Báo cáo khoa học: A novel glycogen-targeting subunit of protein phosphatase 1 that is regulated by insulin and shows differential tissue distribution in humans and rodents pdf

A novel glycogen-targeting subunit of protein phosphatase that is regulated by insulin and shows differential tissue distribution in humans and rodents Shonagh Munro1, Hugo Ceulemans2, Mathieu Bollen2, Julie Diplexcito1 and Patricia T.W Cohen1 Medical Research Council Protein Phosphorylation Unit, University of Dundee, UK Katholieke Universiteit Leuven, Faculteit Geneeskunde, Afdeling Biochemie, Belgium Keywords diabetes; glycogen metabolism; glycogen synthase; insulin; PP1 Correspondence P T W Cohen, MRC Protein Phosphorylation Unit, School of Life Sciences, MSI ⁄ WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK Fax: +44 1382 223778 Tel: +44 1382 344240 E-mail: p.t.w.cohen@dundee.ac.uk (Received 23 September 2004, revised 16 December 2004, accepted 26 January 2005) doi:10.1111/j.1742-4658.2005.04585.x Stimulation of glycogen-targeted protein phosphatase (PP1) activity by insulin contributes to the dephosphorylation and activation of hepatic glycogen synthase (GS) leading to an increase in glycogen synthesis The glycogen-targeting subunits of PP1, GL and R5 ⁄ PTG, are downregulated in the livers of diabetic rodents and restored by insulin treatment We show here that the mammalian gene PPP1R3E encodes a novel glycogen-targeting subunit of PP1 that is expressed in rodent liver The phosphatase activity associated with R3E is slightly higher than that associated with R5 ⁄ PTG and it is downregulated in streptozotocin-induced diabetes by 60– 70% and restored by insulin treatment Surprisingly, although mRNA for R3E is most highly expressed in rat liver and heart muscle, with only low levels in skeletal muscle, R3E mRNA is most abundant in human skeletal muscle and heart tissues with barely detectable levels in human liver This species-specific difference in R3E mRNA expression has similarities to the high level of expression of GL mRNA in human but not rodent skeletal muscle The observations imply that the mechanisms by which insulin regulates glycogen synthesis in liver and skeletal muscle are different in rodents and humans Insulin-stimulated glycogen synthesis is decreased in type diabetes [1,2] One of the routes by which insulin stimulates this pathway is through activation of the rate-limiting enzyme, glycogen synthase (GS), via the phosphatidylinositol-3-kinase ⁄ protein kinase B pathway, which leads to the inhibition of glycogen synthase kinase (GSK3) [3,4] Activation of GS results from a net dephosphorylation of serine residues that are phosphorylated by GSK3 and dephosphorylated by glycogen-associated protein phosphatase (PP1) [5–8] In order to determine how insulin modifies GS activity, it is therefore crucial to understand the mechanisms by which insulin may activate glycogen-targeted PP1 The latter mainly exists as heterodimeric complexes of the catalytic subunit, PP1c, bound to a regulatory subunit [9] In striated muscles the most abundant glycogenbinding subunit GM (124–126 kDa, encoded by the gene PPP1R3A) targets PP1c to the sarcoplasmic reticulum as well as to glycogen particles [10–12] A much smaller protein, GL (33 kDa, encoded by the gene PPP1R3B), is the most abundant glycogen-targeting subunit of PP1 in liver, although it is only 23% identical to the N-terminal region of GM [13,14] Two other glycogen-binding subunits, R5 ⁄ PTG (36 kDa, encoded by PPP1R3C) with  40% identity to GL and R6 (33 kDa, encoded by PPP1R3D) with  30% identity Abbreviations GL, hepatic glycogen-targeting subunit of PP1 encoded by the gene PPP1R4(3B); GM (also termed RGL), skeletal muscle glycogen-targeting subunit of PP1 encoded by the gene PPP1R3(3A); GS, glycogen synthase; GSK3, glycogen synthase kinase-3; GSP, glycogen synthase phosphatase; GST, glutathione S-transferase; MBP, maltose-binding protein; NCBI, National Center for Biotechnology Information USA; PCR, PP1, protein phosphatase 1; PP1c, protein phosphatase catalytic subunit; R5 (also termed PTG), regulatory subunit of PP1 encoded by the gene PPP1R5(3C); R6, regulatory subunit of PP1 encoded by the gene PPP1R6(3D) 1478 FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS S Munro et al to GL have a wide tissue distribution [15–17] Interestingly, although GL is expressed at only very low levels in rodent skeletal muscles, it is found in human skeletal muscles at levels comparable with those in human liver [18] The four glycogen-targeting subunits bind to PP1c via a short highly conserved motif (-RVXF-) This motif is also responsible for the interaction of many other regulatory subunits with PP1, explaining why the binding of targeting regulatory subunits to PP1c is mutually exclusive [19] However, certain inhibitor proteins have also been noted to form ternary structures with PP1c-targeting subunit complexes [10,20–22] In addition to the PP1 and glycogen-binding motifs, the PP1 glycogen-regulatory subunits possess a motif for the interaction with substrates [23] The glycogen-targeting subunits can modulate the activity of PP1c towards different substrates; for example, GL enhances PP1c activity towards GS while suppressing its activity towards phosphorylase There is evidence that PP1-GM and PP1-GL may be regulated acutely by insulin Assay of PP1 following insulin infusion of skeletal muscle and immunopelleting of PP1-GM showed a 1.5–2-fold increase in phosphatase activity with insulin [24] In GM null mice, this activity was absent and GS could not be fully activated by insulin [24] In contrast, studies on an independently derived GM null mouse model found that insulin activation of GS was in the normal range, indicating that the PP1-GM is not required for the insulin activation of GS [25] These workers postulated the existence of a novel insulin-activated form of glycogen-targeted PP1 [25] In the case of hepatic glycogen-targeted PP1, insulin is thought to exert its acute activating effect on PP1-GL mainly through modulation of cAMP levels and decrease of phosphorylase a, which is a potent inhibitor of hepatic glycogen synthase phosphatase (GSP) activity [26–32] Phosphorylase a binds to 16 amino acids at the extreme C-terminus of GL, a sequence that is absent from the other three glycogentargeting subunits [18,31] R5 ⁄ PTG and R6 ⁄ PPP1R3D are not known to be acutely regulated by insulin Insulin exerts a longer term regulation on hepatic GL and R5 ⁄ PTG [33,34] Diabetic rats exhibit a loss of hepatic glycogen-bound synthase phosphatase activity that can be restored by insulin administration [35,36] The main underlying defects are decreased expression of the two PP1 glycogen-targeting subunits, GL and R5 ⁄ PTG, in the diabetic state [33,34] Downregulation of both the protein and mRNA levels of hepatic GL and R5 ⁄ PTG are restored by insulin treatment, but the skeletal muscle R5 ⁄ PTG level is not altered by insulin [33,34,37] The expression of hepatic R6 ⁄ PPP1R3D is also unaffected in diabetic animals [34] FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS Novel glycogen-targeting subunit of PP1 Ceulemans et al [38], undertook a bioinformatic approach in order to trace the evolution of regulatory subunits of PP1 Searching completed genome sequences, with the sequences of known PP1 regulatory subunits, including the conserved PP1 and glycogen-binding regions of the glycogen-targeting subunits, they identified nine new potential regulatory subunits of PP1 Of these nine sequences, three were deduced to encode for putative human glycogen-targeting subunits, and were given the nomenclature PPP1R3E, PPP1R3F and PPP1R3G These potential human proteins all contained the canonical -RVXF- motif that mediates interaction with PP1, as well as putative modules for targeting to glycogen and facilitating interaction with PP1 substrates such as GS Here we show that the phosphatase activity of one of these novel targeting subunits, R3E, is under long-term control by insulin in rodent liver while being virtually absent from rodent skeletal muscle; yet surprisingly, PPP1R3E mRNA is found at appreciable levels in human skeletal muscle Results Cloning of human PPP1R3E and PPP1R3G from human cDNA libraries Interrogation of the NCBI databases revealed no fulllength mammalian cDNAs sharing similarities to the human genomic sequences PPP1R3E, PPP1R3F and PPP1R3G (Accession nos: AL049829, NM_033215, AL035653) with chromosomes locations 14q11.2, Xp11.23 and 6p24.3-25.3, respectively In order to establish whether these sequences were functional genes or pseudogenes, attempts were made to amplify their putative cDNAs from human libraries, using primers designed from the genomic sequences For PPP1R3E, a single cDNA of  800 bp was amplified by two rounds of polymerase chain reaction (PCR) from both human testis and brain cDNA libraries The size of these products was consistent with the expected size for a putative sequence for a glycogen-targeting subunit of PP1 assuming the genomic sequence has two coding exons separated by one intron (Fig 1A) A cDNA corresponding to the genomic sequence of PPP1R3G was obtained by PCR from the human brain library, but no full-length cDNA products were obtained for PPP1R3F Subcloning and sequencing of the PCR products confirmed the identity of the testis and brain cDNAs for PPP1R3E and showed that the protein translated from this sequence was composed of 279 amino acids with a predicted molecular mass of 30.6 kDa (Fig 1B) The PPP1R3G cDNA sequence 1479 Novel glycogen-targeting subunit of PP1 A S Munro et al Gene -272 417 719 E1 1279 1713 1819 E2 3036 3176 E3 3660 E4 6656 E5 mRNA (ATG) E1 837 (STOP) E2 E3 E4 E5 B -105 gaagcggacccagcgacttctgcgctgacgcggggcgggcgggagagaggaagagaggggagcgcggtggcgctgcgagctggccccgccggggaaggggctgcc -1 1 ATG TCC CGT GAG CGG CCC CCG GGC ACC GAC ATT CCC CGC AAC CTG AGC TTC ATC GCC GCG CTA ACG GAG CGC GCC M S R E R P P G T D I P R N L S F I A A L T E R A 75 25 76 26 TAC TAC CGT AGC CAG CGG CCC AGC CTC GAG GAG GAG CCG GAG GAG GAG CCA GGC GAG GGC GGG ACG CGG TTC GGG Y Y R S Q R P S L E E E P E E E P G E G G T R F G 150 50 151 51 GCC CGA TCC CGC GCT CAC GCA CCG AGT CGG GGC CGC CGG GCC CGA TCT GCA CCA GCC GGA GGC GGC GGG GCC CGG A R S R A H A P S R G R R A R S A P A G G G G A R 225 75 226 76 GCG CCC CGC AGC CGT AGC CCA GAC ACC CGC AAG AGA GTG CGT TTC GCC GAC GCA CTG GGG TTG GAG CTG GCT GTC A P R S R S P D T R K R V R F A D A L G L E L A V 300 100 301 101 GTG CGC CGC TTC CGT CCC GGT GAG CTG CCC CGG GTG CCC CGC CAC GTG CAG ATC CAA TTG CAG AGG GAC GCC CTC V R R F R P G E L P R V P R H V Q I Q L Q R D A L 375 125 376 126 CGC CAC TTC GCG CCC TGC CAG CCC CGC GCC CGC GGC CTC CAG GAG GCG CGC GCC GCC CTG GAG CCG GCC AGC GAG R H F A P C Q P R A R G L Q E A R A A L E P A S E 450 150 451 151 CCC GGC TTC GCC GCC CGC TTG CTG ACG CAG CGC ATC TGC CTG GAA CGC GCC GAG GCG GGC CCG CTG GGC GTG GCC P G F A A R L L T Q R I C L E R A E A G P L G V A 525 175 526 176 GGG AGC GCG CGC GTG GTG GAC CTG GCC TAC GAG AAG CGC GTG AGC GTG CGC TGG AGC GCC GAC GGC TGG CGG AGC G S A R V V D L A Y E K R V S V R W S A D G W R S 600 200 601 201 CAA CGC GAG GCG CCA GCC GCC TAC GCC GGT CCG GCC CCG CCC CCG CCG CGC GCC GAC CGC TTC GCC TTC CGC CTG Q R E A P A A Y A G P A P P P P R A D R F A F R L 675 225 676 226 CCC GCG CCG CCG ATT GGG GGC GCC CTG CTC TTC GCC TTG CGC TAC CGT GTG ACA GGT CAC GAG TTC TGG GAC AAC P A P P I G G A L L F A L R Y R V T G H E F W D N 750 250 751 251 AAC GGC GGC CGT GAC TAT GCT CTA CGT GGG CCC GAG CAC CCG GGC AGT GGC GGA GCT CCG GAG CCG CAG GGC TGG N G G R D Y A L R G P E H P G S G G A P E P Q G W 825 275 826 276 ATC CAC TTT ATC TGA gacgaggcgcctgcggccgacggcggaaaacaccaaaggcacccgggggcggggcgacccgatgtggcggggaggagtag 920 I H F I * 279 921 gagagaccaggattggcgggagcggtccaagggagtc 957 Fig (A) Diagram of human PPP1R3E mRNA compared with PPP1R3E gene (Accession no: ENSG00000129525) Nucleotide numbers at the start and end of each exon are given relative to the first nucleotide of the initiating methionine codon The exon ⁄ intron structure within the coding region was determined experimentally by PCR of cDNA libraries, whereas that for the untranslated region is predicted from the genomic sequence and partial cDNAs in the database (B) Human PPP1R3E cDNA and the encoded protein determined by PCR of human brain and testis cDNA libraries The PP1 binding motif is underlined, glycogen-targeting domain is double underlined, and the substrate-binding sequence is underscored by a wavy line Oligonucleotides primers used for PCR are indicated by arrows was verified as being identical to the genomic sequence with one coding exon specifying a protein of 358 amino acids and a molecular mass of 38 kDa (data not shown) Comparison of the glycogen-targeting subunits of PP1 Searching mouse and rat genomic sequences in the NCBI databases identified predicted rodent cDNAs from genes homologous to human PPP1R3E and 1480 PPP1R3G The encoded rat and mouse R3E proteins share around 97% amino acid identity and are 89% identical to their human orthologue, indicating that this regulatory protein is very well conserved in mammals (Fig 2A) R3G is slightly less highly conserved; the rodent orthologues are 90% identical and they are 11 amino acids shorter than their human orthologue, sharing 67% identity (Fig 2B) A phylogenetic tree depicting the relationship between known glycogen-targeting subunits of PP1 and the novel glycogen-targeting subunits is shown in Fig 2D Although all seven FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS S Munro et al Novel glycogen-targeting subunit of PP1 A C MOUSE R3E RAT R3E HUMAN R3E MSPERPPRTDIPRNLSFIAALTERAYYRSQRPSLEEESEEEPGEGGTRPGARSRAHVPG MSHERPPRNDIPRNLSFIAALTERAYYRSQRPSLEEESEEEPGEGGTRPGARSRAHVPG MSRERPPGTDIPRNLSFIAALTERAYYRSQRPSLEEEPEEEPGEGGTRFGARSRAHAPS MOUSE R3E RAT R3E HUMAN R3E 60 RGRRARSAPAGGGGARTARSRSPDTRKRVRFADALGLELAVVRRFRPGEPPRVPRHVQV 60 RGRRARSAPAGGGGARTARSRSPDTRKRVRFPDALGLELAVVRRFRPGEPPRVPRHVQV 60 RGRRARSAPAGGGGARAPRSRSPDTRKRVRFADALGLELAVVRRFRPGELPRVPRHVQI MOUSE R3E 119 QLQRDALRHFAPCPPRARGLQEARVALEPALEPGFAARLQAQRICLERADAGPLGVAGS RAT R3E 119 QLQRDALRHFAPCPPRTRGLQDARIALEPALEPGFAARLQAQRICLERADAGPLGVAGS HUMAN R3E 119 QLQRDALRHFAPCQPRARGLQEARAALEPASEPGFAARLLTQRICLERAEAGPLGVAGS PP1 binding motif GM/ RGL/R3A GL/R3B R5/PTG/R3C R6/R3D R3E R3F R3G 60 58 81 99 84 167 128 GTRRVSFAD VKKRVSFAD AKKRVVFAD QKLRVRFAD TRKRVRFAD APRRVLFAD CKKRVQFAD Glycogen binding domain MOUSE R3E 178 ARVLDLAYEKRVSVRWSADGWRSLRESPASYAGPAPSPPRADRFAFRLPAPPVGGTLLF RAT R3E 178 ARVLDLAYEKRVSVRWSADGWRSLRESPASYAGPAPAPPRADRFAFRLPAPPVGGALLF HUMAN R3E 178 ARVVDLAYEKRVSVRWSADGWRSQREAPAAYAGPAPPPPRADRFAFRLPAPPIGGALLF GM/ RGL/R3A GL/R3B R5/PTG/R3C R6/R3D R3E R3F R3G MOUSE R3E 237 ALRYRVTGREFWDNNGGRDYALLGPEHPAGAGAAEPQGWIHFI 279 RAT R3E 237 ALRYRVTGREFWDNNGGRDYALLGPEHPGGAGAAEPQGWIHFI 279 HUMAN R3E 237 ALRYRVTGHEFWDNNGGRDYALRGPEHPGSGGAPEPQGWIHFI 279 Substrate binding domain B Rat R3G Mouse R3G Human R3G MEASGEQLHRSEASSSTSSEDPPPAEELSVPEVLCVESG -TSEVPI MDPSGEQLHRSEASSSTSSGDPQSAEELSVPEVLCVESG -TSETPI MEPIGARLS-LEAPGPAPFREAPPAEELPAPVVPCVQGGGDGGGASETPS Rat R3G Mouse R3G Human R3G 46 PDDQLQDRLLSAQKVAALPEQEELQEYRR-SRVRSFSLPADPILQAAKLL 46 PDAQLQDRPLSPQKGAALPEQEELQEYRR-SRARSFSLPADPILQAAKLL 50 PDAQLGDRPLSPKEEAAPQEQEELLECRRRCRARSFSLPADPILQAAKFL GM/ RGL/R3A GL/R3B R5/PTG/R3C R6/R3D R3E R3F R3G 144 146 171 191 176 300 235 219 221 246 267 248 407 339 GIIRVLNVSFEKLVYVRMSLDDW GTVKVQNLAFEKTVKIRMTFDTW GTVKVKNVSFEKKVQIRITFDSW GTVRVCNVAFEKQVAVRYTFSGW GSARVVDLAYEKRVSVRWSADGW GLVRVLNRSFEKAVHVRASHDGW GSGRVLSCPGPRAVTVRYTFTEW WSNNNGTNY WDSNRGKNY WDNNDGQNY WDNNDHRDY WDNNGGRDY WANNHGRNY WDNNAGANY D hR3F mR3F GM/RGL Rat R3G 95 QQRQQ -AGQPSSEGGEPAGDCCSKCKKRVQFADSLGLSLASVKHFS Mouse R3G 95 QQRQQ -AGQPSSEGGAPAGDCCSKCKKRVQFADSLGLSLASVKHFS Human R3G 100 QQQQQQAVALGGEGAEDAQLGPGGCCAKCKKRVQFADTLGLSLASVKHFS Rat R3G 140 EAEEPQVPPAVLSRLHSFPLRAEDLQQLGELLAVAKVPAPLLTPRAQLRP Mouse R3G 140 EAEEPQVPPAVLSRLHSFPLRAEDLQQLGGLLAVATMPDPLLVPCARLRP Human R3G 150 EAEEPQVPPAVLSRLRSFPMRAEDLEQLGGLLAAAAVAAPLSAPPSRLRP Rat R3G 190 LFQLPGLIAAEERLRRQRVCLERVQCSQPPRAEVTGSGRVISCPGPRAVA Mouse R3G 190 HFQLPELRAAEERLRRQRVCLERVQCSQPPRAEVTGSGRVISCPGPRAVA Human R3G 200 LFQLPGPSAAAERLQRQRVCLERVQCSTASGAEVKGSGRVLSCPGPRAVT rbR3A mR3A hR3A R6 mR3DrR3D hR3D mR3E rR3E hR3E hR3C R5/PTG rR3C mR3C hR3B rR3B mR3B mR3G hR3G rR3G GL Rat R3G 240 VRYTFTEWRTFLDVPAELHPESLEPLSP-VRSGNSGPGAEDSEGEPGTER Mouse R3G 240 VRYTFTEWRTFLDVPAELDPESLEPLPP-LQSGDSGSKAEDSEEGPGTER Human R3G 250 VRYTFTEWRSFLDVPAELQPEPLEPQQPEAPSGASEPGSGDAKKEPGAEC Rat R3G 289 FCFSLCLPPGLQPKEGEDADTWGVAIHFAVCYRCEQGEYWDNNEGANYTL Mouse R3G 289 FHFSLCLPPGLQPKEGEDAGAWGVAIHFAVCYRCEQGEYWDNNEGANYTL Human R3G 300 FHFSLCLPPGLQPEDEEDADERGVAVHFAVCYRCAQGEYWDNNAGANYTL Rat R3G 339 RYVCSTDPL 347 Mouse R3G 339 RYVCSTDPL 347 Human R3G 350 RYARPADAL 358 Fig Amino acid alignment of human proteins with their rat and mouse homologues (A) R3E, (B) R3G Identities are shaded in black and similarities are shaded in grey The PP1-binding motif is indicated by a single underline The sequences were aligned using CLUSTALW (http:// www.clustalw.genome.ad.jp/) and shading was performed using BOXSHADE (v3.21 K.Hofmann and M.Baron) NCBI Accession nos for predicted cDNAs are: XM_193763 (mouse R3E), XM_344406 (rat R3E), XM_225280 (rat R3G) Mouse R3G cDNA Accession no is AK049829 (C) Amino acid alignment of the conserved regions of the glycogen-targeting subunits of PP1 Identification of the PP1-binding motif was described in Egloff et al [43], the glycogen-binding domain in [15,31,44] and the substrate binding domain in [23,45] (D) Phylogenetic relationship between the glycogen-targeting subunits of PP1 The unrooted tree is derived by the neighbour-joining method in CLUSTAL W from pairwise sequence distances between the conserved PP1, glycogen and substrate-binding domains (corresponding to amino acids 85–258 of R3E) of human (h), mouse (m), rat (r) and rabbit (rb) glycogen-targeting subunits The proteins aligned and their database Accession nos are R3A(PPP1R3AG ⁄ RGL) NP_002702 (h), NP_536712 (m), A40801 (rb); R3B(PPP1R3B ⁄ GL) [18] and NP_078883 (h), NP_808409 (m), NP_620267 (r); R3C(PPP1R3C ⁄ R5 ⁄ PTG) NP_005389 (h), NP_058550 (m), XP_220048 (r); R3D(PPP1R3D ⁄ R6) Y18206 ⁄ NP_006233 (h), XP_141580 (m), XP_230940 (r); R3E(PPP1R3E) this study (h, m, r); R3F(PPP1R3F) XP_372210 (h), AAH59275 (m); R3G(PPP1R3G) this study (h, m, r) FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS 1481 Novel glycogen-targeting subunit of PP1 Pancreas Kidney Skeletal muscle Liver Placenta Lung Brain Human PPP1R3E mRNA blot Heart A S Munro et al 9.5 kb 7.5 kb 7.2 kb 5.9 kb 4.4 kb Tissue distribution of PPP1R3E and PPP1R3G mRNA 2.4 kb 1.35 kb β-actin α-actin 2.0 kb 1.8 kb Kidney Testis Skeletal muscle Lung Liver Spleen Brain Rat PPP1R3E mRNA blot Heart B 9.5 kb 7.5 kb 6.0 kb 5.0 kb 4.5 kb 4.4 kb 2.4 kb 1.35 kb 2.0 kb 1.8 kb β-actin α-actin 9.5 kb 7.5 kb Pancreas Kidney Skeletal muscle Liver Placenta Lung Brain Human PPP1R3G mRNA blot Heart C 2.4 kb 1.35 kb 0.24 kb 1482 The human PPP1R3E cDNA probe hybridized to two mRNA species on a human multiple tissue northern blot with sizes 7.2 and 5.9 kb (Fig 3A) These transcripts were predominantly present in skeletal muscle and heart, although the smaller transcript was also present in pancreas and placenta and was detectable at very low levels in liver and kidney The sizes and the tissue distribution of these transcripts are not consistent with those encoding any of the other characterized glycogen-targeting subunits In addition, the extremely low level of sequence similarity at the nucleotide level implies that cross-hybridization with the mRNAs for the other subunits is unlikely Hybridization of a northern blot with the human PPP1R3G cDNA probe revealed a single PPP1R3G mRNA transcript of  kb that was present exclusively in brain (Fig 3C) Unfortunately, attempts to amplify mouse or rat PPP1R3E cDNA from tissue specific libraries were unsuccessful However, the rat PPP1R3E exons showed a high level of conservation (86% identity) with the coding region of human PPP1R3E cDNA This coupled with the lack of sequence similarity to the coding regions of other glycogen-targeting subunits, allowed the human cDNA probe to be used to establish the tissue distribution of rat PPP1R3E mRNA on a northern blot (Fig 3B) Following a series of stringent washes and autoradiography, the probe hybridized predominantly to 6.0 kb and 5.0 kb mRNA species in heart ~ 9.0 kb 4.4 kb 2.0 kb 1.8 kb human subunits and their rodent orthologues possess known or putative PP1, glycogen and substrate-binding domains (Fig 2C), no two subunits share more than 40% amino acid identity Despite this, each glycogen-targeting subunit is particularly well conserved between rodents and humans, suggesting that each subunit may serve an important, nonredundant function in mammals β-actin α-actin Fig Tissue distribution of (A) human PPP1R3E mRNA (B) rat PPP1R3E mRNA (C) human PPP1R3G mRNA Blots contained  lg of poly(A)+ RNA from different tissues The upper panels of (A) and (B) were hybridized with a probe corresponding to the entire coding region (837 bp) of human PPP1R3E and the upper panel of (C) was hybridized with a probe corresponding to the entire coding region (1074 bp) of human PPP1R3G Following autoradiography, the membranes were stripped in 0.5% (w ⁄ v) SDS at 100 °C for and subsequently re-probed with a b-actin in order to assess whether equal amounts of the samples were loaded In heart and skeletal muscle the b-actin probe cross-hybridizes with a-actin FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS S Munro et al Novel glycogen-targeting subunit of PP1 A B Fig Specificity and characterization of PPP1R3E antibodies (A) Recognition of 0.1–10 ng of bacterially expressed GST-R3E by anti-R3E(8–23) and anti-GST-R3E(1–98) sera Both antibodies were used at a concentration of 0.2 lgỈmL)1 (B) Specificity of anti-R3E(1–98) sera for R3E The immunoblot of several glycogen-targeting subunits was probed with 0.2 lgỈmL)1 affinity purified antibody Lane 1, rat liver glycogen pellet; lane 2, rat liver lysate; lane 3, ng GST-R3E(full-length); lane 4, ng GST-R3E, lane 5, ng GST-R3E; lane 6, 100 ng GST-GM(1–243); lane 7, 100 ng GSTGL; lane 8, 100 ng GST-R5 ⁄ PTG; lane 9, 100 ng GST-R6 In the lower panel, the blot was stripped and reprobed with ant-GST sera to show the loading of the samples C and to a 4.5 kb RNA mRNA in liver Surprisingly, the probe hybridized only weakly to the 6.0 and 5.0 kb transcripts in skeletal muscle The 5.0 kb transcript was also present in brain, spleen, lung, liver, kidney and testis, albeit at very low levels R3E protein is present in the rat liver glycogen fraction and phosphatase activity associated with R3E is higher than that associated with R5/PTG Anti-R3E(8–23) sera were raised against amino acids 8–23 in the N-terminus of human R3E, as this is the region that shares no similarity with other glycogentargeting subunits These antibodies and anti-GSTR3E(1–98) sera recognized as little as 0.2 ng of bacterially expressed GST-R3E(full length, human) (Fig 4A) Anti-GST-R3E(1–98) was virtually specific for R3E as it did not recognize 100 ng of GM, GL, R6 or R5 ⁄ PTG (Fig 4B) The peptide antibody was extremely specific as it did not cross-react with 100 ng of GM, GL, R5 ⁄ PTG or R6 (data not shown) FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS The presence of a fairly well-conserved glycogenbinding motif in R3E suggested that it may interact with glycogen To test this hypothesis, a rat liver lysate, microsomal fraction and a glycogen fraction were prepared and the proteins in these fractions separated by SDS ⁄ PAGE, transferred to nitrocellulose and immunoblotted A single R3E band was detected in the glycogen fraction, which was consistent with the predicted size of R3E ( 31 kDa) (Fig 5A) This band was sometimes detectable at low levels in rat liver lysates (Figs 4B and 5A) In order to establish whether R3E could bind (and therefore be regulated allosterically by phosphorylase a, lg of GST-R3E was transferred to nitrocellulose membrane and tested for its ability to bind to [32P]phosphorylase a [31] The 32P-labelled phosphorylase a was found to bind to GST-GL, but not to GSTR3E or GST-R5 ⁄ PTG (data not shown) A specific and sensitive phosphatase immunoadsorption assay has been developed [34,39], which allows characterization of the activities of the different glyco1483 S Munro et al Glycogen Lysate A Microsomal Novel glycogen-targeting subunit of PP1 39 R3E 28 C 0.03 Phosphorylase phosphatase activity (mU/mg) Phosphorylase phosphatase activity (mU/mg) B 0.02 0.01 0.1 0.075 0.050 0.025 0 -RVXF peptide +RVXF peptide GL R5 R3E Fig Detection of PPP1R3E and its associated phosphatase activity in liver (A) Rat liver lysate (20 lg protein), microsomal fraction (20 lg protein) and glycogen fraction (2 lg protein) were subjected to electrophoresis on 10% SDS ⁄ polyacrylamide gels After transfer to nitrocellulose, the blot was probed with 0.5 lgỈmL)1 anti-GSTR3E(1–98) (B) Phosphorylase phosphatase activity associated with R3E in rat liver lysates (assayed in the presence of nM okadaic acid) The R3E complex was immunoadsorped from 100 lg of rat liver lysate The immune pellets were then assayed for spontaneous phosphorylase phosphatase activity (in the absence of dissociating peptide) and total phosphorylase phosphatase activity (assayed in the presence of the PP1c-dissociating RVXF containing peptide) Phosphatase activity is expressed in mmg)1 total protein in the rat liver lysate The phosphatase activity in control IgG protein G-Sepharose immune pellets (0.001 mmg)1) was subtracted Error bars indicate the SEM for assay of three liver lysates, each assay being performed in triplicate (C) Comparison of the total phosphorylase phosphatase activity associated with GL, R5 and R3E (measured in the presence of the PP1-dissociating peptide) gen-targeted forms of PP1 Essentially, using specific antibodies to a glycogen-targeting subunit of choice, it is possible to pellet the bound PP1c activity in an immune complex However, the interaction of regulatory subunits with PP1c may modify substrate specificity, decreasing the activity of PP1c against some substrates while increasing it against others The immune pellet is therefore assayed for protein phosphatase activity in the absence and presence of a peptide that dissociates the interaction between PP1c and glycogen-targeting subunits [40] Inclusion of the dissociating peptide relieves the modification of phosphatase activity imposed by the glycogen-targeting subunit and provides a means to calculate the actual amount of 1484 PP1c bound to each subunit After immunoadsorption of R3E with anti-R3E(8–23) serum, the spontaneous phosphorylase phosphatase activity associated with PPP1R3E (measured in the absence of the dissociating peptide) was 0.006 ± 0.0008 mmg)1 Addition of the dissociating peptide to the assay increased the activity by approximately fourfold to 0.024 ± 0.005 mmg)1 This provided evidence that R3E does indeed interact with PP1c and suggests that the interaction of R3E with PP1 inhibits its activity substantially with phosphorylase a as a substrate (Fig 5B) In contrast, the activity of PP1-R3E using GS as substrate was similar in the presence and absence of dissociating peptide, demonstrating that R3E exhibited little or no inhibition of PP1c activity towards this substrate (Fig 6B) The glycogen synthase phosphatase ⁄ phosphorylase phosphatase (GSP ⁄ PhP) activity ratio for R3E-PP1c of 3.7 is substantially higher than that calculated for GL (1.9), R5 (0.9) and R6 ( 2) [34] Comparison of the level of phosphorylase phosphatase activity associated with PPP1R3E with that associated with GL and R5 in rat liver, shows that the activity associated with PPP1R3E is  30% of that bound to GL, and is slightly higher than that associated with R5 ⁄ PTG (Fig 5C) Effect of induced diabetes and insulin treatment on the expression and activity of PPP1R3E in vivo Previous studies [33,34] have shown that streptozotocin-induced diabetes in rats causes 75 and 60% decreases in the hepatic protein phosphatase activity associated with GL and R5 ⁄ PTG, respectively This response is accompanied by a corresponding decrease in the hepatic levels of GL and R5 ⁄ PTG proteins All of these effects were restored by the intravenous administration of insulin The finding that R3E appears to be most highly expressed in rodent liver prompted investigation into whether this subunit may be regulated in vivo in liver by streptozotocin-induced diabetes and changes in insulin levels Figure 6(A,B) illustrates the results of assays of antiR3E(8–21)–protein G–Sepharose immunopellets from liver lysates of control, diabetic and insulin-treated diabetic rats The phosphorylase phosphatase and GSP activities associated with R3E are decreased by  65–70% in the diabetic rat liver Furthermore, the phosphatase activities associated with R3E could be restored to that of control levels following intravenous administration of insulin for 96 h The same percentage decrease in phosphorylase phosphatase and GSP activities in diabetic livers and restoration by insulin treatment was observed in the presence of the dissociFEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS S Munro et al ating peptide (Fig 6) The activity in IgG control immune pellets was < 5% of the phosphatase activity associated with R3E Analysis of the RNA in the livers of control and streptozotocin diabetic rats showed that the R3E mRNA levels varied in parallel with the phosphatase activities of PP1-R3E (Fig 6C) The data demonstrate that, like GL and R5 ⁄ PTG, R3E is downregulated in type diabetic animals A 0.03 Phosphorylase phosphatase activity (mU/mg) -RVXF +RVXF 0.02 0.01 diabetic +96 h i nsulin diabetic control B Glycogen synthase phosphatase activity (mU/mg) Fig Effect of streptozotocin-induced diabetes on R3E-associated phosphorylase and GSP activities and R3E mRNA in rat liver.R3E immune pellets assayed for phosphorylase phosphatase activity (A) and GSP (B) activities assayed in the absence and presence of the PP1c RVXF-containing dissociating peptide The activities are expressed as mmg)1 of total protein in the rat liver lysate Error bars indicate the SEM Control rats (n ¼ 3), diabetic rats (n ¼ 5), diabetic rats +96 h insulin treatment (n ¼ 4).The differences in spontaneous phosphorylase phosphatase activities (P < 0.01 for control and diabetic livers, P < 0.001 for diabetic and insulin treated livers), and the total phosphorylase phosphatase activities in the presence of the PP1c dissociating peptide (P < 0.02 for control and diabetic livers, P < 0.001 for diabetic and insulin-treated livers) are statistically significant The differences in spontaneous GSP activities (P < 0.05) and total GSP activities in the presence of the PP1c-dissociating peptide (P < 0.05) are also statistically significant (C) Analysis R3E mRNA levels in the livers of control and streptozotocin-induced diabetic rats The R3E and control b-actin DNA bands obtained by multiplex RT–PCR using rat R3E-specific and b-actin-specific primers are stained with ethidium bromide and visualized under UV light Novel glycogen-targeting subunit of PP1 0.0004 -RVXF +RVXF 0.0003 0.0002 0.0001 The novel gene PPP1R3E encoding a putative glycogen-targeting subunit of PP1 is shown here to express R3E protein in rodent liver R3E shows 86% identity), suggesting that it may serve an important nonredundant function The R3E protein was found to be present in the hepatic glycogen fraction and to bind to PP1 The phosphorylase phosphatase activity associated with R3E in rat liver was slightly higher than that bound to R5 ⁄ PTG and  30% of that bound to the most abundant hepatic glycogen-targeting subunit GL However, the GSP ⁄ PhP activity ratio associated with R3E is 3.7 compared with 1.9 for GL and 0.9 for R5 ⁄ PTG indicating that PP1cR3E has the potential to contribute 60% of the GSP activity of PP1c-GL in rat liver The data also indicate that PP1c-R3E, like PP1c-GL, would be expected to function mainly as a GSP, whereas PP1c-R5 ⁄ PTG is more likely to function predominantly as phosphorylase phosphatase FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS C control diabetic + 96 h insulin Discussion diabetic control diabetic R3E β-actin Although analysis of the mRNA encoding rat R3E revealed that the main tissues of expression are liver and heart, with only very low levels being present in skeletal muscle, analysis of the human tissues indicated that PPP1R3E mRNA is most highly expressed in skeletal muscle and heart Very low levels of PPP1R3E mRNA were detected in most other human tissues examined, including liver The difference in tissue distribution between humans and rats reflects in part that 1485 Novel glycogen-targeting subunit of PP1 seen for the GL glycogen-targeting subunit of PP1 [18] GL, which is highly expressed in rodent liver but only present at very low levels in rodent skeletal muscle, is found at appreciable levels in human skeletal muscle (as well as in human liver) The finding that two glycogen-targeting subunits are highly expressed in human skeletal muscle while being present at only very low levels in rodent skeletal muscle may underlie a fundamental difference in the regulation and function of glycogen-bound PP1 in skeletal muscle in humans and rodents The observation that R3E appeared to be predominantly expressed in insulin-sensitive tissues, led to investigation of whether this protein is regulated by insulin in vivo Although no evidence was found for acute regulation via phosphorylase a as seen for PP1-GL, PP1-R3E associated phosphorylase phosphatase and GSP activities were substantially decreased in the livers of diabetic rats and these activities were restored by insulin treatment The similar decreases in activity observed for PP1-GL and PP1-R5 ⁄ PTG in the livers of diabetic animals was found to correspond to a decrease in protein and mRNA levels for their glycogen-targeting subunits [34] Because R3E protein was barely detectable in liver lysates (Figs 4B and 5A) by either of two different antibodies, it was not possible to directly confirm a decrease in R3E protein in the livers of streptozotocin diabetic rats by immunoblotting However, examination of R3E mRNA levels demonstrated a decrease to below detectable levels in the livers of diabetic rats It therefore appears that hepatic R3E, like GL and hepatic R5 ⁄ PTG, is regulated at the transcriptional level by insulin and that R3E mRNA and consequently protein levels are decreased in streptozotocin diabetic animals The novel PPP1R3G appears to be expressed at low levels exclusively in brain as judged from mRNA blotting and detection in brain cDNA libraries This situation is unusual, in that other PP1 glycogen-targeting subunits are either expressed at low levels ubiquitously or are present at significant levels in insulin-sensitive tissues such as liver and skeletal muscle However, glycogen is a major energy reserve in brain astrocytes and glycogen mobilization is tightly coupled to neuronal activity [41] Conservation of the amino acid sequence of R3G from human to rodents suggests that, like R3E, it may perform a distinct and critical function The generation of mice lacking the gene encoding the major striated muscle glycogen-targeting subunit of PP1, GM, has provided evidence to suggest that there is insufficient compensatory response from other subunits because mice lacking the GM subunit have only 10% muscle glyco1486 S Munro et al gen compared with their wild-type littermates [24,25] The homozygous deletion of PTG ⁄ R5 ⁄ PPP1R3C has recently been reported to be embryonic lethal [42] Mice heterozygous for this deletion have decreased glycogen stores and GS activity in muscle, liver and adipose tissue Glucose intolerance, hyperinsulinaemia and insulin resistance were also observed to develop with increasing age These results indicate that PTG performs a critical role that cannot be undertaken by the other glycogentargeting subunits The development of mice lacking particular subunits may, therefore, uncover whether there is any functional redundancy among the other glycogen-targeting subunits of PP1 The high levels of GL and PPP1R3E mRNA in human compared with rodent skeletal muscle indicates that rodents may not be appropriate models from which to gain an understanding of the hormonal regulation of human skeletal muscle GSP In addition, this species-specific difference in the expression of PP1 regulatory subunits is likely to be relevant to the study of the mechanism of action of insulin on human skeletal muscle and liver glycogen synthesis and the pathophysiology of human type diabetes Materials and methods Amplification of PPP1R3E and PPP1R3G from human cDNA libraries Full-length coding sequences of PPP1R3E and PPP1R3G were amplified from human brain and testis Matchmaker cDNA libraries (Clontech, Palo Alto, CA, USA) by two rounds of PCR using the Advantage GC-cDNA polymerase and instructions (Clontech) PPP1R3E was amplified by an initial PCR with the forward primer (nucleotides )105 to )84) 5¢-GAAGCGGACCCACGGACTTCTG-3¢ and the reverse primer (complementary to nucleotides 957–937 5¢-GA CTCCCTTGGACCGCTCCCG-3¢), followed by a second round of PCR with the forward primer (nucleotides 1–21 5¢-ATGTCCGCTGAGCGGCCCCCG-3¢) and the reverse primer (complementary to nucleotides 837–815 5¢-GATA AAGTGGATCCAGCCCCATAGGGGCGCGG-3¢) and the reverse primer (complementary to nucleotides 1074–1058 5¢-GAGCGCGTCCGCAGGGCACGC-3¢) PCR products were resolved on 1% (w ⁄ v) agarose gels, gel-purified, cloned into pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA, USA) and sequenced in both directions using M13 forward and reverse primers DNA sequencing was performed in conjunction with the Sequencing Service managed by Dr Nick Helps (School of Life Sciences, University of Dundee; http://www.dnaseq.co.uk) using an Applied Biosystems 373 A DNA sequencer or Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS S Munro et al RNA analyses Northern blots (Clontech) contained  lg poly(A)+ RNA from different tissues of fed rats post mortem and human tissues collected from fed individuals no more than h after death Blots were hybridized a32P-labelled cDNA probes according to the manufacturer’s instructions with the final wash in 10 mmolỈL)1 NaCl, 1.5 mmolỈL)1 sodium citrate, 0.1% SDS, at 55 °C Levels of R3E and control b-actin mRNA transcripts were assessed in total rat liver RNA by multiplex RT-PCR (Promega, Madison, WI, USA) as described previously [34] The rat R3E specific forward and reverse primers were 5¢-ATGTCCCGTGAGCGGCCCCCG-3¢ and 5¢-GAT AAAGTGGATCCAGCCCTGCG-3¢, respectively Treatment of animals Diabetes was induced with either intravenous or intraperitoneal injection of streptozotocin into male Wistar rats and insulin was subsequently administered intravenously into some of the rats for 96 h [34] Blood glucose levels were elevated ‡ fourfold in diabetic animals prior to insulin treatment The rats were killed by suffocation in CO2 and tissues were excised, freeze-clamped, and stored at )80 °C All procedures were performed in accordance with the guidelines of the ethical committees of the University of Dundee or the Katholieke Universiteit Leuven Immunological techniques Homogenization of tissues was performed as detailed in Munro et al [18] Homogenates were centrifuged at 16 000 g for 10 min, and the supernatants were snap-frozen in liquid nitrogen and stored at )80 °C Preparation of subcellular fractions was performed as detailed in Browne et al [34] Proteins were separated by 10% SDS ⁄ PAGE, transferred to nitrocellulose, and probed with affinity purified antibodies Peptides were synthesized by G Bloomberg (University of Bristol, UK); antibodies were raised in sheep by Diagnostics Scotland (Penicuik, Midlothian, UK) and affinity purified in conjunction with the Division of Signal Transduction Therapy, University of Dundee coordinated by H McLauchlan and J Hastie Antibodies to human PP1b peptide (amino acids 316–327) and human PPP1R3E(8–23) were affinity purified against their respective peptides Antibodies to human GST-PPP1R3E(1–98) were affinity purified against MBP-PPP1R3E Immunoblotting followed by detection of immunoreactive bands by enhanced chemiluminescence was performed as described in Munro et al [18] Protein phosphatase assays PP1 activities were determined by release of [32P]phosphate from phosphorylase a (10 mmolỈL)1, phosphorylated by FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS Novel glycogen-targeting subunit of PP1 phosphorylase kinase) and GS (1 mmolỈL)1, phosphorylated by GSK3) in the presence of nm okadaic acid for 10 at 30 °C For immunoadsorption of PP1-GL, PPP1R5 and PPP1R3E with anti-GL, anti-R5 and antiR3E sera, respectively, lysates were prepared in the presence of 100 nm okadaic acid Immune pellets from 100 lg of liver lysate were washed five times in the presence of nmolỈL)1 okadaic acid, and PP1 activities in the immune pellets were assayed as described above either before (‘spontaneous’ activity) or after (‘total’ activity) preincubation with 0.1 mgỈmL)1 ‘dissociating’ peptide (GKRTNLR KTGSERIAHGMRVKFNPLALLLDSC) that causes the release of free PP1c from the glycogen-targeting subunit [34,39] One unit of activity is the amount of enzyme that catalyses the release of mmol of [32P]phosphate per minute Statistical significance was assessed using the Student’s t-test Acknowledgements The work was supported by the UK Medical Research Council, UK and Diabetes UK SM was initially the recipient of a Cooperative Awards in Science and Engineering postgraduate studentship from the Biotechnology and Biological Research Sciences Council, UK and Novo Nordisk, Bagsvaerd, Denmark Subsequently, SM was supported on a postdoctoral research assistantship by Diabetes UK HC is a postdoctoral fellow of the Fund for 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synthase association with the striated muscle glycogen-targeting subunit of protein phosphatase-1 J Biol Chem 275, 26074–26081 1489 ... phosphorylase There is evidence that PP1-GM and PP1-GL may be regulated acutely by insulin Assay of PP1 following insulin infusion of skeletal muscle and immunopelleting of PP1-GM showed a 1. 5–2-fold increase... TGA gacgaggcgcctgcggccgacggcggaaaacaccaaaggcacccgggggcggggcgacccgatgtggcggggaggagtag 920 I H F I * 279 9 21 gagagaccaggattggcgggagcggtccaagggagtc 957 Fig (A) Diagram of human PPP1R3E mRNA compared... RGRRARSAPAGGGGARAPRSRSPDTRKRVRFADALGLELAVVRRFRPGELPRVPRHVQI MOUSE R3E 11 9 QLQRDALRHFAPCPPRARGLQEARVALEPALEPGFAARLQAQRICLERADAGPLGVAGS RAT R3E 11 9 QLQRDALRHFAPCPPRTRGLQDARIALEPALEPGFAARLQAQRICLERADAGPLGVAGS HUMAN R3E 11 9