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

2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês

10.1074/jbc.m009677200

1083-351X

Kenji Yoshimura, Hiroshi Takeuchi, Osamu Satō, Kiyoshi Hidaka, Naoko Doira, Miho Terunuma, Kae Harada, Yasuo Ogawa, Yushi Ito, Takashi Kanematsu, Masato Hirata,

Cell death mechanisms and regulation

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-δ1 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 1α (PP1cα) as a p130-binding protein. The association between p130 and PP1cα was also confirmedin vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1cα 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 PP1cα also detected the presence of a complex of p130 and PP1cα. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1cα, 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 PP1cα. 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-δ1 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 1α (PP1cα) as a p130-binding protein. The association between p130 and PP1cα was also confirmedin vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1cα 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 PP1cα also detected the presence of a complex of p130 and PP1cα. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1cα, 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 PP1cα. 4,5)P3, inositol 1,4,5-trisphosphate 5)P2, phosphatidylinositol 4,5-bisphosphate phospholipase C pleckstrin homology PLC-related catalytically inactive protein catalytic subunit of protein phosphatase 1α polyvinylidene difluoride cAMP-dependent protein kinase nucleotide glutathione S-transferase bovine serum albumin polyacrylamide gel electrophoresis d-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3),1a 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 (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar). We previously isolated two Ins(1,4,5)P3-binding proteins with molecular masses of 130 and 85 kDa from rat brain (2Kanematsu T. Takeya H. Watanabe Y. Ozaki S. Yoshida M. Koga T. Iwanaga S. Hirata M. J. Biol. Chem. 1992; 267: 6518-6525Abstract Full Text PDF PubMed Google Scholar, 3Yoshida M. Kanematsu T. Watanabe Y. Koga T. Ozaki S. Iwanaga S. Hirata M. J. Biochem. ( Tokyo ). 1994; 115: 973-980Crossref PubMed Scopus (61) Google Scholar) with the use of an Ins(1,4,5)P3 affinity column (4Hirata M. Sasaguri T. Hamachi T. Hashimoto T. Kukita M. Koga T. Nature. 1985; 317: 723-725Crossref PubMed Scopus (36) Google Scholar, 5Hirata M. Watanabe Y. Ishimatsu T. Yanaga F. Koga T. Ozaki S. Biochem. Biophys. Res. Commun. 1990; 168: 379-386Crossref PubMed Scopus (50) Google Scholar). Partial amino acid sequencing revealed that the 85-kDa molecule was PLC-δ1 (2Kanematsu T. Takeya H. Watanabe Y. Ozaki S. Yoshida M. Koga T. Iwanaga S. Hirata M. J. Biol. Chem. 1992; 267: 6518-6525Abstract Full Text PDF PubMed Google Scholar). Identification of the pleckstrin homology (PH) domain of PLC-δ1 as the site of Ins(1,4,5)P3 binding helped to define the PH domain as an inositol compound binding module (6Hirata M. Kanematsu T. Sakuma K. Koga T. Watanabe Y. Ozaki S. Yagisawa H. Biochem. Biophys. Res. Commun. 1994; 205: 1563-1571Crossref PubMed Scopus (28) Google Scholar, 7Yagisawa H. Hirata M. Kanematsu T. Watanabe Y. Ozaki S. Sakuma K. Tanaka H. Yabuta N. Kamata H. Hirata H. Nojima H. J. Biol. Chem. 1994; 269: 20179-20188Abstract Full Text PDF PubMed Google Scholar). The Ins(1,4,5)P3-binding protein with a molecular mass of 130 kDa, termed p130, was a previously unidentified molecule (2Kanematsu T. Takeya H. Watanabe Y. Ozaki S. Yoshida M. Koga T. Iwanaga S. Hirata M. J. Biol. Chem. 1992; 267: 6518-6525Abstract Full Text PDF PubMed Google Scholar, 3Yoshida M. Kanematsu T. Watanabe Y. Koga T. Ozaki S. Iwanaga S. Hirata M. J. Biochem. ( Tokyo ). 1994; 115: 973-980Crossref PubMed Scopus (61) Google Scholar). The predicted amino acid sequence of rat p130 shares 38.2% identity with that of rat PLC-δ1; the five identified domains of PLC-δ1 (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-δ1, a notion that is supported by the results of limited proteolysis with trypsin (8Kanematsu T. Yoshimura K. Hidaka K. Takeuchi H. Katan M. Hirata M. Eur. J. Biochem. 2000; 267: 2731-2737Crossref PubMed Scopus (56) Google Scholar). However, p130 exhibits some distinct characteristics. It is larger than the PLC-δ 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-δ that are critical for enzyme activity (His356and Glu390) are not conserved in p130 (9Kanematsu T. Misumi Y. Watanabe Y. Ozaki S. Koga T. Iwanaga S. Ikehara Y. Hirata M. Biochem. J. 1996; 313: 319-325Crossref PubMed Scopus (90) Google Scholar). The PH domain of p130, like that of PLC-δ1, is important for the binding of Ins(1,4,5)P3 (10Takeuchi H. Kanematsu T. Misumi Y. Yaakob H.B. Yagisawa H. Ikehara Y. Watanabe Y. Tan Z. Shears S.B. Hirata M. Biochem. J. 1996; 318: 561-568Crossref PubMed Scopus (61) Google Scholar). Other molecules that show sequence similarity to p130, including human PLC-L (11Kohno T. Otsuka T. Takano H. Yamamoto T. Hamaguchi M. Terada M. Yokota J. Hum. Mol. Genet. 1995; 4: 667-674Crossref PubMed Scopus (44) Google Scholar) and the K10F12.3 gene product of Caenorhabditis elegans (12Koyanagi M. Ono K. Suga H. Iwabe N. Miyata T. FEBS Lett. 1998; 439: 66-70Crossref PubMed Scopus (47) Google Scholar), have also been described. Otsuki et al. (13Otsuki M. Fukami K. Kohno T. Yokota J. Takenawa T. Biochem. Biophys. Res. Commun. 1999; 266: 97-103Crossref PubMed Scopus (51) Google Scholar) 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 (14Kikuno R. Nagase T. Ishikawa K. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Obara O. DNA Res. 1999; 6: 197-205Crossref PubMed Scopus (175) Google Scholar). 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 (10Takeuchi H. Kanematsu T. Misumi Y. Yaakob H.B. Yagisawa H. Ikehara Y. Watanabe Y. Tan Z. Shears S.B. Hirata M. Biochem. J. 1996; 318: 561-568Crossref PubMed Scopus (61) Google Scholar, 15Hirata M. Kanematsu T. Takeuchi H. Yagisawa H. Jpn. J. Pharmacol. 1998; 76: 255-263Crossref PubMed Scopus (33) Google Scholar, 16Takeuchi H. Kanematsu T. Misumi Y. Sakane F. Konishi H. Kikkawa U. Watanabe Y. Katan M. Hirata M. Biochim. Biophys. Acta. 1997; 1359: 275-285Crossref PubMed Scopus (90) Google Scholar, 17Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (618) Google Scholar). 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 (18Takeuchi H. Oike M. Paterson H.F. Allen V. Kanematsu T. Ito Y. Erneux C. Katan M. Hirata M. Biochem. J. 2000; 349: 357-368Crossref PubMed Scopus (54) Google Scholar). 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 1α (PP1cα). To characterize the interaction between p130 and PP1cα, 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 PP1cα, and we examined the effect of such binding on the enzymatic activity of PP1cα. Cloning vectors pGBT9 and pACT2, a human brain cDNA library, and yeast strains HF7c and SFY526 were obtained fromCLONTECH (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). [γ-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 (19Ozaki S. Watanabe Y. Ogasawara T. Kondo Y. Shiotani N. Nishii H. Matsuki T. Tetrahedron Lett. 1986; 27: 3157-3160Crossref Scopus (93) Google Scholar). The catalytic subunit of cAMP-dependent protein kinase (PKA) was obtained fromPromega (Madison, WI), and wild-type rabbit PP1cα 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. For construction of p130 bait plasmids, pcMT3 (9Kanematsu T. Misumi Y. Watanabe Y. Ozaki S. Koga T. Iwanaga S. Ikehara Y. Hirata M. Biochem. J. 1996; 313: 319-325Crossref PubMed Scopus (90) Google Scholar) was digested with XhoI, and the released fragment (nucleotides (nt) 535 to 5233) was cloned into theSalI site of pGBT9. The resulting plasmid, pGBT9-p130Full (p130 plasmid 1; amino acid residues 24 to 1096) (see Fig.1 A), was digested with PstI, rendered blunt-ended with DNA polymerase, and then self-ligated at theSmaI 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 theBamHI 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), aSpeI site (nt 3011) was introduced into pcMT3 by site-directed mutagenesis, and the resulting plasmid was digested withSpeI (nt 3011) and XhoI (nt 5233). The released 2.2-kilobase pair fragment was then ligated into theSpeI-SalI sites of pGBT9. For construction of pACT2-PP1cα(Δ29–163) a positive clone obtained from the yeast two-hybrid screening, pACT2-PP1cα, was digested with PvuII (nt 116 and 815) and self-ligated. For expression of recombinant PP1cα in Escherichia coli, the BamHI fragment of pACT2-PP1cα was ligated into the BamHI site of pGEX-3X; the resulting construct encodes a fusion protein of glutathioneS-transferase (GST) and PP1cα. 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 PP1cα were investigated by expression of various combinations of bait and target plasmids in yeast SFY526 cells and measurement of β-galactosidase activity. The recombinant GST-PP1cα 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 (8Kanematsu T. Yoshimura K. Hidaka K. Takeuchi H. Katan M. Hirata M. Eur. J. Biochem. 2000; 267: 2731-2737Crossref PubMed Scopus (56) Google Scholar, 10Takeuchi H. Kanematsu T. Misumi Y. Yaakob H.B. Yagisawa H. Ikehara Y. Watanabe Y. Tan Z. Shears S.B. Hirata M. Biochem. J. 1996; 318: 561-568Crossref PubMed Scopus (61) Google Scholar). For "pull-down" assays, GST-PP1cα was incubated for 1 h at 4 °C with glutathione-Sepharose 4B beads in binding buffer (50 mm Tris-HCl (pH 7.5), 5 mmMgCl2, 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-PP1cα) 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 mmreduced glutathione and were then subjected to SDS polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis with antibodies to p130 (2F9) or to p130PH (3Yoshida M. Kanematsu T. Watanabe Y. Koga T. Ozaki S. Iwanaga S. Hirata M. J. Biochem. ( Tokyo ). 1994; 115: 973-980Crossref PubMed Scopus (61) Google Scholar, 8Kanematsu T. Yoshimura K. Hidaka K. Takeuchi H. Katan M. Hirata M. Eur. J. Biochem. 2000; 267: 2731-2737Crossref PubMed Scopus (56) Google Scholar). 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. COS-1 cells, COS-1p130cells (COS-1 cells stably expressing p130) (18Takeuchi H. Oike M. Paterson H.F. Allen V. Kanematsu T. Ito Y. Erneux C. Katan M. Hirata M. Biochem. J. 2000; 349: 357-368Crossref PubMed Scopus (54) Google Scholar), and mouse brain extract were subjected to immunoprecipitation with a specific monoclonal antibody to p130 (2F9) or polyclonal antibodies to PP1cα (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 mmHEPES-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 PP1cα 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 PP1cα or to p130. Protein-protein interaction was examined in real time with a BIACORE 2000 surface plasmon resonance analyzer (Biacore International, Uppsala, Sweden). Recombinant GST-PP1cα was immobilized on the surface of a CM5 sensor chip that had been activated withN-hydroxysuccinimide andN-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. COS-1 or COS-1p130 cells (2 × 106) were lysed by three freeze-thaw cycles in a solution containing 50 mmNaCl, 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 (20Cori G.T. Illingworth B. Keller P.J. Methods Enzymol. 1955; 1: 200-205Crossref Scopus (121) Google Scholar). Phosphatase activity was determined in a reaction mixture (40 μl) containing 139.2 mm KCl, 20 mm4-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 PP1cα, and various concentrations of recombinant full-length p130 or p130PH, in the absence or presence of 10 μmIns(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 (21Sato O. Ogawa Y. J. Biochem. ( Tokyo ). 1999; 126: 787-797Crossref PubMed Scopus (6) Google Scholar). Screening of a human brain cDNA library with a bait plasmid (2Kanematsu T. Takeya H. Watanabe Y. Ozaki S. Yoshida M. Koga T. Iwanaga S. Hirata M. J. Biol. Chem. 1992; 267: 6518-6525Abstract Full Text PDF PubMed Google Scholar) 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. 1 A), 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 PP1cα, a 37,510-Da protein composed of 330 amino acids. To delineate more precisely the region of p130 required for binding to PP1cα, we used plasmids encoding smaller portions of p130 as baits in two-hybrid analysis with the plasmid encoding full-length PP1cα (Fig. 1 A). Positive signals were obtained with a bait plasmid (3Yoshida M. Kanematsu T. Watanabe Y. Koga T. Ozaki S. Iwanaga S. Hirata M. J. Biochem. ( Tokyo ). 1994; 115: 973-980Crossref PubMed Scopus (61) Google Scholar) encoding amino acids 24 to 222, as well as with that (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar) encoding full-length p130. Neither a bait plasmid (4Hirata M. Sasaguri T. Hamachi T. Hashimoto T. Kukita M. Koga T. Nature. 1985; 317: 723-725Crossref PubMed Scopus (36) Google Scholar) encoding amino acids 24 to 82 nor one (5Hirata M. Watanabe Y. Ishimatsu T. Yanaga F. Koga T. Ozaki S. Biochem. Biophys. Res. Commun. 1990; 168: 379-386Crossref PubMed Scopus (50) Google Scholar) encoding residues 222 to 298 yielded a positive signal (Fig. 1 B). A plasmid encoding a PP1cα 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 PP1cα comprising residues 30 to 162. We next examined the interaction of p130 and PP1cα in vitro by several methods. The association was first analyzed with an overlay assay (Fig.2 A). Extracts of nontransformed E. coli and of bacteria expressing a GST-PP1cα fusion protein, as well as recombinant GST-PP1cα 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 PP1cα to confirm that the prominent band that migrated at a position corresponding to a molecular size of 37 kDa was indeed PP1cα. 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-PP1cα 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 PP1cα. The GST-PP1cα fusion protein was also subjected to a pull-down assay with recombinant p130 or p130PH (Fig. 2 B). Incubation of a GST-PP1cα resin with p130PH and subsequent immunoblot analysis of bead-bound proteins with antibodies to p130PH revealed that p130PH was precipitated by GST-PP1cα and that this interaction was sensitive to the presence of low concentrations of full-length p130 but not to PLC-δ1 (Fig. 2 B, panel a). In contrast, although full-length p130 also bound to GST-PP1cα (but not to GST alone), this interaction was not sensitive to the presence of p130PH (Fig. 2 B, panel b). These results indicate that, although the PH domain of p130 is primarily responsible for the binding of this protein to PP1cα, other regions of p130 also contribute to the interaction between these two proteins. Analysis of the interaction of various regulatory subunits with PP1cα has led to the identification of a consensus sequence for binding, (K/R)(K/R)(V/I)XF (22Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T.W. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (537) Google Scholar). The sequence VSF (residues 95 to 97) is present in the region of p130 shown to bind to PP1cα. To determine whether this sequence participates in the interaction of p130 with PP1cα, we examined the effect of a peptide (GMpeptide, GRRVSFADNFGFN) that has been shown to inhibit the association between PP1cα and several regulatory subunits (22Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T.W. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (537) Google Scholar). This peptide inhibited the interaction of PP1cα with either full-length p130 or p130PH, whereas a random peptide with the same amino acid composition had no such effect (Fig. 2 B, panel c). To confirm the role of the VSF sequence of p130 in the interaction of this protein with PP1cα, we expressed in and purified from E. coli p130 fragments comprising amino acids 82 to 232. Whereas the wild-type fragment bound to PP1cα, fragments containing either V95L or F97A mutations bound to the lesser extent (Fig. 2 B, panel d). Given that p130 contains four consensus motifs for phosphorylation by PKA (74Arg Arg Thr Ser77,90Arg Lys Lys Thr93,104Lys Ile Ser107, and567Arg Arg Val Ser570 [underlining refers to phosphorylatable residues] one of which (104Lys Lys Ile Ser 107) is present in p130PH, it was possible that p130 associates with PP1cα because it is a substrate for phosphatase activity of this enzyme. Indeed, p130 was phosphorylated by PKA (Fig. 2 C, lane 2), although the precise site (or sites) phosphorylated remains to be determined. However, this explanation for the interaction between p130 and PP1cα is unlikely, because phosphorylated p130 did not associate with PP1cα, whereas p130 treated with ATP alone (without PKA) bound to PP1cα (Fig. 2 C, lane 1). The association between p130 and PP1cα was further confirmed by surface plasmon resonance analysis. Full-length p130 was introduced into the analysis chamber after immobilization of GST-PP1cα 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.2 D). 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.2 B, panels a and b). We next investigated whether the activity of PP1cα is affected by the association with p130. The dephosphorylation of phosphorylated smooth muscle myosin light chain (21Sato O. Ogawa Y. J. Biochem. ( Tokyo ). 1999; 126: 787-797Crossref PubMed Scopus (6) Google Scholar) by recombinant rabbit skeletal muscle PP1cα was inhibited by full-length p130 in a concentration-dependent manner (Fig.3). Recombinant p130PH also inhibited the activity of PP1cα, although higher concentrations of p130PH than of full-length p130 were required for this effect. The effects of Ins(1,4,5)P3 and water-soluble (short-chain) PtdIns(4,5)P2 on the association of p130 with PP1cα, as well as on the inhibition of PP1cα activity by p130, were also examined, given that the site of p130 responsible for the association with PP1cα was shown to be located immediately upstream of the PH domain and that PH domains mediate binding to Ins(1,4,5)P3or PtdIns(4,5)P2. The presence of Ins(1,4,5)P3or 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 PP1cα (data not shown), and 10 μm Ins(1,4,5)P3 had no effect on p130-induced inhibition of PP1cα activity (Fig. 3). To determine whether p130 and PP1cα interact in living cells, we first examined COS-1 cells that stably express recombinant p130 (COS-1p130 cells) (18Takeuchi H. Oike M. Paterson H.F. Allen V. Kanematsu T. Ito Y. Erneux C. Katan M. Hirata M. Biochem. J. 2000; 349: 357-368Crossref PubMed Scopus (54) Google Scholar). Immunoblot analysis of extracts of both control COS-1 cells (which lack endogenous p130) and COS-1p130 cells with antibodies to PP1cα revealed that both cell lines express similar amounts of PP1cα (Fig.4 A, a). Cell extracts were then subjected to immunoprecipitation with antibodies to either p130 (Fig. 4 A, b) or PP1cα (Fig.4 A, c), and the resulting precipitates were subjected to immunoblot analysis with the same two types of antibodies. Stable association of p130 with PP1cα was apparent in COS-1p130 cells but not in control COS-1 cells (Fig.4 A). We also examined whether these two proteins interact in mouse brain, which contains both molecules (Fig. 4 B). PP1cα was detected in p130 immunoprecipitates (Fig. 4 B,b), and p130 was detected in PP1cα immunoprecipitates (Fig. 4 B, c) prepared from mouse brain. PP1cα is thought to catalyze protein dephosphorylation reactions that underlie many aspects of cell function (23Barford D. Trends Biochem. Sci. 1996; 21: 407-412Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 24Cohen P.T.W. Trends Biochem. Sci. 1997; 22: 245-251Abstract Full Text PDF PubMed Scopus (462) Google Scholar). Glycogen phosphorylase, which catalyzes the conversion of glycogen to glucose 1-phosphate, is a substrate for PP1cα in a wide variety of cell types (25Stryer L. Stryer L. Biochemistry. 4th Ed. Freeman, New York1995: 581-602Google Scholar); 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. 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 PP1cα as one such protein. PP1 is a widely expressed serine-threonine protein phosphatase that exists in several isoforms, including α, α2, γ1, γ2, and δ (23Barford D. Trends Biochem. Sci. 1996; 21: 407-412Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 24Cohen P.T.W. Trends Biochem. Sci. 1997; 22: 245-251Abstract Full Text PDF PubMed Scopus (462) Google Scholar). Various regulatory subunits have been shown to associate with PP1cα and thereby to influence its catalytic activity (26Aggen J.B. Nairn A.C. Chamberlin R. Chem. Biol. 2000; 7: 13-23Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). For example, GM and GL subunits function to target PP1cα to glycogen granules; phosphorylation of these subunits by PKA induces their dissociation from PP1cα, whereas that triggered by insulin promotes their association with and activation of PP1cα, resulting in inhibition of glycogen breakdown. The association of I-1 (inhibitor 1) or DARPP-32 (dopamine- and cAMP-regulatedphosphoprotein of 32 kDa) with PP1cα 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 PP1cα 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 PP1cα. 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 (18Takeuchi H. Oike M. Paterson H.F. Allen V. Kanematsu T. Ito Y. Erneux C. Katan M. Hirata M. Biochem. J. 2000; 349: 357-368Crossref PubMed Scopus (54) Google Scholar). PRIP-1 may therefore serve not only to inhibit the activity of PP1cα 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 PP1cα. The fragment of PRIP-1 comprising residues 24 to 222 interacted with PP1cα in the yeast two-hybrid assay, and p130PH (PRIP-1PH) (residues 95 to 232) as well as the full-length molecule, associated with PP1cαin vitro, as demonstrated with a variety of binding assays. A GM peptide that disrupts the interaction of PP1cα 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 PP1cα. Furthermore, mutation of residues 95 or 97 of PRIP-1 prevented the association of this protein with PP1cα. Other regions of the PRIP-1 molecule may also interact with PP1cα, as suggested by the observations that the full-length molecule bound to PP1cα 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 PP1cα 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 PP1cα 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 PP1cα. Phosphorylation of PRIP-1 by PKA resulted in inhibition of the association between PRIP-1 and PP1cα. 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 PP1cα, is a likely candidate. PP1cα 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) (24Cohen P.T.W. Trends Biochem. Sci. 1997; 22: 245-251Abstract Full Text PDF PubMed Scopus (462) Google Scholar, 25Stryer L. Stryer L. Biochemistry. 4th Ed. Freeman, New York1995: 581-602Google Scholar). It remains to be determined which of these cellular activities are physiologically regulated by PRIP-1 through its interaction with PP1cα. Our data do suggest, however, that the association between PRIP-1 and PP1cα 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, PP1cα. Glycogen phosphorylase may therefore be a physiological target for regulation by the interaction of PRIP-1 with PP1cα. In summary, we have shown that (i) p130, which belongs to the PRIP family of proteins and is here renamed PRIP-1, associates with PP1cα 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 PP1cα 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 PP1cα and accumulation of the phosphorylated, active form of glycogen phosphorylase. In addition to its role in Ins(1,4,5)P3 and Ca2+ signaling (18Takeuchi H. Oike M. Paterson H.F. Allen V. Kanematsu T. Ito Y. Erneux C. Katan M. Hirata M. Biochem. J. 2000; 349: 357-368Crossref PubMed Scopus (54) Google Scholar), 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 PP1cα or on its inhibition of PP1cα activity, PRIP-1 may contribute to both Ca2+ signaling and regulation of protein dephosphorylation simultaneously and, in some instances, cooperatively. We thank M. Katan for helpful comments.