Interaction of p130 with, and Consequent Inhibition of, the Catalytic Subunit of Protein Phosphatase 1alpha *

Kenji YoshimuraDagger §, Hiroshi TakeuchiDagger §, Osamu Sato, Kiyoshi HidakaDagger , Naoko Doira||, Miho TerunumaDagger , Kae HaradaDagger , Yasuo Ogawa, Yushi Ito||, Takashi KanematsuDagger , and Masato HirataDagger **

From the Dagger  Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, and Station for Collaborative Research and the || Laboratory of Pharmacology, Faculty of Medical Science, Kyushu University, Fukuoka 812-8582 and the  Department of Pharmacology, Juntendo University School of Medicine, Tokyo 113-8421, Japan

Received for publication, October 23, 2000, and in revised form, February 1, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta 1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1alpha (PP1calpha ) as a p130-binding protein. The association between p130 and PP1calpha was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1calpha resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1calpha also detected the presence of a complex of p130 and PP1calpha . The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1calpha , was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca2+ signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1calpha .


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3),1 a product of receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C (PLC), plays an important role as an intracellular second messenger by mobilizing Ca2+ from nonmitochondrial stores (1). We previously isolated two Ins(1,4,5)P3-binding proteins with molecular masses of 130 and 85 kDa from rat brain (2, 3) with the use of an Ins(1,4,5)P3 affinity column (4, 5). Partial amino acid sequencing revealed that the 85-kDa molecule was PLC-delta 1 (2). Identification of the pleckstrin homology (PH) domain of PLC-delta 1 as the site of Ins(1,4,5)P3 binding helped to define the PH domain as an inositol compound binding module (6, 7).

The Ins(1,4,5)P3-binding protein with a molecular mass of 130 kDa, termed p130, was a previously unidentified molecule (2, 3). The predicted amino acid sequence of rat p130 shares 38.2% identity with that of rat PLC-delta 1; the five identified domains of PLC-delta 1 (PH, EF-hand, putative catalytic (X and Y), and C2 domains) are all present in p130. The domain organization of p130 suggests that the protein is likely to possess a fold similar to that of PLC-delta 1, a notion that is supported by the results of limited proteolysis with trypsin (8). However, p130 exhibits some distinct characteristics. It is larger than the PLC-delta isozymes, and it possesses unique regions both at the NH2 terminus, preceding the PH domain, and at the COOH terminus. Moreover, the residues within the catalytic domain of PLC-delta that are critical for enzyme activity (His356 and Glu390) are not conserved in p130 (9). The PH domain of p130, like that of PLC-delta 1, is important for the binding of Ins(1,4,5)P3 (10). Other molecules that show sequence similarity to p130, including human PLC-L (11) and the K10F12.3 gene product of Caenorhabditis elegans (12), have also been described. Otsuki et al. (13) recently isolated a cDNA from mouse brain that encodes a protein with 66% sequence identity to PLC-L; they therefore termed this protein PLC-L2 and renamed the original PLC-L as PLC-L1. Furthermore, the gene for human type2 p130 (PLC-L2) has also been cloned (14). All of these proteins exhibit characteristic NH2- and COOH-terminal extensions and replacement of critical catalytic residues. The identification of a p130-related molecule in such a simple organism as C. elegans suggests that this family of proteins diverged early from other PLC isozymes. We propose that this distinct family of PLC-related proteins be designated the PLC-related catalytically inactive protein (PRIP) family (comprising PRIP-1 and -2 subfamilies).

To investigate the physiological functions of PRIP family proteins, we previously examined the possible role of the binding of inositol compounds to the PH domain of p130 (10, 15-17). Our results suggested that p130, which is localized predominantly in the cytoplasm, contributes to Ins(1,4,5)P3-mediated Ca2+ signaling. The high affinity binding of Ins(1,4,5)P3 to the PH domain of p130 might also serve to sequester Ins(1,4,5)P3 and therefore prevent its interaction with Ins(1,4,5)P3 receptors and metabolizing enzymes (18).

We have now applied the yeast two-hybrid system to identify proteins that interact with p130. With the unique NH2-terminal region of p130 as the bait for screening a human brain cDNA library, we isolated two positive clones, one of which was shown to encode the catalytic subunit of protein phosphatase 1alpha (PP1calpha ). To characterize the interaction between p130 and PP1calpha , we studied the association of these two proteins both in vitro and in living cells, we delineated further the region of p130 that is responsible for binding to PP1calpha , and we examined the effect of such binding on the enzymatic activity of PP1calpha .

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Cloning vectors pGBT9 and pACT2, a human brain cDNA library, and yeast strains HF7c and SFY526 were obtained from CLONTECH (Palo Alto, CA). All restriction endonucleases and DNA-modifying enzymes were from Toyobo (Tokyo, Japan). Dropout yeast selection medium and dropout base medium were from BIO101 (Vista, CA). YPD medium for yeast and bacterial medium were obtained from Becton Dickinson (Sparks, MD). Polyvinylidene difluoride (PVDF) membranes were from Millipore (Bedford, MA). [gamma -32P]ATP (222 terabecquere/mmol) was obtained from DuPont-New England Nuclear. A large scale plasmid preparation kit, QIAfilter Plasmid Giga kit, and nitrilotriacetic acid-agarose beads for purification of His6-tagged proteins were from Qiagen (Chatsworth, CA). Protein G-Sepharose, glutathione-Sepharose 4B beads, and pGEX vectors were from Amersham Pharmacia Biotech. Ins(1,4,5)P3 was synthesized as described (19). The catalytic subunit of cAMP-dependent protein kinase (PKA) was obtained from Promega (Madison, WI), and wild-type rabbit PP1calpha was from Calbiochem-Novabiochem (La Jolla, CA). A soluble form of PtdIns(4,5)P2, diC8-PtdIns(4,5)P2, was obtained from Echelon Research Laboratories (Salt Lake City, UT). GM peptide (GRRVSFADNFGFN) and its random sequence (GNFRGFRSADFVN) were synthesized using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) cleavage method on an Advanced ChemTech 348MPS peptide synthesizer, and the purity was checked by applying the sample to a µBondashere 5-µ C18 column mounted on a high-performance liquid chromatography column (more than 90%). Other reagents used were of the highest grade available.

Plasmid Construction-- For construction of p130 bait plasmids, pcMT3 (9) was digested with XhoI, and the released fragment (nucleotides (nt) 535 to 5233) was cloned into the SalI site of pGBT9. The resulting plasmid, pGBT9-p130Full (p130 plasmid 1; amino acid residues 24 to 1096) (see Fig. 1A), was digested with PstI, rendered blunt-ended with DNA polymerase, and then self-ligated at the SmaI site (nt 1359), thereby generating p130 plasmid 2 (amino acids 24 to 298). In the same manner, pGBT9-p130Full was self-ligated between the KpnI site (nt 1130) or the BamHI site (nt 710) and the PstI site (all sites were blunt-ended) to generate p130 plasmids 3 (amino acids 24 to 222) and 4 (amino acids 24 to 82), respectively. The p130 plasmid 5 (amino acids 222 to 298) was constructed by self-ligation between the blunt-ended XhoI (nt 535) and KpnI (nt 1130) sites of plasmid 2.

For construction of pGBT9-p130D (amino acids 848 to 1096), a SpeI site (nt 3011) was introduced into pcMT3 by site-directed mutagenesis, and the resulting plasmid was digested with SpeI (nt 3011) and XhoI (nt 5233). The released 2.2-kilobase pair fragment was then ligated into the SpeI-SalI sites of pGBT9. For construction of pACT2-PP1calpha (Delta 29-163) a positive clone obtained from the yeast two-hybrid screening, pACT2-PP1calpha , was digested with PvuII (nt 116 and 815) and self-ligated. For expression of recombinant PP1calpha in Escherichia coli, the BamHI fragment of pACT2-PP1calpha was ligated into the BamHI site of pGEX-3X; the resulting construct encodes a fusion protein of glutathione S-transferase (GST) and PP1calpha .

Yeast Two-hybrid Screening and beta -Galactosidase Assay-- Yeast two-hybrid screening of a human brain cDNA library cloned in the pACT2 vector was performed in yeast strain HF7c with the bait plasmids pGBT9-p130PH or pGBT9-p130D. Transformants (total of 2 × 106) were plated and selected with a combination of tryptophan, leucine, and histidine. The positive clones identified by two-hybrid screening were sequenced with an ABI 373A automated DNA sequencer. The domains required for the interaction between p130 and PP1calpha were investigated by expression of various combinations of bait and target plasmids in yeast SFY526 cells and measurement of beta -galactosidase activity.

GST Fusion Protein Precipitation and Protein Overlay Analyses-- The recombinant GST-PP1calpha fusion protein was purified from E. coli by affinity chromatography, and recombinant full-length p130 (amino acids 24 to 1096) and the PH domain of p130 (p130PH; amino acids 95 to 232) were prepared as described previously (8, 10). For "pull-down" assays, GST-PP1calpha was incubated for 1 h at 4 °C with glutathione-Sepharose 4B beads in binding buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 100 mM NaCl, 10% glycerol, 0.5 mg/ml bovine serum albumin (BSA), 5 mM 2-mercaptoethanol). The beads were washed with 50 volumes of binding buffer and then incubated (6 µg of GST-PP1calpha ) for 1 h at 4 °C, with gentle rotation, in a total volume of 150 µl with recombinant full-length p130 or p130PH. After washing of the beads five times with 500 µl of binding buffer, bound proteins were eluted with 50 µl of a solution containing 50 mM Tris-HCl (pH 8.0) and 10 mM reduced glutathione and were then subjected to SDS polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis with antibodies to p130 (2F9) or to p130PH (3, 8).

For overlay analysis, samples were fractionated by SDS-PAGE, and the separated proteins were transferred electrophoretically to a PVDF membrane. After the blocking of nonspecific sites with 5% dried skim milk, the membrane was incubated for 1 h at room temperature with a protein probe (10 µg/ml). The membrane was then washed and incubated with antibodies to the probe protein followed by alkaline phosphatase-conjugated secondary antibodies, after which immune complexes were detected by enzymatic reaction.

Immunoprecipitation-- COS-1 cells, COS-1p130 cells (COS-1 cells stably expressing p130) (18), and mouse brain extract were subjected to immunoprecipitation with a specific monoclonal antibody to p130 (2F9) or polyclonal antibodies to PP1calpha (Santa Cruz Biotechnology, Santa Cruz, CA). Cells (5 × 106) or mouse brain (wet weight, 0.2 g) were homogenized in 0.5 ml of a solution containing 20 mM HEPES-NaOH (pH 7.4), 130 mM NaCl, 5 mM EDTA, and a mixture of protease inhibitors. The homogenate was centrifuged (14,000 × g, 20 min, 4 °C), and the resulting supernatant was incubated, with gentle rotation, for 1 h at 4 °C with 30 µg of antibodies to p130 or to PP1calpha that had been premixed with 10 µl of a 50% slurry of protein G-Sepharose in phosphate-buffered saline containing 0.1% BSA. The beads were then washed twice with a homogenizing solution (described above) containing 0.2% Triton X-100, boiled in SDS sample buffer, and subjected to SDS-PAGE and immunoblot analysis with antibodies to PP1calpha or to p130.

Analysis of Protein-Protein Interaction in Real Time-- Protein-protein interaction was examined in real time with a BIACORE 2000 surface plasmon resonance analyzer (Biacore International, Uppsala, Sweden). Recombinant GST-PP1calpha was immobilized on the surface of a CM5 sensor chip that had been activated with N-hydroxysuccinimide and N-ethyl-N'-(3-diethylaminopropyl) carbodiimide. Recombinant full-length p130 (0.23, 2.3, 23, 230, or 2300 nM) was injected over the chip surface at a rate of 10 µl/min in a solution containing 10 mM HEPES-NaOH (pH 7.4), 0.15 M NaCl, 3.4 mM EDTA, and 0.005% Tween 20.

Assay of Glycogen Phosphorylase Activity-- COS-1 or COS-1p130 cells (2 × 106) were lysed by three freeze-thaw cycles in a solution containing 50 mM NaCl, 10 mM MES-NaOH (pH 6.0), 1 mM EDTA, and 10 mM 2-mercaptoethanol. The lysate was subjected to centrifugation at 15,000 × g for 30 min, and the resulting supernatant was assayed for glycogen phosphorylase activity as described (20).

Assay of Phosphatase Activity-- Phosphatase activity was determined in a reaction mixture (40 µl) containing 139.2 mM KCl, 20 mM 4-morpholinepropanesulfonic acid-KOH (pH 7.0), 0.1 mM MnCl2, 0.5 mM dithiothreitol, BSA (0.5 mg/ml), 2 µM phosphorylated myosin light chain (from bovine stomach), 3.4 nM recombinant rabbit skeletal muscle PP1calpha , and various concentrations of recombinant full-length p130 or p130PH, in the absence or presence of 10 µM Ins(1,4,5)P3. The mixture minus the substrate was incubated for 10 min at 25 °C, and the reaction was started by the addition of phosphorylated myosin light chain and stopped after 20 min by the addition of 0.2 ml of ice-cold 10% trichloroacetic acid. The unphosphorylated and phosphorylated myosin light chains were separated by two-dimensional electrophoresis, and the density of each spot was determined as described (21).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two-hydrid Screening for Proteins That Interact with p130-- Screening of a human brain cDNA library with a bait plasmid (2) encoding the unique NH2-terminal region of rat p130 (amino acids 24 to 298), including the PH domain and a portion of the EF-hand motif (Fig. 1A), yielded 51 positive clones of a total of 2 million clones examined; no positive clones were obtained with a bait plasmid encoding the COOH-terminal region (amino acids 848 to 1096) of p130. 10 of the 51 clones identified proved to be false positives, and the remaining 41 clones were divided into two groups on the basis of analysis of their inserts by polymerase chain reaction amplification and restriction enzyme digestion. Sequencing revealed that one of these 41 clones encoded full-length PP1calpha , a 37,510-Da protein composed of 330 amino acids. To delineate more precisely the region of p130 required for binding to PP1calpha , we used plasmids encoding smaller portions of p130 as baits in two-hybrid analysis with the plasmid encoding full-length PP1calpha (Fig. 1A). Positive signals were obtained with a bait plasmid (3) encoding amino acids 24 to 222, as well as with that (1) encoding full-length p130. Neither a bait plasmid (4) encoding amino acids 24 to 82 nor one (5) encoding residues 222 to 298 yielded a positive signal (Fig. 1B). A plasmid encoding a PP1calpha mutant lacking amino acids 30 to 162 did not yield a positive signal with any of the p130 bait plasmids examined. These results thus suggested that the region of p130 composed of residues 83 to 222 interacts with that of PP1calpha comprising residues 30 to 162. 


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Fig. 1.   Yeast two-hybrid analysis of the interaction between p130 and PP1calpha . A, schematic representation of plasmids encoding various regions of p130 (left) and PP1calpha (right) that were used for analysis of the domains required for binding. The initial screening of the human brain cDNA library for p130-binding proteins was performed with p130 bait plasmid 2 (pGBT9-p130PH(24-298)). Bait plasmids 3 (XhoI-KpnI fragment of p130 cDNA), 4 (XhoI-BamHI), and 5 (KpnI-SmaI) encode amino acids 24 to 222, 24 to 82, and 222 to 298 of p130, respectively. These constructs were introduced into yeast strain SFY526, together with pACT2-PP1calpha or pACT2-PP1calpha (Delta 30-162), the latter of which was prepared from the former by digestion with PvuI and self-ligation. B, beta -Galactosidase assay of protein-protein interaction. Two-hybrid analysis was performed with SFY256 cells transformed with the indicated p130 (1 to 6) and PP1calpha plasmids. The activity of beta -galactosidase was determined with a filter assay.

Association of p130 with PP1calpha in Vitro-- We next examined the interaction of p130 and PP1calpha in vitro by several methods. The association was first analyzed with an overlay assay (Fig. 2A). Extracts of nontransformed E. coli and of bacteria expressing a GST-PP1calpha fusion protein, as well as recombinant GST-PP1calpha purified from such a latter extract, were fractionated by SDS-PAGE, and the separated proteins were transferred to a PVDF membrane and probed with antibodies to PP1calpha to confirm that the prominent band that migrated at a position corresponding to a molecular size of 37 kDa was indeed PP1calpha . Duplicate membranes were incubated in the presence of a recombinant p130 fragment containing the PH domain (p130PH; amino acids 95 to 232), recombinant full-length p130 (residues 24 to 1096), or BSA (negative control). After washing, the membranes were exposed to the corresponding antibodies to p130PH or to p130. Both the recombinant GST-PP1calpha present in the bacterial extract and the purified protein interacted with both full-length p130 and p130PH. Together with the results from the yeast two-hybrid analysis, these data indicate that residues 95 to 222 of p130 (which include the entire PH domain and the 20 residues preceding it) mediate the interaction of this protein with PP1calpha .


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Fig. 2.   Association between p130 and PP1calpha in vitro. A, overlay assay. Extracts (10 µg of protein) of either nontransformed E. coli (lane 3) or E. coli expressing recombinant GST-PP1calpha (lane 1), or 1 µg of purified recombinant GST-PP1calpha (lane 2), were subjected to SDS-PAGE. The separated proteins were transferred to a PVDF membrane, which was subsequently subjected to immunoblot analysis with antibodies to PP1calpha (panel a). Alternatively, the membranes were first incubated in the presence of p130PH (panel b), full-length p130 (panel c), or BSA (panel d), each at a concentration of 10 µg/ml, and were then subjected to immunoblot analysis with antibodies to p130PH or to p130 as indicated. B, pull-down assay. Recombinant GST-PP1calpha (or GST) attached to glutathione-Sepharose 4B beads was incubated with various protein samples, after which proteins that had bound to the beads were eluted with reduced glutathione and subjected to SDS-PAGE and immunoblot analysis with appropriate antibodies. Panel a, beads were incubated with 1 µM p130PH in the absence or presence of 0.01 or 0.1 µM full-length p130 or of 0.1 or 1 µM PLC-delta 1 (prepared as described in Ref. 7); immunoblot analysis was performed with antibodies to p130PH. Panel b, beads were incubated with 0.1 µM full-length p130 in the absence or presence of 0.1, 1, or 10 µM p130PH; immunoblot analysis was performed with antibodies to full-length p130. Panel c, beads were incubated in the presence of 1 µM p130PH or full-length p130, in the absence or presence of 50 µM GM peptide (GRRVSFADNFGFN) or a peptide of the same amino acid composition but of random sequence (GNFRGFRSADFVN); immunoblot analysis was performed with antibodies to p130PH or to full-length p130. Panel d, beads were incubated in the presence of 3 µM wild-type (wt) or mutant (V95L or F97A) fragments of p130 spanning residues 82 to 232; immunoblot analysis was performed with antibodies to p130PH(95-232) or to PP1calpha . C, phosphorylation of p130 by PKA and its effect on association with PP1calpha Panel a, recombinant full-length p130 (150 pmol) was incubated in a volume of 50 µl with 100 µM [gamma -32P]ATP in the absence (lane 1) or presence (lane 2) of 0.1 µg of the catalytic subunit of PKA, after which the reaction mixtures were subjected to SDS-PAGE and autoradiography. Panels b and c, recombinant p130 phosphorylated by PKA (lane 2) or treated with ATP alone (lane 1) was incubated with recombinant GST-PP1calpha immobilized on glutathione-Sepharose 4B beads, after which bead-bound protein was eluted with reduced glutathione and subjected to SDS-PAGE and immunoblot analysis with antibodies to p130 (b) or to PP1calpha (c). D, surface plasmon resonance analysis. GST-PP1calpha was immobilized on a sensor chip and exposed to various concentrations of full-length p130 (0.23, 2.3, 23, 230, and 2300 nM) for 360 s before the application of buffer alone; data are expressed in relative units (RU).

The GST-PP1calpha fusion protein was also subjected to a pull-down assay with recombinant p130 or p130PH (Fig. 2B). Incubation of a GST-PP1calpha resin with p130PH and subsequent immunoblot analysis of bead-bound proteins with antibodies to p130PH revealed that p130PH was precipitated by GST-PP1calpha and that this interaction was sensitive to the presence of low concentrations of full-length p130 but not to PLC-delta 1 (Fig. 2B, panel a). In contrast, although full-length p130 also bound to GST-PP1calpha (but not to GST alone), this interaction was not sensitive to the presence of p130PH (Fig. 2B, panel b). These results indicate that, although the PH domain of p130 is primarily responsible for the binding of this protein to PP1calpha , other regions of p130 also contribute to the interaction between these two proteins.

Analysis of the interaction of various regulatory subunits with PP1calpha has led to the identification of a consensus sequence for binding, (K/R)(K/R)(V/I)XF (22). The sequence VSF (residues 95 to 97) is present in the region of p130 shown to bind to PP1calpha . To determine whether this sequence participates in the interaction of p130 with PP1calpha , we examined the effect of a peptide (GM peptide, GRRVSFADNFGFN) that has been shown to inhibit the association between PP1calpha and several regulatory subunits (22). This peptide inhibited the interaction of PP1calpha with either full-length p130 or p130PH, whereas a random peptide with the same amino acid composition had no such effect (Fig. 2B, panel c). To confirm the role of the VSF sequence of p130 in the interaction of this protein with PP1calpha , we expressed in and purified from E. coli p130 fragments comprising amino acids 82 to 232. Whereas the wild-type fragment bound to PP1calpha , fragments containing either V95L or F97A mutations bound to the lesser extent (Fig. 2B, panel d).

Given that p130 contains four consensus motifs for phosphorylation by PKA (74Arg Arg Thr Ser77, 90Arg Lys Lys Thr93, 104Lys Ile Ser107, and 567Arg Arg Val Ser570 [underlining refers to phosphorylatable residues] one of which (104Lys Lys Ile Ser107) is present in p130PH, it was possible that p130 associates with PP1calpha because it is a substrate for phosphatase activity of this enzyme. Indeed, p130 was phosphorylated by PKA (Fig. 2C, lane 2), although the precise site (or sites) phosphorylated remains to be determined. However, this explanation for the interaction between p130 and PP1calpha is unlikely, because phosphorylated p130 did not associate with PP1calpha , whereas p130 treated with ATP alone (without PKA) bound to PP1calpha (Fig. 2C, lane 1).

The association between p130 and PP1calpha was further confirmed by surface plasmon resonance analysis. Full-length p130 was introduced into the analysis chamber after immobilization of GST-PP1calpha onto the sensor chip. Positive signals indicative of protein-protein interaction were generated in a p130 concentration-dependent manner and were abolished by washing away of the applied p130 (Fig. 2D). The dissociation constant was calculated to be 1.2 ± 0.1 nM (mean ± S.E. of values from five independent determinations). Replacement of the full-length p130 molecule with p130PH yielded a dissociation constant in the micromolar range, consistent with the results obtained with pull-down assays (Fig. 2B, panels a and b).

We next investigated whether the activity of PP1calpha is affected by the association with p130. The dephosphorylation of phosphorylated smooth muscle myosin light chain (21) by recombinant rabbit skeletal muscle PP1calpha was inhibited by full-length p130 in a concentration-dependent manner (Fig. 3). Recombinant p130PH also inhibited the activity of PP1calpha , although higher concentrations of p130PH than of full-length p130 were required for this effect.


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Fig. 3.   Effect of p130 on PP1calpha activity. The phosphatase activity of recombinant PP1calpha toward phosphorylated smooth muscle myosin light chain was assayed in the presence of the indicated concentrations of p130 (circles) or p130PH (triangles) and in the absence (open symbols) or presence (closed circles) of 10 µM Ins(1,4,5)P3. Data are means of triplicates from a representative experiment that was repeated 4 times with similar results.

The effects of Ins(1,4,5)P3 and water-soluble (short-chain) PtdIns(4,5)P2 on the association of p130 with PP1calpha , as well as on the inhibition of PP1calpha activity by p130, were also examined, given that the site of p130 responsible for the association with PP1calpha was shown to be located immediately upstream of the PH domain and that PH domains mediate binding to Ins(1,4,5)P3 or PtdIns(4,5)P2. The presence of Ins(1,4,5)P3 or short-chain PtdIns(4,5)P2 at a concentration of 10 µM in the reaction mixture for the pull-down assay had no effect on the interaction of p130 with PP1calpha (data not shown), and 10 µM Ins(1,4,5)P3 had no effect on p130-induced inhibition of PP1calpha activity (Fig. 3).

Association between p130 and PP1calpha in Intact Cells-- To determine whether p130 and PP1calpha interact in living cells, we first examined COS-1 cells that stably express recombinant p130 (COS-1p130 cells) (18). Immunoblot analysis of extracts of both control COS-1 cells (which lack endogenous p130) and COS-1p130 cells with antibodies to PP1calpha revealed that both cell lines express similar amounts of PP1calpha (Fig. 4A, a). Cell extracts were then subjected to immunoprecipitation with antibodies to either p130 (Fig. 4A, b) or PP1calpha (Fig. 4A, c), and the resulting precipitates were subjected to immunoblot analysis with the same two types of antibodies. Stable association of p130 with PP1calpha was apparent in COS-1p130 cells but not in control COS-1 cells (Fig. 4A). We also examined whether these two proteins interact in mouse brain, which contains both molecules (Fig. 4B). PP1calpha was detected in p130 immunoprecipitates (Fig. 4B, b), and p130 was detected in PP1calpha immunoprecipitates (Fig. 4B, c) prepared from mouse brain.


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Fig. 4.   Association between p130 and PP1calpha in intact cells and mouse brain. A, panel a, extracts prepared from COS-1 or COS-1p130 cells were subjected to immunoblot analysis (IB) with antibodies to p130 or to PP1calpha , as indicated. Panels b and c, extracts of COS-1 (lanes 1) and COS-1p130 (lanes 3) cells were subjected to immunoprecipitation (IP) with antibodies to p130 (b) or to PP1calpha (c), and the resulting precipitates were subjected to immunoblot analysis with the same two types of antibodies. Lanes 2 correspond to COS-1p130 cell extract subjected to immunoprecipitation with protein G-Sepharose in the absence of antibodies. Upper bands in lanes 1 and 3 in the blot analyzed with anti-PP1calpha in panel b are immunoglobulin heavy chains. B, mouse brain extract was analyzed as described in A; lanes 2 correspond to brain extract subjected to immunoprecipitation with the indicated antibodies, whereas lanes 1 correspond to extract subjected to precipitation with protein G-Sepharose in the absence of antibodies.

PP1calpha is thought to catalyze protein dephosphorylation reactions that underlie many aspects of cell function (23, 24). Glycogen phosphorylase, which catalyzes the conversion of glycogen to glucose 1-phosphate, is a substrate for PP1calpha in a wide variety of cell types (25); its dephosphorylation by this phosphatase results in inhibition of phosphorylase activity. Measurement of glycogen phosphorylase activity in extracts of COS-1 and COS-1p130 cells yielded values of 68 ± 6 and 130 ± 9 nmol per milligram of protein per 5 min (means ± S.E. of six independent determinations), respectively. These results thus indicate that glycogen phosphorylase is phosphorylated to a greater extent in COS-1p130 cells than in COS-1 cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

With the use of its specific NH2-terminal region as a bait, we applied the yeast two-hybrid screen to identify human brain proteins that bind to p130, designated henceforth as PRIP-1. This approach identified PP1calpha as one such protein. PP1 is a widely expressed serine-threonine protein phosphatase that exists in several isoforms, including alpha , alpha 2, gamma 1, gamma 2, and delta  (23, 24). Various regulatory subunits have been shown to associate with PP1calpha and thereby to influence its catalytic activity (26). For example, GM and GL subunits function to target PP1calpha to glycogen granules; phosphorylation of these subunits by PKA induces their dissociation from PP1calpha , whereas that triggered by insulin promotes their association with and activation of PP1calpha , resulting in inhibition of glycogen breakdown. The association of I-1 (inhibitor 1) or DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) with PP1calpha appears not to affect phosphatase activity, whereas phosphorylation of I-1 or DARPP-32 by PKA induces marked inhibition of such activity. Our results now suggest that PRIP-1 also functions as a regulatory subunit of PP1calpha that inhibits phosphatase activity. The binding of Ins(1,4,5)P3 or PtdIns(4,5)P2 to PRIP-1 had no effect on its association with or inhibition of PP1calpha . Our previous observations suggested that Ins(1,4,5)P3 may be a physiological ligand for PRIP-1 and that this protein is localized predominantly to the cytosol (18). PRIP-1 may therefore serve not only to inhibit the activity of PP1calpha but also to target this enzyme to the cytosol.

Amino acid residues 95 to 97 of PRIP-1, located upstream of the PH domain, appear to contribute to the binding site for PP1calpha . The fragment of PRIP-1 comprising residues 24 to 222 interacted with PP1calpha in the yeast two-hybrid assay, and p130PH (PRIP-1PH) (residues 95 to 232) as well as the full-length molecule, associated with PP1calpha in vitro, as demonstrated with a variety of binding assays. A GM peptide that disrupts the interaction of PP1calpha with several regulatory subunits and that contains the VSF (residues 95 to 97) sequence of PRIP-1 also inhibited the association of PRIP-1 with PP1calpha . Furthermore, mutation of residues 95 or 97 of PRIP-1 prevented the association of this protein with PP1calpha . Other regions of the PRIP-1 molecule may also interact with PP1calpha , as suggested by the observations that the full-length molecule bound to PP1calpha was not displaced by an excess amount of PRIP-1PH and that the dissociation constant obtained by surface plasmon resonance analysis for the interaction with PP1calpha was smaller for the full-length molecule than for PRIP-1PH. However, the observation that the GM peptide was similarly effective in inhibiting the association of PP1calpha with full-length PRIP-1 and with PRIP-1PH suggests rather that other regions of PRIP-1 promote the interaction of the region containing residues 95 to 97 with PP1calpha .

Phosphorylation of PRIP-1 by PKA resulted in inhibition of the association between PRIP-1 and PP1calpha . Although the phosphorylated residues of PRIP-1 that underlie this effect remain to be identified, T93, which is located immediately upstream of the putative binding site for PP1calpha , is a likely candidate.

PP1calpha contributes to the regulation of many aspects of cellular metabolism, including glycogen metabolism (through dephosphorylation of phosphorylase kinase, glycogen phosphorylase, and glycogen synthase) and lipid metabolism (through dephosphorylation of acetyl-CoA carboxylase, hormone-dependent lipase, and hydroxymethylglutaryl-CoA reductase). Furthermore, it participates in the regulation of Ca2+ transport (through dephosphorylation of phospholamban and Ca2+ channel proteins), smooth muscle contraction (through dephosphorylation of myosin light chain), DNA replication (through dephosphorylation of histones H2B and H1), and protein synthesis (through dephosphorylation of initiation factor eIF-2, RNA-dependent protein kinase, heat shock protein, S6 protein, and S6 kinase) (24, 25). It remains to be determined which of these cellular activities are physiologically regulated by PRIP-1 through its interaction with PP1calpha . Our data do suggest, however, that the association between PRIP-1 and PP1calpha occurs in living cells, and we have shown that the activity of glycogen phosphorylase, which is regulated exclusively by phosphorylation, was increased in COS-1 cells by the expression of PRIP-1, probably as a result of the interaction of PRIP-1 with, and the consequent inhibition of, PP1calpha . Glycogen phosphorylase may therefore be a physiological target for regulation by the interaction of PRIP-1 with PP1calpha .

In summary, we have shown that (i) p130, which belongs to the PRIP family of proteins and is here renamed PRIP-1, associates with PP1calpha through a GM peptide-like region located upstream of the PH domain; (ii) association with PRIP-1 results in inhibition of the catalytic activity of PP1calpha as measured in vitro with phosphorylated myosin light chain as substrate; and (iii) glycogen phosphorylase activity was increased by expression of PRIP-1 in intact cells, likely as a result of inhibition of PP1calpha and accumulation of the phosphorylated, active form of glycogen phosphorylase. In addition to its role in Ins(1,4,5)P3 and Ca2+ signaling (18), PRIP-1 might therefore also contribute to the regulation of protein dephosphorylation. Given that the binding of Ins(1,4,5)P3 to the PH domain of PRIP-1 had no effect on the association of PRIP-1 with PP1calpha or on its inhibition of PP1calpha activity, PRIP-1 may contribute to both Ca2+ signaling and regulation of protein dephosphorylation simultaneously and, in some instances, cooperatively.

    ACKNOWLEDGEMENTS

We thank M. Katan for helpful comments.

    FOOTNOTES

* This work was funded in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (to H. T., T. K., and M. H.), Kyushu University interdisciplinary programs in education and projects in research development (to M. H.), the Fujisawa Foundation (to M. H.), the Fugaku Trust for medicinal research (to M. H.), Kowa Life Science Foundation (to T. K.), and the Uehara Memorial Foundation (to H. T.).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.

§ Contributed equally to this work.

** To whom correspondence should be addressed. Tel.: 81-92-642-6317. Fax: 81-92-642-6322; E-mail: hirata1@dent.kyushu-u.ac.jp.

Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M009677200

    ABBREVIATIONS

The abbreviations used are: Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PH, pleckstrin homology; PRIP, PLC-related catalytically inactive protein; PP1calpha , catalytic subunit of protein phosphatase 1alpha ; PVDF, polyvinylidene difluoride; PKA, cAMP-dependent protein kinase; nt, nucleotide; GST, glutathione S-transferase; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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