Molecular Determinants of Nuclear Protein Phosphatase-1 Regulation by NIPP-1*

Monique BeullensDagger §, Aleyde Van EyndeDagger §, Veerle VulstekeDagger , John Connor, Shirish Shenolikar, Willy StalmansDagger , and Mathieu BollenDagger parallel

From the Dagger  Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium and the  Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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NIPP-1 is a subunit of the major nuclear protein phosphatase-1 (PP-1) in mammalian cells and potently inhibits PP-1 activity in vitro. Using yeast two-hybrid and co-sedimentation assays, we mapped a PP-1-binding site and the inhibition function to the central one-third domain of NIPP-1. Full-length NIPP-1 (351 residues) and the central domain, NIPP-1143-217, were equally potent PP-1 inhibitors (IC50 = 0.3 nM). Synthetic peptides spanning the central domain of NIPP-1 further narrowed the PP-1 inhibitory function to residues 191-200. A second, noninhibitory PP-1-binding site was identified by far-Western assays with digoxygenin-conjugated catalytic subunit (PP-1C) and included a consensus RVXF motif (residues 200-203) found in many other PP-1-binding proteins. The substitutions, V201A and/or F203A, in the RVXF motif, or phosphorylation of Ser199 or Ser204, which are established phosphorylation sites for protein kinase A and protein kinase CK2, respectively, prevented PP-1C-binding by NIPP-1191-210 in the far-Western assay. NIPP-1191-210 competed for PP-1 inhibition by full-length NIPP-11-351, inhibitor-1 and inhibitor-2, and dissociated PP-1C from inhibitor-1- and NIPP-1143-217-Sepharose but not from full-length NIPP-11-351-Sepharose. Together, these data identified some of the key elements in the central domain of NIPP-1 that regulate PP-1 activity and suggested that the flanking sequences stabilize the association of NIPP-1 with PP-1C.

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Reversible protein serine/threonine phosphorylation in higher eukaryotes is catalyzed by protein kinases and phosphatases (1). The functional diversity of protein phosphatases is increased by the presence of isoforms of the catalytic subunits and numerous noncatalytic or regulatory subunits. For example, there are four mammalian isoforms of the catalytic subunit of type-1 protein phosphatases (2, 3). These catalytic subunits (PP-1C)1 are anchored at various subcellular locations by their association with targeting subunits that also define the substrate specificity and activity of the enzyme. Of the nearly 20 PP-1-targeting subunits thus far identified in mammalian cells, the best characterized is the G subunit which targets PP-1C to glycogen and enhances its glycogen-synthase phosphatase activity (4-6). Interaction of the skeletal muscle GM subunit with PP-1C is in part mediated by an RVXF motif that binds in an extended conformation within a hydrophobic channel near the C terminus of PP-1C (5, 6). Phosphorylation of GM by protein kinase A (PKA) at a threonine in the X position abolished PP-1 binding. Variants of the RVXF motif are present in many other PP-1 regulators, including the myosin-binding or M subunit, the p53 binding-protein-2, inhibitor-1, DARPP-32, and inhibitor-2 (5-10). Moreover, the regulation of PP-1C by these proteins was impaired by mutations within the RVXF motif or by the presence of competing RVXF-containing peptides. However, these regulators also possessed other interaction sites with PP-1C in addition to the RVXF motif (4, 5, 8).

Biochemical (11) and immunofluorescence (12) studies showed that a large fraction of PP-1 is present in the nucleus. Nuclear PP-1 (PP-1N) has been implicated in the regulation of mRNA splicing (13), in the cell cycle-dependent control of the retinoblastoma protein (14) and lamin B (15), and in the dephosphorylation of the transcription factors CREB (16) and Sp1 (17). However, the PP-1 holoenzymes involved and their regulation remain poorly understood. The two major nuclear targeting subunits of PP-1 have been identified (11) as NIPP-1 and R111, which is also known as p99 (18) or PNUTS (19). NIPP-1 and R111 are each associated with 30-50% of PP-1C in mammalian nuclei and may target PP-1 to RNA (19-21). NIPP-1 potently inhibits PP-1C activity in vitro (Ki = 1-10 pM), and the NIPP-1·PP-1C complex (PP-1NNIPP-1) is essentially inactive. However, PP-1NNIPP-1 can be reactivated by phosphorylation with PKA and protein kinase CK2, without disruption of the holoenzyme (21). Two phosphorylation sites, Ser199 for PKA and Ser204 for CK2, flank an RVXF motif that may contribute to PP-1 binding. Thus, the phosphorylation of NIPP-1 may impair or disrupt the interaction of PP-1 with the RVXF motif and thereby enhance nuclear PP-1 activity. On the other hand, there must be yet other interactions between PP-1 and NIPP-1 that maintain the association of these two proteins in the active complex. It is also highly unlikely that the RVXF motif itself mediates PP-1 inhibition, because this sequence is present in many PP-1 regulators that do not inhibit enzyme activity. Thus, the precise molecular basis for PP-1 inhibition by NIPP-1 remains unknown.

In the studies described, we have analyzed the association of NIPP-1 and PP-1C by the yeast two-hybrid assay as well as by co-sedimentation. Further studies used numerous synthetic peptides based on NIPP-1 to identify two sequences in the central domain that independently mediate PP-1 binding and inhibition. A synthetic peptide encompassing these two sequences effectively competed for PP-1 inhibition by full-length NIPP-1 as well as other PP-1 inhibitors. Our studies established that phosphorylation of the synthetic NIPP-1 peptide on the two serines flanking the RVXF motif resulted in the dissociation of this motif from PP-1C and decreased the potency of the peptide as a PP-1 inhibitor. Other experiments suggested that sequences outside the central domain were also required for the continued and stable association of PP-1C with full-length NIPP-1 following its phosphorylation and inactivation by PKA and CK2. Together, these studies provided the first insights into the molecular determinants for the regulation of nuclear PP-1 by NIPP-1.

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Materials-- PP-1C (22) and phosphorylase b (23) were purified from rabbit skeletal muscle. Digoxygenin-labeled PP-1C was prepared according to Beullens et al. (24) using the digoxygenin protein labeling kit purchased from Roche Molecular Biochemicals. CNBr-activated Sepharose, glutathione-Sepharose, and Blue Sepharose were purchased from Amersham Pharmacia Biotech. Peptides were synthesized on Milligen 9050, using the N-(9-fluorenyl)methoxycarbonyl method. For the PP-1-binding assays, the synthetic peptides were coupled to bovine serum albumin using glutaraldehyde (25). Phosphorylase b was phosphorylated in the presence of [gamma -32P]ATP by purified phosphorylase kinase (26).

Preparation of Recombinant Proteins-- Bovine NIPP-1 was expressed in Sf9 cells using the baculovirus system and purified according to Vulsteke et al. (21). Oligonucleotides 5'-ATCGAACATATGGGTGGAGAGGATGATGAAC-3' (NdeI site underlined) and 5'-CTGCACGGATCCTCAGTTCCGAAAGCGACCAACC-3' (BamHI site underlined) were used as sense and antisense primers in a PCR reaction using the NIPP-1 cDNA as template to yield a product encoding NIPP-1143-217, which was verified by double-stranded sequencing. The PCR product was digested with NdeI/BamHI and subcloned into pET-16b (Novagen) for expression in bacteria as a polyhistidine-tagged protein. Escherichia coli BL21(DE3) cells, transformed with the expression vector, were grown at 37 °C in LB medium supplemented with 200 µg/ml ampicillin, to reach an A600 of 0.6-0.8. NIPP-1 protein was induced by the addition of 1 mM isopropyl-1-thio-beta -D-galactopyranoside. After 2 h at 30 °C, bacteria were collected by centrifugation (10 min at 5,000 × g) and resuspended in lysis buffer (pH 7.9) containing 20 mM Tris/HCl, 5 mM imidazole, 0.5 M NaCl, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM benzamidine, and 5 µM leupeptin. Cells were disrupted by three cycles of freezing in liquid N2, followed by boiling for 15 min. After centrifugation for 20 min at 20,000 × g, the supernatant was applied to a 5-ml Ni2+-IDA-Sepharose column (Sigma) equilibrated in lysis buffer. The column was washed with 25 ml of lysis buffer containing in addition 100 mM imidazole, and the protein was eluted with 50 ml of lysis buffer containing 400 mM imidazole. Fractions containing NIPP-1143-217 were pooled, concentrated in a Vivaspin ultrafiltration unit (VivaScience), and dialyzed against 20 mM Tris/HCl at pH 7.5.

Human inhibitor-1 was expressed in E. coli as a fusion protein with glutathione S-transferase and purified on glutathione-Sepharose as described by Endo et al. (27). Glutathione S-transferase inhibitor-1 was digested with thrombin (1 units/ml for 60 min) and boiled for 5 min, and inhibitor-1 was separated from the denatured proteins by centrifugation (5 min at 10,000 × g). Purified inhibitor-1 was phosphorylated with PKA catalytic subunit (27). Inhibitor-2 was purified from heat-treated lysates from BL21(DE3) cells transformed with pET8d inhibitor-2 plasmid (kindly provided by Prof. A. A. DePaoli-Roach) using chromatography on Blue Sepharose (28).

Coupling of Inhibitor-1 and NIPP-1 to Sepharose-- NIPP-11-351, NIPP-1143-217, and phosphorylated inhibitor-1 (80 µg of protein) were each coupled to 0.5 ml of CNBr-activated Sepharose as described by the manufacturer. The gels were incubated with 10 pmol of PP-1C for 30 min at 4 °C on a rotating wheel. The unbound PP-1C was removed by washing with buffer A (50 mM glycylglycine, pH 7.4, containing 0.5 mM dithiothreitol and 5 mM beta -mercapthoethanol). The matrices were incubated with an equal volume of buffer A containing 150 mM NaCl and 10 µM NIPP-1191-210 for 10 min at 25 °C. Following centrifugation (5 min at 15,000 × g), the gel was washed twice with buffer A containing 150 mM NaCl and resuspended in the same buffer. The pellet and supernatant were assayed for phosphorylase phosphatase activity before and after incubation with trypsin as described (29). One unit of phosphatase releases 1 nmol of phosphate/min at 30 °C.

Assays-- NIPP-1, its fragments, inhibitor-1, and inhibitor-2 were assayed as inhibitors of the phosphorylase phosphatase activity of PP-1C (29). The inhibitor-2-mediated inactivation of PP-1C was monitored by the time-dependent decrease in trypsin-revealed phosphorylase phosphatase activity (2). The inhibitors were diluted in buffer A containing bovine serum albumin (1 mg/ml) and 0.05% Triton X-100. All phosphatase assays were carried out in buffer A, except for the inhibitor-2-induced inactivation, which was undertaken in 50 mM imidazole, pH 7.4, containing 0.5 mM dithiothreitol, 5 mM beta -mercapthoethanol, and 1 mg/ml bovine serum albumin. Phosphatase assays at pH 5.0 and 6.0 were carried out in buffer containing 20 mM Tris, 20 mM MES, 20 mM CAPS, and 0.1% of Triton X-100, adjusted to the desired pH at room temperature.

For the far-Western with digoxygenin-labeled PP-1C, the albumin-coupled peptides were slot-blotted on polyvinylidene difluoride membrane (Bio-Rad), and the bound PP-1 was visualized using anti-digoxygenin antibodies (24). Protein concentrations were measured according to Bradford (30) with bovine serum albumin as standard. Statistics are given as the means ± S.E. for the indicated number (n) of experiments.

Yeast Two-hybrid Assays-- The plasmid pAS1-Glc7 (31) encodes the yeast PP-1 catalytic subunit fused to the Gal4 DNA-binding domain (residues 1-147). The cDNAs encoding full-length NIPP-1 (351 residues) and various fragments (NIPP-11-169, NIPP-1143-169, NIPP-1143-224, NIPP-1143-351, NIPP-1167-224, and NIPP-1225-351) were subcloned into pACT2 (32), in-frame with the Gal4 activation domain (residues 768-881) and the hemagglutinin (HA) epitope. Full-length NIPP-1 cDNA was cloned into the pACT2 vector in two steps. First, PCR was carried out using pBl-2175 as template (33) and 5'-ATCGAACCGCGGCCATGGAGGCCATGGCGGCAGCCGCGAACTC-3' (the SacII site is shown in bold and the SfiI site is underlined) and 5'-AGAAAGTGCCGTGTGTAC-3' as primers. The PCR product (300 base pairs) was digested with SacII and ClaI, and the resulting 118-base pair fragment was exchanged for the SacII-ClaI fragment of the original pBl-2175 vector. The second step involved digestion of this vector with SfiI and XhoI, which excised the entire NIPP-1 cDNA, which was then cloned into the SfiI-XhoI sites of the pACT2 vector. The NIPP-1143-351, NIPP-1225-351, and NIPP-1143-224 deletion mutants were also constructed using PCR. The sense oligonucleotides were: 5'-ATCGAACCGCGGCCATGGAGGCCATGGGTGGAGAGGATGATGAAC-3' for NIPP-1143-351 and NIPP-1143-224 and 5'-ATCGAACCGCGGCCATGGAGGCCATGGTGCAGACTGCAGTGGTC-3' for NIPP-1225-351. The SfiI sites are underlined. The antisense oligonucleotides were: 5'-CATTCCCTCGAGATCCCACCCTCCTC-3' for NIPP-1143-351 and NIPP-1225-351 and 5'-CTGCACCTCGAGCCGAAAGCGACCAACC-3' for NIPP-1143-224. The XhoI sites are underlined. The PCR products were digested with SfiI and XhoI and cloned into the SfiI-XhoI sites of pACT2. PCR was performed using standard conditions and Pfu DNA polymerase (Stratagene). NIPP-11-169 was obtained by subcloning the SfiI-EcoRI fragment of NIPP-11-351 into pACT2. NIPP-1143-169 was obtained by subcloning the SfiI-EcoRI fragment of NIPP-1143-224 into pACT2. To obtain NIPP-1167-224, cDNA for NIPP-1143-224 was digested with SfiI and EcoRI (to eliminate a 75-base pair 5'-fragment), gel-purified, filled in with Klenow, and blunt-ended into pACT2. All constructs were verified by nucleotide sequencing.

In the yeast two-hybrid assay, Saccharomyces cerevisiae strains Y190 and Y187 were transformed by the lithium acetate method of Gietz et al. (34) with pACT2-NIPP-1-fragments and pAS1-Glc7, respectively. Individual transformants were purified on selection media (synthetic complete medium lacking leucine for Y190 or tryptophan for Y187) and grown at 30 °C. After mating of the appropriate yeast transformants, diploids were selected on a medium deficient in both Trp and Leu. Expression of beta -galactosidase was determined in duplicate after 8 h, using the 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside filter assay described in the CLONTECH Yeast Protocols Handbook.

Co-sedimentation of NIPP-1 and PP-1C-- Overnight cultures (20 ml) of Y190 yeast transformed with pACT2-NIPP-1 constructs were inoculated into 200 ml of synthetic complete medium minus leucine. Cells were grown to an A600 of 0.5-0.8, harvested by centrifugation, washed with water, and resuspended in 500 µl of buffer B, containing 50 mM Tris/HCl, pH 7.5, 0.3 M NaCl, 0.1% Triton X-100, 1 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, 5 µM leupeptin, 50 µM 1-chloro-3-tosylamido-7-amino-2-heptanone, and 50 µM L-1-tosylamido-2-phenylethyl chloromethyl ketone. Cells were lysed by vortexing with glass beads (six times for 30 s). The homogenates were centrifuged for 10 min at 2000 × g to remove cell debris. The pellets were extracted once with 500 µl of buffer B, and the supernatants were combined with the extracts. Immunoprecipitation of Gal4-HA-NIPP-1 fusion proteins from 250 µl of yeast extract was performed with monoclonal antibodies (clone 12CA5) against the HA tag, and protein A-TSK® (Affiland). The immunoprecipitates were washed once with 0.25 M LiCl in TBS (10 mM Tris/HCl, pH 7.5, 150 mM NaCl) and twice with TBS and resuspended in 225 µl of buffer C containing 10 mM Tris/HCl, pH 7.5, 0.1 M NaCl, 0.05% Triton, 1 mM dithiothreitol, and the mixture of protease inhibitors present in buffer B. Aliquots (50 µl) of the immunoprecipitates were incubated for 10 min with either buffer or with 1 pmol PP-1C on a rotating wheel, washed twice with buffer C, resuspended in 45 µl buffer C, and assayed for spontaneous and trypsin-activated phosphorylase phosphatase activity. The phosphatase activities (derived from endogenous Glc7p and added muscle PP-1C) were normalized for the amount of NIPP-1 fusion proteins in the immunoprecipitates, as determined by Western blotting with antibodies against the Gal4 activation domain (CLONTECH).

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Identification of PP-1 Binding Sites-- Initial studies of NIPP-1 interactions with PP-1C were undertaken using the yeast two-hybrid assay. For this purpose, the cDNAs encoding bovine NIPP-1 and various fragments were subcloned in the pACT2 vector containing the Gal4 transcriptional activation domain fused at its C terminus to a HA tag (Fig. 1A). The cDNA encoding the budding yeast PP-1 catalytic subunit (Glc7p) was subcloned in the pAS vector containing the Gal4 DNA-binding domain. Additional experiments showed that purified Glc7p was inhibited by bovine NIPP-1 with similar potency to mammalian PP-1 catalytic subunit (data not shown). Mating single transformants containing pAS-Glc7 with those containing pACT2-NIPP-1 yielded double tranformants that contained the beta -galactosidase reporter gene under the control of the Gal4 promoter. Analysis of numerous NIPP-1 constructs (Fig. 1B) showed that only strains that expressed both Glc7p and NIPP-1 fragments containing residues 167-224 were stained blue with 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (Fig. 1B).


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Fig. 1.   Analysis of PP-1C interaction with NIPP-1 by yeast two-hybrid and co-sedimentation assays. A, a scheme of the Gal4-NIPP-1 constructs used in the yeast two-hybrid assay. B, interaction of Glc7p and NIPP-1 in the two-hybrid assay. The 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside filter assay was carried out in duplicate for 8 h. Diploids containing pACT2-NIPP-1 and the empty pAS1 vector were white in this assay (data not shown). C, co-immunoprecipitation of PP-1C with Gal4-HA (control) or Gal4-HA-NIPP-1 fusion proteins. The fusion proteins were immunoprecipitated from yeast extracts with anti-HA antibodies and protein A-TSK, and the immunoprecipitates were washed with 0.25 M LiCl (see "Experimental Procedures"). Subsequently, aliquots of the immunoprecipitates were incubated with either buffer (- exogenous PP1C) or muscle PP-1C (+ exogenous PP1C) and resedimented. The figure shows the spontaneous (open bar fragments) and trypsin-revealed (open plus closed bar fragments) phosphorylase phosphatase activities that co-sedimented with the same amount of NIPP-1 fusion proteins, as determined by densitometry of Western blots with antibodies against the Gal4 tag. The figure shows a representative profile of five different experiments.

In additional experiments, we used the monoclonal anti-HA antibodies to immunoprecipitate Gal4-NIPP-1 fusion proteins from extracts of single-transformed yeast. Subsequently, it was investigated whether endogenous Glc7p had co-sedimented with the fusion proteins and whether additional catalytic subunit could be bound by the immunoprecipitates, when preincubated with an excess of PP-1C from rabbit skeletal muscle. The "spontaneous" phosphatase activity in these immunoprecipitates was assayed using phosphorylase a as substrate. Because the complex of intact NIPP-1 and PP-1C is inactive (21), we also assayed for "total" PP-1 activity following the trypsin digestion of NIPP-1 present in the immunoprecipitates. Because PP-1C is fully active after trypsinization, the total activity can be taken as a measure of the amount of catalytic subunit. Thus, the assay of spontaneous and total activity enabled us to differentiate between fragments of NIPP-1 that bound PP-1C and those that also inhibited enzyme activity (2). In comparison with the endogenous Glc7p activity that was co-immunoprecipitated, four to seven times more phosphorylase phosphatase activity co-sedimented with the NIPP-1 fusion proteins when the immunoprecipitates were preincubated with an excess of muscle PP-1C (Fig. 1C). This suggests that the immunoprecipitated NIPP-1 fusion proteins were only partially saturated with endogenous Glc7p, either because the level of Glc7p was limiting or because Glc7p was dissociated during the washing of the immunoprecipitates with 0.25 M LiCl. Fig. 1C compares the binding and inhibition of endogenous Glc7p or Glc7p plus muscle PP-1C by equivalent molar amounts of NIPP-1 fusion proteins, as measured by Western analysis with anti-Gal4 antibodies. Compared with full-length NIPP-11-351, the immunoprecipitates with the Gal4 fusions containing either the N terminus, NIPP-11-169 or the C terminus, NIPP-1225-351, showed no significant PP-1C binding as assessed after trypsin digestion. On the other hand, the Gal4-NIPP-1143-224 fusion protein bound PP-1C and effectively inhibited its activity. Interestingly, a further N-terminal deletion to produce NIPP-1167-224 yielded a higher spontaneous PP-1 activity in the immunoprecipitates than obtained with either NIPP-11-351 or NIPP-1143-224. Moreover, this activity was further increased by only 2-fold following trypsin treatment. This suggested that residues 143-167, albeit not inhibitory by themselves (Fig. 1C), were also required for effective PP-1 inhibition. Essentially the same results were obtained for the binding and inhibition of Glc7p or muscle PP-1C by these NIPP-1 fusion proteins (Fig. 1C), suggesting that similar structural determinants mediate the interactions of NIPP-1 with the mammalian and yeast catalytic subunits.

To more precisely delineate the PP-1 binding sites in NIPP-1, we synthesized a series of overlapping 20-mer peptides that together encompassed the central domain of NIPP-1 defined by the sequence (residues 141-230). Of the eight peptides analyzed, only two, NIPP-1181-200 and NIPP-1191-210, inhibited PP-1C, with IC50 values of 32 and 11 µM, respectively (Fig. 2). A decapeptide representing the overlapping sequence, NIPP-1191-200, was then synthesized and shown to inhibit PP-1 activity with an IC50 of 8 µM (Table I). This peptide lacked an RVXF consensus sequence and thus clearly established that enzyme inhibition was not mediated by this motif. Substitution of pairs of adjacent basic residues with Ala increased the IC50 of the decapeptide by 2-3-fold (Table I). On the other hand, substitution of the three Lys residues for Arg did not significantly alter the inhibitory potency of the decapeptide. Collectively, these data suggested that the overall basic nature of the peptide, rather than other features, such as the occurrence of alternating Lys and Arg residues, mediates PP-1C inhibition.


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Fig. 2.   Synthetic peptides identify PP-1C-binding sites in the NIPP-1 central domain. Full-length NIPP-1 is represented as a bar in which the central domain is defined by the two methionines, Met143 and Met225. A series of overlapping 20-mer peptides encompassing the entire central domain were tested for their ability to inhibit PP-1C purified from rabbit skeletal muscle. IC50 is the concentration of peptide (means ± S.E., n = 4) that inhibited the phosphorylase phosphatase activity of PP-1C by 50%. Also shown is the ability of albumin-coupled peptides to bind digoxygenin-labeled PP-1C (DIG-PP-1C) in a far-Western assay.

                              
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Table I
Determinants of PP-1C inhibition by NIPP-1191-200
IC50 refers to the peptide concentration required for 50% inhibition of PP-1C (means ± S.E. for four assays). The first line shows unmodified NIPP-1191-200. In the other peptides the substituted residues are underlined.

We also investigated the direct association of the synthetic peptides with PP-1C using a far-Western assay. The synthetic peptides directly immobilized on polyvinylidene difluoride membranes failed to bind the digoxygenin-conjugated PP-1C (data not shown). However, when first coupled to albumin, the peptide NIPP-1191-210 readily bound PP-1C (Fig. 2). Surprisingly, NIPP-1181-200, already shown to inhibit PP-1C activity, failed to bind PP-1C in the far-Western assay. This suggested that the inhibitory site (residues 191-200) was not recognized in this assay and that the digoxygenin-conjugated PP-1C preferentially associated with the region of NIPP-1 containing the RVXF motif. Support for this hypothesis comes from the observation that the majority of PP-1-binding proteins that can be visualized by far-Western analysis contain this highly conserved PP-1-binding motif. To check whether the binding of NIPP-1191-210 to PP-1C in the far-Western assay was indeed mediated via the RVXF motif (residues 200-203), we analyzed NIPP-1191-210 peptides containing the substitutions V201A and/or F203A (Fig. 3B). These studies showed that like NIPP-1181-200 and NIPP-1201-220 (which do not contain the complete RVXF motif), the mutant peptides failed to bind PP-1C in the far-Western assay. This suggested that three of the four residues in the RVXF motif, Arg200, Val201, and Phe203, may be required for PP-1C binding in the far-Western assay. On the other hand, the mutations of both Val201 and Phe203 did not diminish the inhibitory potency of the NIPP-1191-210 peptide (Fig. 3A), further emphasizing that the RVXF motif did not mediate PP-1 inhibition. These data also showed that the far-Western assay visualized the interaction of NIPP-1 with PP-1C via the RVXF motif but could not be used as an indicator for the binding to PP-1C via other NIPP1 site(s).


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Fig. 3.   Importance of Val201 and Phe203 for the interaction of NIPP-1191-210 with PP-1C. A shows the effects of NIPP-1191-210 and the peptide with Val201 and/or Phe203 mutated to Ala on the phosphorylase phosphatase activity of PP-1C (n = 3). B shows the ability of the same peptides coupled to albumin to bind digoxigenin-labeled PP-1C (DIG-PP-1C) in the far-Western assay.

The above data identified at least two distinct interactions of PP-1C with the central domain, NIPP-1143-217, that inhibited PP-1C with essentially identical potency to full-length NIPP-11-351 (IC50 = 0.3 nM). However, the synthetic peptide NIPP-1191-210, which retained these two PP-1 binding sites, was a much weaker inhibitor of PP-1 (IC50 = 11 µM). This could point to the existence of additional interaction sites in the central domain that could not be identified by two-hybrid analysis, far-Westerns, and phosphatase inhibition assays. Alternately, the reduced potency of the NIPP-1191-210 peptide may arise from the inability of the PP-1 regulatory sequences to assume the conformation found in NIPP-1143-217. In this regard, it is interesting to note that the deletion of residues 143-166, which did not directly mediate PP-1 binding, also increased the spontaneous activity of the PP-1C·NIPP-1167-224 complex (Fig. 1C).

Competition between NIPP-1191-210 and Endogenous PP-1 Inhibitors-- The inactive complex of NIPP-11-351 and PP-1C, previously shown to be reactivated by phosphorylation with PKA and/or CK2 (21), was also activated by the addition of NIPP-1191-210 (Fig. 4A). This "deinhibition" effect was observed at peptide concentrations that were themselves only marginally inhibitory, indicating effective competition for PP-1 binding between NIPP-1 and the less potent NIPP-1191-210 peptide that alleviated PP-1 inhibition.


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Fig. 4.   NIPP-1191-210 competes for PP-1C inhibition by full-length NIPP-1, inhibitor-1, and inhibitor-2. Inhibition of PP-1C by varying concentrations of NIPP-1 (A), PKA-phosphorylated inhibitor-1 (B), and inhibitor-2 (C) was measured in the presence or absence of NIPP-1191-210. The assays with NIPP-1 were done in the presence of 150 mM NaCl. The results (n = 3) are expressed as a percentage of control activity without the inhibitors. The right panel of C also shows the effect of NIPP-1191-210 on the time-dependent inactivation of PP-1C by inhibitor-2 (I2), as assessed by trypsin-activated phosphorylase phosphatase activity (n = 3). The control (100%) value was not significantly affected by up to 2.5 µM NIPP-1191-210. The 100% value with 3.1, 5, and 10 µM NIPP-1191-210 was 24, 34, and 40% lower than that without peptide.

The deinhibition of PP-1NNIPP-1 by NIPP-1191-210 was, however, only seen at physiological salt concentration, i.e. 150 mM NaCl (data not shown). A similar salt requirement was previously noted for the activation of PP-1NNIPP-1 by PKA and CK2 (21). On the other hand, 150 mM NaCl was not required for the activation of a complex formed with PP-1C and the central domain, NIPP-1143-217, by either phosphorylation or addition of the NIPP-1191-210 competitor (data not shown). This indicated that despite their similar inhibitory potency, the central domain forms a less stable complex with PP-1C than does full-length NIPP-1. Our data also suggested that in the absence of salt, there are additional phosphatase binding site(s) in the N- and/or the C-terminal domains of NIPP-1.

Because the RVXF motif did not mediate PP-1 inhibition but was a determinant of PP-1C interaction with NIPP-1191-210, this raised the question of whether the NIPP-1 peptide also competed with other PP-1 inhibitors that contained a RVXF motif. Thus, we examined the competition between NIPP-1191-210 and two other PP-1 inhibitors, inhibitor-1 and inhibitor-2, which contain variants of the consensus RVXF motif (8, 10). Inhibitor-2 also mediates a time-dependent inactivation of PP-1C that can be readily distinguished from enzyme inhibition by the inability to recover total enzyme activity following the proteolytic degradation of inhibitor-2 (2). Our data showed that PP-1 inhibition by inhibitor-1 and inhibitor-2 as well as the inhibitor-2-mediated inactivation of PP-1C were reversed by NIPP-1191-210 (Fig. 4, B and C), suggesting potential competition between these inhibitors at the RVXF-binding site. This hypothesis was strengthened by the finding that mutant NIPP-1191-210 peptides with the substitutions V201A and/or F203A within the RVXF motif, even at much higher concentrations, failed to deinhibit PP-1 in the presence of NIPP-1, inhibitor-1, or inhibitor-2 (data not shown).

NIPP-1191-210 Disrupts PP-1 Complexes-- To further investigate the ability of NIPP-1191-210 to disrupt PP-1 binding to other inhibitors, the NIPP-1 peptide was added to phosphoinhibitor-1-Sepharose saturated with PP-1C. Fig. 5 shows that NIPP-1191-210 dissociated virtually all the inhibitor-1-bound PP-1 catalytic subunit. Similarly, NIPP-1191-210 eluted most of the PP-1C bound to NIPP-1143-217-Sepharose. On the other hand, NIPP-1191-210 failed to release PP-1C bound to full-length NIPP-1-Sepharose. These data are consistent with our earlier findings that the phosphorylation of the central one-third domain of NIPP-1 (35), but not full-length NIPP-1 (21), released the bound PP-1C and suggest once again that the complex of the catalytic subunit with full-length NIPP-1 is more stable.


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Fig. 5.   NIPP-1191-210 competes for PP-1C association with inhibitor-1 or NIPP-1. Phosphoinhibitor-1, NIPP-1, and NIPP-1143-217 were covalently bound to CNBr-activated Sepharose and saturated with rabbit skeletal muscle PP-1C. The washed resins were incubated either with buffer or 10 µM NIPP-1191-210. The figure shows the trypsin-activated phosphorylase phosphatase activity that was released from the matrix as a percentage of the total activity associated with the column (n = 3).

Functional Effects of NIPP-1191-210 Phosphorylation-- Our previous studies had shown that the potency of NIPP-1 as a PP-1 inhibitor was reduced by its phosphorylation by PKA and/or CK2 and had established the phosphorylation sites for PKA (Ser199) and CK2 (Ser204) that flanked the RVXF motif (21). In the present study, we investigated the functional impact of these phosphorylations on the NIPP-1191-210 peptide. Synthesis of NIPP-1191-210 with phosphoserine at either position 199 or 204 significantly reduced its activity as a PP-1 inhibitor (Fig. 6A). The doubly phosphorylated peptide showed an even greater reduction in its PP-1 inhibitory activity. We have also found that phosphoserine-199 and/or phosphoserine-204 abolished PP-1C binding to NIPP-1191-210 in far-Western assays (Fig. 6B). Likewise, the phosphorylated NIPP-1191-210 was less efficient in competing for inhibition of PP-1C by inhibitor-1 and -2 (data not shown) and in reversing the inhibitor-2-mediated inactivation of PP-1C (Fig. 7).


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Fig. 6.   Phosphorylation of NIPP-1191-210 reduces its affinity for PP-1C. A shows PP-1C inhibition by indicated concentrations of NIPP-1191-210 and the peptides phosphorylated on Ser199 and/or Ser204 (n = 4). B shows the binding of these peptides, after coupling to albumin, to digoxigenin-labeled PP-1C in the far-Western assay.


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Fig. 7.   Phosphorylation of NIPP-1191-210 abrogates its ability to antagonize inhibitor-2-mediated inactivation of PP-1C. PP-1C was incubated at 25 °C with 0.3 nM inhibitor-2 (control) and with 1 µM NIPP-1191-210 or NIPP-1191-210 phosphorylated on Ser199 and/or Ser204. At indicated times, aliquots were taken and assayed for trypsin-activated phosphorylase phosphatase activity (n = 3).

The above data suggested that phosphorylation of the flanking serines impaired PP-1 binding at or near the RVXF motif and reduced the activity of NIPP-1191-210 as a PP-1 inhibitor. We speculate that the reduced inhibitory potency of phosphorylated NIPP-1191-210 is accounted for by an electrostatic interaction of the phosphate groups with basic residues in the upstream inhibitory binding site, which we have shown to be positive determinants of the inhibitory potency (Table I). This would also explain why the substitutions V201A or F203A, albeit causing a disruption of the RVXF motif, did not affect the potency of NIPP-1191-210 as a PP-1 inhibitor (Fig. 3).

Conclusions-- The present study identified two interactions of PP-1C with the central domain of NIPP-1. One, mediated by the consensus RVXF motif found in many other PP-1C-binding proteins, did not inhibit enzyme activity. The second interaction occurred through a highly basic sequence immediately N-terminal to the RVXF motif and inhibited enzyme activity. However, a peptide encompassing both sequences (NIPP-1191-210) did not inhibit PP-1 activity at subnanomolar concentrations as seen with NIPP-1143-217. This implied that other sequences within the central domain were also required to enhance the potency of NIPP-1 as a PP-1 inhibitor. Because no additional inhibitory fragments could be detected in the central domain in our experiments (Fig. 2), we have postulated that NIPP-1143-217 is a more potent inhibitor because of its unique conformation.

NIPP-1191-210 also harbored the determinants for NIPP-1 regulation by PKA and CK2. Our data showed that the phosphorylation of two serines in NIPP-1191-210 decreased its affinity for PP-1 and concomitantly reduced its activity, both as a direct PP-1 inhibitor and as a competitor with other PP-1 inhibitors. Finally, we have provided evidence suggesting that the N-terminal and/or C-terminal thirds of NIPP-1 are required for its stable association with PP-1C following phosphorylation by PKA and CK2.

The crystal structure of a complex between PP-1C and a synthetic fragment of the muscle GM subunit has revealed that the RVXF motif binds in a hydrophobic channel within the C-terminal region of PP-1C, which is located some distance from the catalytic-site channel (5, 6). The inhibitory sequence (residues 191-200) in the central domain of NIPP-1 would appear to be too close to the RVXF motif to bridge the distance between the hydrophobic channel and the catalytic site. We therefore propose that the inhibition of PP-1C by NIPP-1 does not result from its direct interaction with the catalytic site but rather results from interactions of the basic inhibitory NIPP-1 site (residues 191-200) with acidic residues immediately adjacent to the RVXF-binding site in PP-1C. This mechanism would be different from that proposed for the inhibition of PP-1C by the cytoplasmic proteins, inhibitor-1, and DARPP-32, which only become potent PP-1 inhibitors after their phosphorylation by PKA (5, 8, 9). It is worth noting that two PP-1 binding sites have also been mapped within the N-terminal 30 residues of inhibitor-1 and DARPP-32. One of these is the KIQF sequence, similar to RVXF in other PP-1 regulators, which probably binds in the same hydrophobic groove in the PP-1 catalytic subunit. The second interaction most likely occurs through the PKA-phosphorylated threonine, which associates with the PP-1 catalytic site and functions as a pseudosubstrate to inhibit enzyme activity. Hence, multiple interactions between PP-1C and its endogenous inhibitors may be a recurrent mechanism for PP-1 regulation.

In summary, we have defined a complex interaction of PP-1C with a major nuclear regulatory subunit, NIPP-1, and identified a unique polybasic sequence in NIPP-1 that is essential for PP-1 inhibition. We have also gained better insight into the phosphorylation mechanism that activates the nuclear phosphatase PP-1NNIPP-1. Although the present studies hint at the existence of additional regulatory sequences in NIPP-1 that greatly enhance its activity as a PP-1 inhibitor, it raises the intriguing possibility that mutations can be introduced into the PP-1 catalytic subunit that will selectively impair its regulation by NIPP-1. Thus, these structure-function studies open the way for addressing the physiological role of NIPP-1 in the control of nuclear PP-1 activity.

    ACKNOWLEDGEMENTS

Stefaan Wera and Juan Colomer are acknowledged for contributions to the identification of inhibitory NIPP-1 peptides. Karl Beckers, AnneMie Hoogmartens, Nicole Sente, Peter Vermaelen, and Cindy Verwichte provided expert technical assistance. Valère Feytons is acknowledged for synthesis of peptides and oligonucleotides.

    FOOTNOTES

* This work was supported by the Fund for Scientific Research Grant G.0179.97, funds from ASLK, funds from Flemisch Concerted Research Action, Prime Minister's office Grant IUAP P4/23, and National Institutes of Health Grant DK52044 (to S. S.).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.

§ Postdoctoral fellow of the National Fund for Scientific Research-Flanders.

parallel To whom correspondence should be addressed: Afdeling Biochemie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-34-57-01; Fax: 32-16-34-59-95, E-mail: Mathieu.Bollen{at}med.kuleuven.ac.be.

    ABBREVIATIONS

The abbreviations used are: PP-1, protein phosphatase-1; PP-1C, catalytic subunit of PP-1; PP-1N, nuclear PP-1; NIPP-1, nuclear inhibitor of PP-1; PKA, protein kinase A; CK2, protein kinase CK2, previously called casein kinase-2; HA, hemagglutinin; PCR, polymerase chain reaction; MES, 4-morpholineethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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