction of I-2 with CS1 readily accommodates uncoupling of binding and inhibition. A single site is sufficient for binding but not for inhibition, which appears to require interaction at two or more sites straddling the masking region. Furthermore, as suggested by the peptide competition and the CS1 overlay studies, interaction at the individual sites may be of low affinity but together create high affinity for CS1.

Huang et al. (32) reported that a peptide containing the KLHY sequence of I-2 did not compete for inhibition of CS1 by DARPP32, but increased the IC₅₀ for I-2 by 3-fold. Although these results are in apparent disagreement with ours, which show that a similar peptide antagonized inhibition by DARPP-32 and I-2 with the same potency, other data in the same report (32) implied the presence of a CS1-binding site in the region comprising Lys¹⁴⁴-Leu-His-Tyr¹⁴⁷. Notably, CS1 could bind to I-2(1-160) but not to I-2(1-140). Furthermore, the 5-fold increase in the IC₅₀ for I-2 that we observed in the presence of I-2(135-151) is similar to effects reported by Huang et al. (32) using the DARPP-32(6-38) or the M110-(1-40) peptides, both of which contain the canonical RVXF motif. Additional evidence that the KLHY sequence is analogous to the consensus CS1-binding motif comes from our finding that the I-2(135-151) peptide also attenuated inhibition of CS1 by Glc8p (data not shown), the yeast homolog of I-2 (62). Most importantly, a peptide in which the Leu¹⁴⁵ and Tyr¹⁴⁷ were replaced by Ala completely lost the ability to antagonize inhibition of CS1 by both I-2 and DARPP-32 (Fig. 4D), as well as by Glc8p (data not shown). Finally, modeling KLHY onto the structure of RVSF complexed with CS1 showed that the KLHY can be readily accommodated in the CS1 binding pocket, contrary to the predictions that a Leu at the position of the Val or Ile would not be favorable (41).

Not all CS1-binding consensus motifs need to bind with equal affinity to site C. A peptide containing KSVTW in p99 showed ~100-fold lower potency in antagonizing p99 inhibition of CS1, compared with a p53BP2 peptide, which harbored the RVKF sequence (18). However, substitution of Trp with Phe in the p99 peptide did not increase its ability to release p99 inhibition, supporting the idea that the affinity of the binding protein for CS1 is not determined solely by the conserved residues. Similarly, the lower efficacy of the I-2(135-151) peptide to affect the IC₅₀ for I-2 as compared with the R_GL(61-80) peptide indicates that binding of the KLHY sequence by itself to CS1 is not very strong. When Leu¹⁴⁵ and Tyr¹⁴⁷ were mutated to Ala in full-length I-2 a more potent inhibitor was created, with 4-fold lower IC₅₀. A similar, ~2-fold higher potency was also observed by Huang et al. (32) for the I-2(Y147A) mutant. The reason that the Ala substitution renders I-2 a more powerful inhibitor is not completely clear. However, R_GL peptides containing the RVSF sequence as well as the I-2(135-151) peptide by themselves slightly activate CS1. One possibility is that interaction at the C site, which is behind the catalytic pocket, affects the conformation of the active site to cause a small activation. Thus, loss of this interaction paradoxically leads to a more potent inhibitor.

In conclusion, it is clear that the interaction of I-2 with CS1 involves multiple sites, some of which are specific for I-2, such as site A where the NH₂-terminal subdomain 1 binds, whereas others are shared with other CS1-binding proteins. This implies that interaction of the various CS1-binding proteins with CS1 at shared as well as unique sites may allow for different modes of regulation of the large number of PP1 holoenzymes and therefore their involvement in diverse cellular functions. The experiments presented support the hypothesis that the KLHY is the I-2 homolog of the RVXF motif. These findings expand the degeneracy of the consensus sequence to (R/K)(V/I/L)X(F/W/Y) to consist of a branched aliphatic residue at position 2 and an aromatic residue at position 4. This more degenerate motif will be important for the identification and analysis of new CS1-binding proteins. However, final definition of the detailed interaction between I-2 and CS1 will have to await the determination of the three-dimensional structure of the complex.

	ACKNOWLEDGEMENTS

We acknowledge the contributions of Erquan Zhang and Marcella Steele for the preparation of the F139A and L145A/Y147A, and the W46A mutant I-2, respectively, and the technical assistance of Lori Cooper. We express our gratitude to Dr. Tania Barshevsky of New England Biolabs for sharing information about the modified I-2 purification procedure. We are particularly grateful to Dr. Peter J. Roach for discussion of the work and for criticism of the manuscript. We thank Dr. David Barford from the Institute of Cancer Research, London, UK, for providing the coordinates of the catalytic subunit of PP1 complexed with the RVSF peptide.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant DK36569.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 317-274-1585; Fax: 317-274-4686; E-mail: adepaoli@iupui.edu.

Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M003082200

² J. Yang and A. A. DePaoli-Roach, unpublished results.

	ABBREVIATIONS

The abbreviations used are: PP1, type 1 serine/threonine protein phosphatase; CS1, catalytic subunit of PP1; I-1, phosphatase inhibitor-1; I-2, phosphatase inhibitor-2; R_GL, regulatory subunit of glycogen-associated PP1; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES