View metadata,FEBS Letters citation 439 and (1998) similar 224^230 papers at core.ac.uk broughtFEBS to you 21118 by CORE provided by Elsevier - Publisher Connector Biophysical interaction between phospholamban and protein phosphatase 1 regulatory subunit GM Isabelle Berrebi-Bertrand*, Michel Souchet, Jean-Claude Camelin, Marie-Paule Laville, Thierry Calmels, Antoine Bril SmithKline Beecham Laboratoires Pharmaceutiques, 4 rue du Chesnay Beauregard, P.O. Box 58, 35762 Saint-Greègoire, France Received 29 September 1998 acid, can increase the activity of the calcium pump [2]. In a Abstract Regulation of the sarco(endo)plasmic reticulum Ca2+- ATPase (SERCA 2a) depends on the phosphorylation state of dephosphorylated form, PLB decreases the a¤nity of SERCA phospholamban (PLB). When PLB is phosphorylated, its 2a for calcium and phosphorylation of PLB removes its in- inhibitory effect towards SERCA 2a is relieved, leading to an hibitory e¡ect towards SERCA 2a [3] which is able to pump enhanced myocardial performance. This process is reversed by a more rapidly the Ca2 from the cytosol into the SR [4]; this sarcoplasmic reticulum (SR)-associated type 1 protein phospha- latter e¡ect is correlated with an enhanced relaxation of the tase (PP1) composed of a catalytic subunit PP1C and a heart [5]. Phosphorylation of PLB at distinct sites by cAMP- regulatory subunit GM. Human GM and PLB have been dependent (at serine 16) and calmodulin-dependent (at threo- produced in an in vitro transcription/translation system and used nine 17) protein kinases is reversed by a sarcoplasmic reticu- for co-immunoprecipitation and biosensor experiments. The lum (SR)-associated protein phosphatase. MacDougall et al. detected interaction between the two partners suggests that [6] have shown that the major protein phosphatase associated cardiac PP1 is targeted to PLB via GM and we believe that this process occurs with the identified transmembrane domains of the with the cardiac SR is a type 1 protein phosphatase, one of two proteins. Thus, the interaction between PLB and GM may the most important serine/threonine protein phosphatases in represent a specific way to modulate the SR function in human eukaryotic cells. Inhibitors of such phosphatases, like calycu- cardiac muscle. lin A, okadaic acid or tautomycin, have been shown to im- z 1998 Federation of European Biochemical Societies. prove cardiac relaxation and calcium uptake by the SR [7,8]. Furthermore, we have recently reported that this e¡ect is di- Key words: G subunit; Protein phosphatase 1 regulatory rectly associated with an increased amount of phosphorylated subunit; Phospholamban; Interaction; PLB [9]. Co-immunoprecipitation; Surface plasmon resonance Although type 1 protein phosphatases are ubiquitously dis- tributed, several lines of evidence suggest that like other en- zymes they do not reach their physiological substrates by 1. Introduction simple di¡usion within cells. In contrast, it appears that phos- phatases are frequently directed to particular loci in the vicin- Phospholamban (PLB) is a 52-amino acid protein that reg- ity of their substrates by interaction with targeting subunits ulates the activity of the cardiac sarco(endo)plasmic reticulum [10]. Type 1 protein phosphatases are typical examples of calcium ATPase (SERCA 2a). The physiological role of PLB heterodimer enzymes involved in controlling diverse cellular on the activity of the calcium ATPase has been demonstrated functions including glycogen metabolism, exit from mitosis, using transgenic models in which the intracellular calcium splicing of RNA and muscle contraction [11] where the transient and the myocardial relaxation are directly related same catalytic subunit (PP1C) is complexed to di¡erent tar- to the level of expressed PLB [1]. Similarly, agents that inhibit geting or regulatory subunits conferring substrate speci¢city. the interaction between PLB and SERCA 2a, such as ellagic In the heart, this heterodimer called PP1 (also known as PP1G) is composed of a 37-kDa catalytic subunit PP1C com- plexed to a larger subunit called GM (also known as G, RG1, PPP1R3) which is responsible for the association of PP1 with SR membranes [6]. In human, GM is one of the several reg- *Corresponding author. Fax: (33) 2 99 28 04 44. ulatory subunits which have been isolated and characterised. E-mail: [email protected] Although GM, a 1122-residue protein, binds directly to gly- cogen, the mechanism by which it interacts with SR mem- Abbreviations: GM, G subunit = RG1 = PPP1R3 = protein phospha- tase 1 (PP1) that targets the catalytic subunit (PP1C) to glycogen branes is still unknown. It has been suggested that it could particles and SR; SR, sarcoplasmic reticulum; SERCA, sarco(endo)- be due to a membrane-associated domain or a domain for plasmic reticulum calcium ATPase; ATPase, adenosine triphospha- interaction with a protein that binds to SR membranes tase; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propane sulfonic [10,12,13]. acid; EGTA, ethylene glycol bis-(L-aminoethyl ether) N,N,NP,NP- The objective of the present study was to investigate tetraacetic acid; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; PBS-T, phosphate buffer saline-Tween 20; Ig, whether PLB, one of the substrates of PP1, can also represent immunoglobulin; Ig-HRP, Ig complexed to horseradish peroxidase; a molecular target for GM. To this end we have produced the ECL, enhanced chemiluminescence; NHS, N-hydroxysuccimide; human GM and PLB recombinant proteins and used co- EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; HBS, HEPES immunoprecipitation and biosensor methodologies to evi- buffer saline; RU, resonance unit; GL, targeting subunit for liver glycogen; PTG, for protein targeting to glycogen; M110, myofibrils; dence the physical interaction between GM and PLB. A meth- PCR, polymerase chain reaction od for predicting transmembrane domains was performed on 0014-5793/98/$19.00 ß 1998 Federation of European Biochemical Societies. All rights reserved. PII: S0014-5793(98)01364-7 FEBS 21118 20-11-98 Cyaan Magenta Geel Zwart I. Berrebi-Bertrand et al./FEBS Letters 439 (1998) 224^230 225 the 1122-residue sequence of GM and con¢rmed that the polymerase coupled with T3 promoter (GM protein synthesis). Neg- protein is able to anchor to the SR membrane via its C-ter- ative control experiments were run by doing antisense translation using alternative T3/T7 promoters with corresponding RNA polymer- minal stretch. Together these data have led to a new way of ase enzymes and a positive control experiment was done using luci- understanding the molecular events involved in SERCA 2a ferase T7 control DNA. In vitro transcription-translation was per- activity. formed in a reaction volume of 25 Wl with 1 Wg of each construct according to conditions recommended by manufacturer. The reaction was incubated at 30³C for 120 min and a 5-Wl aliquot of the synthes- 2. Materials and methods ised proteins was analysed by SDS-PAGE. 2.1. Antibodies and reagents 2.5. Immunoprecipitation of PLB and GM Anti-canine PLB monoclonal antibodies were obtained from Euro- 6 Wlof[35S]PLB lysate (107 cpm) was immunoprecipitated with 5 Wl medex, France and Dr Philip Cohen (University of Dundee, UK) of anti-canine PLB antibodies in a total volume of 1 ml PBS bu¡er at provided sheep antibodies raised against GM subunit. Anti-mouse 4³C for 2 h under stirring conditions. Then 50 Wl of protein A Se- Ig-HRP-linked whole antibody (from sheep) and anti-rabbit Ig- pharose was added to the medium and incubated at 4³C for 45 min HRP-linked whole antibody (from donkey) were purchased from under stirring conditions. Five successive washing steps were done, Amersham Life Science, France. Anti-sheep IgG-HRP-linked whole Laemmli bu¡er (v/v) was added to the pellet, the solution was then molecule (from donkey) was purchased from Sigma. Protein A and G boiled 5 min and ¢nally centrifuged. The supernatant was loaded on Sepharose were purchased from Pharmacia Biotech, France. the gel and run on a 15% polyacrylamide gel. The same protocol was applied for [35S]GM lysate (107 cpm), with sheep antibodies raised 2.2. Human PLB cDNA cloning against GM subunit (anti-GM) and with protein G Sepharose instead PLB encoding cDNA was generated according to the sequence of protein A Sepharose. published by Fujii et al. [14], GenBank accession number m63603. The sequence was modi¢ed by the addition of a HindIII site at the 2.6. Co-immunoprecipitation of PLB and GM 5P end and an EcoRI site at the 3P end. Eight oligonucleotides were The putative interaction between PLB and GM was investigated in synthesised on an Applied Biosystems 380B DNA synthesiser using the following conditions: 2 Wl of PLB T7 lysate and 50 WlGMT3 standard cyanoethyl phosphoramidite chemistry. The oligonucleotides lysate were incubated with 943 Wl PBS at 4³C for 1 h, 5 Wl of PLB were gel-puri¢ed and assembled by pairs (OL1+OL2, OL3+OL4, monoclonal antibody was added to the medium and incubation was OL5+OL6 and OL7+OL8) into the full-length species (159 bp): done at 4³C for 2 h. Then, 50 Wl of protein A Sepharose was added and incubated at 4³C for 45 min under gentle stirring. After three OL1: 5P-AGCTTCCCGGGATGGAGAAAGTCCAATACCTCACT- washes with PBS, the Laemmli bu¡er (v/v) was added to the pellet, CGCTCAGC-3P boiled for 5 min, centrifuged and the supernatant was loaded on the OL2: 5P-CTTCTTATAGCTGAGCGAGTGAGGTATTGGACTTT- gel and electrophoresed. CTCCATCCCGGGA-3P OL3: 5P-TATAAGAAGAGCCTCAACCATTGAAATGCCTCAAC- 2.7. Revelation method for £uorographies AAGCACGTCA-3P Proteins were ¢xed on the gel after migration. To increase detection OL4: 5P-TGTAGCTTTTGACGTGCTTGTTGAGGCATTTCAAT- sensitivity of £uorography, the gel was then incubated with Amplify GGTTGAGGCT-3P reagent from Amersham. After drying, the gel was kept at 370³C OL5: 5P-AAAGCTACAGAATCTATTTATCAATTTCTGTCTCAT- overnight in contact with a hyper¢lm before revelation.