ican Heart Association, Indiana Affiliate, Inc. Present address: .... primed DNA labeling kits were obtained from United States Bio- chemicals .... bar indicates the.
THEJOURNAL OF BIOLOGICAL CHEMISTRY (CJ
Vol. 266, No. 24, Issue of August 25, pp. 15782-15789.1991 Printed in U.S.A.
1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Expression of the Regulatory (RG~) Subunit of the Glycogen-associated Protein Phosphatase* (Received for publication, April 12, 1991)
Pauline M. Tang$, Jeffry A. Bondor, KristineM. Swidereks, and Anna A. DePaoli-Roachn From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122
DNA clones encodingthe glycogen-binding (Rcl)subunit of glycogen-associated protein phosphatase were isolated from rabbit skeletal muscle Xgtll cDNA libraries. Overlapping clones provided an open reading frame of 3327 nucleotides that predicts a polypeptide of 1109 aminoacidswith a molecularweight of 124,257. Northern hybridizationof rabbit RNA identified a major mRNA transcript of 7.5 kilobases present in skeletal, diaphragm, and cardiac muscle, but not in brain,kidney, liver, and lung. Southern analysis of rabbit genomic DNA digested with various restriction endonucleases gave rise to a single hybridizing fragment, suggesting thata single geneis present. Expression of the complete Rcl subunit coding sequence in Escherichia coli generated a protein of apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 160,000, similar to the size of the polypeptide detected byWestern immunoblot in rabbit skeletalmuscle extracts. The Rcl subunit shares significant homology with the Saccharomyces cerevisiae GACl gene product which is involved in activation of glycogen synthase and glycogen accumulation. The homology with GACl substantiates the role of this enzyme in controlof glycogen metabolism. Hydropathy analysis of the Rc, subunit amino acidsequence revealed the presence of a hydrophobicregion in the COOH terminus,suggesting a potential association with membrane. This result suggests that the same phosphatase regulatory component may be involved in targeting the enzyme bothmemto branes andto glycogen.
Protein phosphorylation is a major mechanism by which many cellular functions are regulated. The co-ordinated con-
* This work was supported in part by National Institutes of Health Grants DK36569 and PO1 HL06308proj.7 and by the Grace M. Foundation. The oligonucleotides used in this study were synthesized in the Biochemistry Biotechnology Facility in our department, partly supported by Indiana University Diabetes Research and Training Center Grant DK20542. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence($reported in this paper has been submitted tothe GenBank““/EMBL Data Bank withaccession numbeds) M65109. $ Partially supported by a postdoctoral fellowship from the American Heart Association, Indiana Affiliate, Inc. Present address: Beckman Research Institute of City of Hope, Division of Immunology, 1450E. Duarte Rd., Duarte, CA 91010-0269. 1 Recipient of Research Career Development Award DK01690.To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Indiana University, School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana 46202-5122.
trol of both types of interconverting enzymes, protein kinases and protein phosphatases, determines the phosphorylation and hence modulation of the activity of intracellular target proteins. Regulation of protein kinases has been extensively studied and for some forms it is reasonably well understood (1-6), whereas knowledge and understanding of the potential control of proteinphosphatases is incomplete. The major cellular serine/threonineproteinphosphatases havebeen classified into two categories, type 1and type 2, according to their substrate specificities and their sensitivities to inhibitor proteins (7, 8). The cDNA coding the catalytic subunits of four forms (type 1, 2A, 2B, and 2C) included in this classification have been cloned (9-16), and with the exception of the 2C protein phosphatase, all appear to be derived from the same gene family. At least four forms of type 1 phosphatases differing in the associated regulatory subunit have been identified ( a ) the ATP-Mg-dependent phosphatase (17-19), ( b ) the glycogen-associated phosphatase (20), (c) the sarcoplasmic reticulum-associated phosphatase (21, 22), and ( d ) the myosin-associated phosphatase (23, 24). There is also evidence for nuclear associated type 1 phosphatases (25, 26). They all appear to share a similar 37-kDa catalytic subunit complexed to different regulatory subunits that are responsible for regulation and/or targeting of the enzymes to specific cellular locales (8,9). These regulatory subunits are ( a ) inhibitor-2, ( b ) glycogen-binding or RG,subunit,’ (c) sarcoplasmic reticulum-binding subunit, and ( d ) a putativemyosin-binding subunit. A recent report hasshown that theproperties of the sarcoplasmic reticulum-associatedphosphatase in rabbit skeletal muscle are similar to those of the glycogen-bound phosphatase, suggesting that the &I subunit might play a dual role in targeting type 1 phosphatase to two different subcellular locations, glycogen and membranes (22). Up to 60% of rabbit skeletal muscle phosphorylase phosphatase is associated with glycogen (27, 28). This glycogenbound protein phosphatase was first identified and purified as a 137-kDa heterodimer, consisting of a 37-kDa catalytic subunit and a103-kDa regulatory component, the &I subunit (20). However, the Rcl subunit is extremely sensitive to proteolysis. More recent analysis by Western immunoblotting The abbreviations used are: Rcl, glycogen-bindingregulatory subunit of type 1protein phosphatase. This protein had previously been designated “G subunit,” causing some confusion with GTP binding proteins. We propose the present nomenclature as a means to avoid this confusion. In addition, other type 1 phosphatase regulatory subunits can be named in a parallel fashion, RSRand R M for ~ the sarcoplasmic reticulum and myosin associated forms, respectively, and RI.* for inhibitor-2. The other abbreviations used are: HX, random hexamer-primed DT, oligo(dT)-primed SDS-PAGE,sodium dodecyl sulfate-polyacrylamide gel electrophoresis; kb, kilobase(s); IPTG, isopropyl-(3-thio-galactopyranoside;TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; EGTA, [ethylenebis(oxyethylenenitri1o)tetraacetic acid.
15782
Protein Phosphatase
15783
the random hexamer-primed library and 160,000 recombinants from the oligo(dT)-primed library. Hybridization of the filters was carried out at55 "C in the solution described above. Phages yielding positive signals were isolated and thecDNA inserts subcloned in pTZ19U for double-stranded DNA sequencing by the dideoxy chain termination method (40) utilizing vector- and cDNA-specific oligonucleotide primers. Isolation of Genomic DNA Clones-A rabbit genomic library constructed in X phage Charon 4A (41), kindly provided by Dr. R. C. Hardison (The Pennsylvania State University), was screened (3.8 X lo5plaque-forming units) with the cDNA fragment (995 bp) obtained from one clone (HX 1-1) and an oligonucleotide corresponding to nucleotides 61-79. One positive clone was identified. The DNA was isolated, digested with restriction enzymes, and analyzed by a Southern blot (42). Fragments hybridizing with the cDNA probe were subcloned into pTZ19U vector for sequencing, utilizing vector- and cDNA-specific oligonucleotide primers. Northern Blot Analysis-Total RNA from rabbit brain, kidney, liver, lung and skeletal, diaphragm, and heart muscle was isolated by the method of Chirgwin et al. (43). RNA samples (15 pg)were electrophoresed through a 0.8%agarose/formaldehyde gel and transferred to nitrocellulose membrane in 10 X SSC (44). Prehybridization was carried out at 55 "C in a solution containing 10 X Denhardt's, 6 X SSPE, 0.05% sodium pyrophosphate, 0.1% SDS, and 0.1 mg/ml Torula RNA. The membrane was hybridized at 55 "C with two 32Plabeled cDNA fragments (2 X lo6 cpm/ml) comprising a total of 3854 bp. After hybridization, the filter was washed two times for 10 min at room temperature followed by 30 min a t 68 "C. Autoradiography was performed utilizing Du Pont Quanta 111 intensifying screens. Primer Extension-A 19-mer synthetic oligonucleotide complementary to residues 61-79 of rabbit Rcl subunit cDNA was used as a primer. The "P-end-labeled oligonucleotide (1.5 X lo6 cpm) was EXPERIMENTALPROEDURES annealed to 25 pg of rabbit skeletal muscle, 30ygof diaphragm or Materials-The bacteriophage T7 polymerase expression system lung total RNA. The extension reaction was carried at 37 "C for 1 h was generously provided by Dr. F. W. Studier (Brookhaven National in 50-pl volume containing first strand buffer (50 mM Tris-HC1, pH Laboratories). Oligonucleotides were synthesized in an Applied Bio- 8.3, 75 mM, KCI, 3 mM MgC12, and 10 mM dithiothreitol), 2.5 mM systems DNA synthesizer model 380A. Restriction and other DNA dNTPs, 500 units of mouse Moloneyleukemia virus reverse transcripmodifying enzymes, M13 vectors, and agarose were purchased from tase (Bethesda Research Laboratories), and625 pg of actinomycin D. Bethesda Research Laboratories. Genescribe-Z vectors and random- The resulting cDNA was analyzed and compared with the sequence primed DNA labeling kits were obtained from United States Bio- of the 6.6-kb SphI genomic DNA fragment on a 6% polyacrylamide chemicals Corp. Deoxy- and dideoxy nucleotide triphosphates were sequencing gel. from Pharmacia LKBBiotechnology Inc. Radionucleotides were purSouthern Analysis of Genomic DNA-Rabbit genomic DNA (20 pg) chased from Du Pont-New England Nuclear, and 1251-proteinA was digested with the restriction enzymes EcoRI, HindIII, and XbaI was from ICN. Other general reagents were from Bethesda Research separated on a 1%agarose gel and transferred to a nitrocellulose Laboratories, Sigma, and Boehringer Mannheim. membrane. Following prehybridization at 68 "C in a solution containIsolation and SequenceDetermination of cDNA Clones-Two rabbit ing 10 X Denhardt's, 6 X SSPE, 0.05% sodium pyrophosphate, 0.1% skeletal muscle random hexamer-primed Xgtll cDNA libraries con- SDS, and 0.1 mg/ml Torula RNA, the membrane was hybridized with taining, respectively, lo6 and 6 X lo6 independent recombinants and the 995-bp cDNA insert of clone HX 1-1labeled by the nick transone rabbit muscle oligo(dT)-primed Xgtll cDNA library containing lation method (1 X lo6 cpm/ml)(45). The membrane waswashed lo7 independent recombinants were constructed (35). To isolate twice for 10 min at room temperature and once for 40 min at 68 "C cDNA clones encoding the Rcl subunit, two oligonucleotide probes in 6 X SSC, 0.1% SDS, and 0.05% sodium pyrophosphate followed were synthesized, corresponding to the amino acid sequences around by a further wash at 68 "C in 2 X SSC for 30 min. the CAMP-dependent protein kinase phosphorylation sites: GConstruction of G.pET-8C Expression Vector-Three cDNA clones, COMP1,GGCGGTGTGCACGTACACCTCCTCandG-COMP2,TT-HX 1-1(995 bp), HX 5-1(1412 bp), and HX 1-2 (2951bp), were used (G/A)AA(G/C)CC(G/A)AA(G/A)TT(G/A)TCGGCGAA. The choice to assemble the entire coding sequence. The 341-bp EcoRI-BglII of the codons was based on the most frequent codon usage for rabbit portion of clone HX 1-1 was ligated with the 1102-bp BglII-EcoRI (36). The two oligonucleotides,5'-end-labeled with T4 polynucleotide portion of clone HX 5-1 a t the BglII site to generate a DNA fragment kinase and [ T - ~ ~ P J A Twere P , used to screen on duplicate filters containing the 5'-most sequences. The resulting DNA fragment was approximately 160,000 recombinants from the rabbit skeletal muscle digested with either RsaI and BglII orwith SphI and BgZlI to produce random hexamer-primed unamplified library. Hybridization was per- a 340-bp RsaI-BglII and a 746-bp BglII-SphI DNA fragment. Two formed at 50 "C in a solution containing 10 X Denhardt's (0.2% (w/ complementary oligonucleotides (18-mer and 14-mer), corresponding v) each of Ficoll, polyvinylpyrrolidone, and gelatin), 6 X SSPE ( 1 X to the sequences from the start ATG to the RsaI site at nucleotide SSPE: 0.15 M sodium chloride, 10 mM sodium phosphate, and 1 mM 28, were synthesized and annealed to generate an adaptor containing EDTA, pH 7.4), 0.05% NaPPi, 0.1% SDS, and theradiolabeled probe a cohesive NcoI site at the5'-end and a RsaI site at the 3'-end. This (2 X lo6 cpm/ml). Nitrocellulose filters were washed in 6 X SSC (1 X adaptor was ligated to the RsaI-BglII which was subsequently joined SSC: 0.15 M sodium chloride and 15 mM sodium citrate, pH 7) at to the BglII-SphI fragment. The resulting NcoI-SphI fragment was room temperature for 10 min twicefollowed by 20 min at 50 "C. further ligated to a SphI-BamHI fragment derived from the HX 1-2 Positive clones were plaque-purified by consecutive screening. DNA clone, giving rise to a 3872-bpNcoI-BamHI fragment, which wasthen from positive recombinant phages was isolated (37) and the cDNA inserted into the PET-8c expression vector (46, 47) cleaved at NcoI inserts were subcloned into the GeneScribe-Z vector, pTZ19U, for and BamHI sites. This plasmid will be referred to as G.pET-8c. The restriction endonuclease analysis and into M13 vectors forDNA nucleotide sequences in the promoter region and the adaptor oligosequencing (38). To obtain the entire coding sequences, confirmed nucleotides were confirmed by sequencing. cDNA fragments labeled by the random hexamer priming method Expression of the RCLSubunit in E. coli-The plasmid G.pET-8c (39) were used to screen additional 310,000 recombinant phages from DNA wastransfected into E. coli strain BL21(DE3), which contained the T7 RNA polymerase gene integrated in the chromosome under V. Cerovsky, K. M. Swiderek, J. H. Haseman, C. J. Fiol, R. W. the control of lacUV5 promoter. Cultures were grown at 37 "C in 2 X Roeske, P. J. Roach, and A. A. DePaoli-Roach, manuscript in prep- YT medium containing ampicillin (50 pg/ml). When the culture aration. reached an AeO0of 0.85 units, the T7 RNA polymerase was induced
indicated thattheintact subunithasa M, of 160,000170,000 (28, 29). Its function appears to be targeting the phosphatase to the glycogen particle, where several of the enzymes, such as glycogen synthase and phosphorylase kinase, involved in glycogen metabolism are located. The &l subunit is phosphorylated in vitro by the CAMP-dependent protein kinase at two sites (29). Phosphorylation of site 2 has been proposed to cause dissociation of the catalytic subunit (30). Studies from our (31) and Cohen's (32) laboratories also revealed a complex multisite phosphorylation of the Rcl subunit. Phosphorylation by CAMP-dependent protein kinase formed the recognition sites for other protein kinases such as glycogen synthase kinase-3 andcasein kinase 11.' The CAMPdependent protein kinase sites and one of the glycogen synthase kinase-3 sites (33) have been shown to be phosphorylated in vivo. Epinephrine is reported to enhance significantly the phosphate content of site 2 (33),whereas insulin leads to increased phosphorylation of site 1 (34). To investigate the native structure and the role of the &I subunit in the regulation of the glycogen-associatedphosphatase we have undertaken the molecular cloning of cDNAs coding for this subunit. This paper reportsthe first isolation and characterization of cDNA clones encoding the Rcl subunit. The deduced amino acid sequence of the entire translated region, the tissue specific distribution, and the expression of the coding sequences in Escherichia coli are presented.
Protein Phosphatase
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3339 Bel11
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FIG, 1. Rcl subunit DNA clones. A partial restriction map and all the clones used for DNAsequence analysisare shown. H X denotes clones isolated from a random hexamer-primed rabbit skeletal muscle Xgtll cDNA library and DT clones obtained from an oligo(dT)primed library. GG10-1 was a clone iso-1- lated froma genomic library. The black barindicatesthe coding region; thick lines indicate regions that were sequencedinboth directions; thin lines indicate regions sequenced in one direction;dashed lines indicate regions not sequenced; and the dotted line indicates a portion of an intron in the genomic DNA clone.
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by adding 0.5 or 1.0mM isopropyl-0-D-thiogalactopyranoside (IPTG), and the cell growthwas continued for 3 h a t 30 "C. Cells were harvested by centrifugation a t 7,000 X g for 15 min and resuspended in 10 volumes of buffer containing 50 mM Tris-HC1, p H 7.5, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM TLCK, 2 mM benzamidine, 10 pg/ml leupeptin, 50 mM 0-mercaptoethanol, and1% Triton X-100. Afterfreezing at -80 "C overnight, the cells were thawed and sonicated for 20 s twice. The lysate was centrifuged at 9,000 X g for 20 min. A sample of 1.4 p1 each of the whole cell lysate and the Triton-soluble fraction were analyzed by SDS-PAGE according to Laemmli (48) and by Western immunoblotting (49). Western Blot Analysis-A synthetic peptide KPGFSPQPSRRGSESSEEVYV surrounding theCAMP-dependent protein kinase phosphorylation site 1 on the Rcl subunit was synthesized and used t o raise antibodies(anti-Rcl)in guinea pigs. The antibodies were affinity-purifiedonSepharose 4B coupled tothepeptide.Rabbit skeletal muscle was homogenized in 3 volumes of 50 mM Tris-HC1, p H 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 0.1 mM TLCK, 10 pg/ml leupeptin and centrifuged for 20 min a t 10,000 X g. A 1-pl sample of the rabbit skeletal muscle solubleextract and samples of E. coli extract prepared as described above were subjected to 7.5% SDS-PAGE. For immunoblotting, gels were equilibrated for 20 min in 20 mM Tris, 190 mM glycine, 20% methanol, sandwiched with nitrocellulose membrane, and subjected to ice-cooled transverse electrophoresis a t 100 V for 2 h. The nitrocellulose filter was blockedovernight a t room temperature in 5% powder milk in PBS-T (20 mM sodium phosphate, p H 7.4, 115 mM sodium chloride, and 0.1% Tween 20) and then incubated for 2 h with anti-Rcl antibody(5 pg/ml in PBS-T). Bound antibodieswere detected by incubating the filterfor 1h in PBS-T containing0.2 &i/ ml of "'1-protein A. After removal of unreacted protein A the filter was subjected to autoradiography. RESULTS
Isolation and Characterization of cDNA and Genomic DNA Clones-The initial screening of the rabbit skeletal muscle random hexamer-primed Xgtll cDNA library identified two positive clones that hybridized with both oligonucleotide probes G-COMP1 and G-COMP2. Nucleotide sequencing confirmed that clone HX 1-1 (995 bp) coded for the available amino acid sequences (50) of the R G ~subunit polypeptide. This labeled cDNAfragment was used to rescreen the original filters and additional 160,000 plaque-forming units. Clones isolated from this screen were used for subsequent screening of another random hexamer-primed and an oligo(dT)-primed cDNA libraries. A total of 630,000 independent recombinants were screened, and 74 positive clones were identified, out of which 23 were characterized. Complete nucleotide sequences were determined from six clones (HX 1-1,1-2, 5-1, 11-1, 13-
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2, and DT 6-1), whereas partial sequences were obtained from other clones including HX 11-2, 15-1, 16-1,18-1, 18-2,18B-1, 21-1, and DT 3-1 (Fig. 1).These overlapping clones provided a combined sequence with an open reading frame of 3317 nucleotides and 537 nucleotides downstream of a stop codon. However, no in-frame ATG codon upstream of the known amino acid sequence was found. To search for the translational start site, the HX 1-1cDNA (995 bp), containing the most 5"sequences and a synthetic oligonucleotide from nucleotide 61 to 79, were used to screen a rabbitgenomic library. Southern blot analysis of the single positive clone identified a 6.6-kb SphI genomic fragment which hybridized with the 995-bp cDNA fragment. Sequence analysis revealed that 108 bp of an intron were present at the 3'-end of the SphI fragment, which continued upstream in the coding region at nucleotide 799. By utilizing as primer an oligonucleotide corresponding to nucleotide 61-79, sequences were extended at the 5'-end by 291 bp. In this region were several stop codons in all reading frames and no intronlexon boundary consensus sequence (51). Only two ATGcodons were found, both in the same reading frame. The first was followed by an in-frame stop codon and the second was located 10 bp upstream from the cDNA 5'-end. The GCCCAATGG sequence around the second ATG is in reasonable agreement with the Kozak's consensus sequence (52). Thus,it seems likely that this ATG is the translational initiation codon. The COOH terminus is defined by a TAA codon which is followed by several other stop codons in all reading frames. No polyadenylation signal was found in the 537 bp of the 3"untranslated region. Sequence data from all the clones establish an open reading frame of 3327 bases (Fig. 2) encoding a proteinof 1109 amino acids with a M , of 124,257. The amino acid sequences of six peptides obtained from purified rabbit skeletal muscle RGI subunit, provided by Dr. Philip Cohen (University of Dundee, Dundee, Scotland), were present in the deduced sequences (Fig. 2) with only two mismatches, cysteine residues at positions 26 and 183 for tyrosine and serine, respectively. Hydropathy analysis by the method of Rao and Argos (53) indicated a region, at the COOH terminus between residues 1063 ando 1097, rich in hydrophobic residues and which predicts a transmembrane helix. Examination of the nucleotide sequences of all the cDNA clones characterized indicated the existence of two groups, clones HX 13-2, HX 1-2,HX 11-2, and HX 16-1in one group and clones HX 1-1,HX 11-1, HX 5-1, and HX 15-1 in the
Protein Phosphatase
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CCAGAGAGCCCAATGGAGCCTTCTGMGTACCTGGTCAGAACAGCAAAGATMTTTTTTAGMGTTCCTMTTTGTCTGATTCTCTTTGTGMGATGMGMGTTMAGCTATTTTCMA 120 M
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S
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L
D
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L
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A
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876
GAAAATAGAGATCTTGGGCAGGTTCMGMTTATCAMGAAAACCGACATAGATAACACTGTTCACTCTGCCTTTMCTCAGACACGMCAGAGCTTCTCGGGATGACTCTCTTCTTTCC 2760
E
N
R
D
L
G
Q
V
Q
E
L
S
K
K
T
D
I
D
N
T
V
H
S
A
F
N
S
D
T
N
R
A
S
R
D
D
S
L
L
S
916 2880 S H H T E T S V L S C E Q A N A V K N T V T T T A L O T S A T E S E Y N C S P T 956 AGGGAAACCCAGGGTCMCCTGCATCTAAACCTGMGAAGTTTCCAGAGGTTCMGMGAGTGACATCAGAGACTAGGAAAGA~ATGCGTAGGCCAGATGTTCCAGTCAGGAGMTGC 3000 R E T Q G Q P A S K P E E V S R G S R R V T S E T R K E K C V G Q M F Q S G E C 996 MTGTGGAAATGTCGCMGGGCCAATGATTTTAGTCAGTGAATCTCGTGAGMCGTAGAMGGGAAAGGCATGAAAATGAAMTGAAGGACTGATTAACTCAGGTGACAAGGAATTTGAGAGCTCT 3120 N V E M S Q G P M I L V S E S R E N V E R E R H E N E G L I N S G D K E F E S S l O 3 6 GCTTCTTCTAGTCTACCTGTGCAGGAMCTCAAGATCAAAGCAATGAATCTCTTCTTTCAAMTACACCMCTCT~TACCTTATTTCCTTTTGTTTCTGATGTTTCTTGTAACCGTC 3240 A S S S L P V Q E T Q D Q S N E S L L S K Y T N S K I _ P Y F L L F J , " ~ J L V _ T - ~ 1 0 7 6 TACCACTATGACCTCATGATAGGCTTGGCATTCTACCTTTTCTCATTGTATTGGCTATACTGGGMGAGGGCAGACAAAMGAGTCTGTCA~GMGTMCCTCAGCACTATTATTAT 3360 1109 ~ H Y D L M I G L A F Y L F S L Y W L Y W E E G R Q K E S V K K K ~ TAAAAGATMGCTGTTTAGCTCCAMCATTTGGATTGGTGMGGAGACTATTCATTGTTCAAAGAGCCAGTGCAGTTTTTCTCTTGAAGGATCATTTMAAAGGAATGCCTATGMGTTT3480 GCTCCTTCATATMGTMTTATTCTATATAGGACCATTATGTTTGGATCATTAAATACCTATATGMTATGAGATCTGAAGCACGTCMGTTGMATTAGGTACAGCTGTTGCTCCTTAG 3600 CAGGCTATGAAGTTGCMTGCTTCACGTCTCTTCACTACTT~GTGCTATTTCTTGTGTTCATTTCTTTTGCAGTAAAGCTTCATTTTTTTCCCCCTGAGCACATCTTTCCCTCTATGG 3720 TTTTTAAAMTAGATAMACATGGACMTGGCAGMGATTTTCTTCCTTTTTTTTTGTCTTTTAGGATTGACMTGAAATTTTCATCTACCACTGTATCATTTATTAGCACATAATGATA 3840 GATCAACTATTTCAACTCATATTTCATAGTTTTAAG 3876 AGTCACCATACAGAAACTTCAGTGCTGTCTTGTGMCAGGCAMTGCTGTAMGAATACAGTTACTACCACTGCTCTCCAGACTTCTGCTACTGAGTCAGMTATMTTGTAGTCCMCA
FIG. 2. Nucleotide and deduced amino acid sequences of rabbit skeletal muscle Rcl subunit. The complete sequence was obtained by combining all the clones shown in Fig, 1. A single underline indicates peptide sequences derived from the RGIsubunit polypeptide and a double underline the amino acid sequences used for synthesis of the oligonucleotide probes employed in the screening. The dotted line indicates the region rich in hydrophobic residues.
second. The two groups differed in six nucleotides, at positions 703, 744,1176,1302,944, and 1251. The last two caused changes in amino acid sequences at residue 311 from threonine to methionine and at residue 413 from asparagine to lysine, respectively. The other four differences were silent. Most likely this discrepancy is due to allelic variations. Determination of the Transcriptional Start Site-Primer extension analysis was carried out in order to map the 5'-end of therabbitRclsubunittranscript. Utilizing total RNA prepared from rabbit skeletal muscle, a major transcriptional start point at a C located 12 nucleotides upstream from the translational start codon ATG was identified (Fig. 3). This putative cap site was observed also in RNA prepared from rabbit diaphragm and cardiac muscle but not from lung (data
not shown) in which no homologous RGl subunit appears to be expressed. Tissue Distribution of the Rcl Subunit mRNA and Southern Analysis of Genomic DNA-The tissue distribution and the complexity of the Rcl subunit mRNA was investigated by Northern analyses. Total RNA from various rabbit tissues was hybridized with the labeled cDNA inserts from clone HX 1-1(995 bp) and from clone HX 1-2 (2.95 bp) as probes. A major hybridizing mRNA species at 7.5 kb was observed in skeletal, diaphragm, and cardiac muscle (Fig. 4). Minor species of approximately 3.5 kb were also observed at low stringency. The level of the mRNA appeared to be higher in skeletal than in cardiac muscle. However,none of the mRNA species was present in brain, kidney, liver, and lung, although staining of the gel with ethidium bromide indicated that
F
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Protein Phosphatase
15786 A
C
G
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T -
RSM
5’ 3’
T
A
G
C
A T A
T A T
b c C
G G
A
T
G
C
A
T
G
C
A
T
G
C
A G
T C
kb
-
23.1 12.1 9.4
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T
A
3 ‘ 5’
FIG.3. Mapping of the 5’-end of Rcl subunit mRNA. Primer extension analysis was carried out asdescribed under “Experimental Procedures.” TheprimerextendedDNAfragmentobtained from rabbit skeletal muscle mRNA ( R S M )and the corresponding sequence of the 6.6-kb SphI genomic DNA fragment are presented. Thearrow at the right indicates the position of the majorlabeled extended product and thearrow at the left the transcriptional startsite.
-6.6 5.1 4.13.1 2.0 1.6 1.0 0.5
-
1
2 3 4
FIG.5. Southern blot analysis of rabbit genomic DNA. Rabbit genomic DNA (20 pgllane) digested by the indicated restriction endonucleases was electrophoresed in an agarose gel, transferred to nitrocellulose membrane,and probedwith the ‘lP-labeled 995-bp cDNA fragment of clone HX 1-1. Numbers indicate the size, in kilobases. of markers.
comparable amounts of RNA were loaded in each track. Southern blot analysis of rabbit genomic DNA digested with different restriction enzymes and probed with the 995bp cDNA fragment of clone HX 1-1 gave rise to a single hybridizing band (Fig. 5), suggesting the presence of only one gene. Expression of Recombinant Rc, Subunit in E. coliCellsThe structure of the polypeptide encoded by the composite 9.49 cDNA was examined by expression in E. coli. Construction of 7.46 the expression vector G.pET-8c as described under “Experimental Procedures” is illustrated inFig. 6. E. coli BL21(DE3) 4-40 cells transfected with the G.pET-8c plasmid were grown and lysed as described under “ExperimentalProcedures.” Analysis of the cell extracts by Western immunoblottingindicated the 2.37 presence of three major immunoreactive polypeptides, one of which had an apparentM , of approximately 160,000, similar 1.35 to that observed in rabbit skeletal muscle extracts (Fig. 7). Similar results were also obtained when the E. coli cells were directly treated with 0.5% SDS at 100 “C for 5 min, before Western analysis (not shown). The amount of immunoreactivematerial increased with increasing concentrations of IPTG from 0.5 to 1 mM (Fig. 7, panel B: lunes 2 , 3 , 5 , and 6 ) and was absent in extractsfrom untransfected cells (lunes 7.024 9).The slight amountof polypeptides detected in extracts of FIG.4. Northern blot analysis of RGl subunit mRNA. Total G.pET-8c-harboring cells not induced by IPTG is attributed RNA (15 &lane) prepared from the indicated rabbit tissues was to the basal T7 RNA polymerase activity. Increasing time electrophoresed through an agarose gel under denaturing conditions (data not shown) and induction by IPTG appeared generate to andtransferredto anitrocellulosemembrane.Hybridizationwas carried out as described under “Experimental Procedures.” Numbers proportionally more of the lower molecular weight species. When the cells were lysed in buffer without 1%Triton Xindicate the size of molecular markers in kilobases. kb
Q Y J r c , n I
--
-
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-
Phosphatase
Protein
15787
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B
kD8
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11694-
E
67-
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1
1
2
3
4
5
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FIG.7. Expression of RCilsubunit. E , ro/i strain F 3 1 2 1 ~ D F 3 ~ carrying the (;.pE’l‘-Hc plasmid were grown. induced with II’TG, and processed for gel electrophoresis and li’estern hlnt as descril)etlunder “Experimental Procedures.” I’anrl A , rahhit skeletal muscle extract. P o n d H. whole lysate (lnnrs 1 - 3 ) and Triton X-lf~O-soluhlefraction (lanes 4 - 6 ) from b;. coli transfected with G.pF:T-Rc plasmid and whole lysate (lnnrs 7-9) of cells transfected with control plasmid p F T - H c . Imnrs I , 4 , and 7. cells not induced with IPTG; /ones 2. 5 , and 8.cells induced with 0.5 mM II’TG: antl /nnrs 3, 6 . antl 9. cells intfuced w i t h 1.0 mM II’TG. The numbers at t h e sidrs indicate the apparent molecular mass in kiloclaltons (kI)a) of protein standards separatc.d on t h e same gel. The arrowhcods show t h e position of t hP native ItS-I’AGF:. in 15% glycerolor on agar plates significantly lost their ability However, despite sharing these properties. the three proteins to express immunoreactive polypeptides. show no resernhlance in sequence. DISCUSSION T h e lower molecular weight polypeptides, 58 and 46 kDa of E . coli transfected with Wereporttheisolationandcharacterization of cDNAs (Fig.7), detected in extracts suhunit cDNA, confirm the extreme sensitivitv of the polvencoding the regulatory (E;,) suhunit of the rahhit skeletal peptide to proteolysis. The observation that the same species muscle glycogen-associated type 1 protein phosphatase. Sehy 100 “ C quencing of 23 cDNA clones failed to provide a translational are also present when the cells are disrupted directly of SIX (datanotshown) ATG start codon, which was obtained from the isolation of a heattreatmentinthepresence suggests that degradation might he occurring inside the cell rabbitgenomicclone.Primerextensionanalysisindicated of the extracts. Such an occurrence that the 5’-untranslated region is very short, 12 nucleotides. and not during processing The procedure of Gubler and Hoffman (54), which can lead is not unusual and has heen reported for other mammalian t o t h eloss of the first20-30 nucleotides, was employed in the polypeptides expressed in hacteria (.59, 60). RiIsuhunit degradation products of similar molecular weight hnve heen ohconstruction of our cDNA libraries and could explain the of rahhitskeletalmuscleglvcogenabsence of the initiation codon in the cDNA clones isolated. servedinpreparations associated phosphatase (27, 61),whichmightindicatethat Combined overlapping nucleotide sequences established an open reading frame coding for 1109 amino acids with a M , specific regions of t h e pol-ypeptide are especiallv sensitive to
15788
Protein Phosphatase
proteolytic cleavage. We can exclude that the58- and 46-kDa forms are generated by initiation of translation at a downstream ATG, since the antibodies used for the detection were raised to theregion corresponding to residues 37 to 56 and no other ATGs are present in thisNHz-terminal region. Recently Hubbard et al. (22) reported that a sarcoplasmic reticulum-associatedphosphatase contains a polypeptide similar, if not identical, to theRG,subunit. Hydrophathy analysis of the deduced amino acid sequencereported here indicated a potential transmembrane region between residues 1063 and 1097 (Fig. 2), which could be responsible for anchoring the protein to the membrane, Thus, the same regulatory subunit might function to target the phosphatase to membranes and glycogen. Several lines of evidence argue against the existence of distinct muscle isoforms. The low molecular weight polypeptides observed in glycogen-associatedphosphatase purified from rabbit skeletal muscle are notalways detected by Western immunoblotting analysis (Fig. 7) and Northern hybridization, utilizing 3,850 bp of cDNA sequence, indicated one major mRNA species, The minor differences observed in the two groups of cDNA clones, four silent nucleotide substitutions and two changing the amino acid residues couldbe explained by allelic variations. Southern analysis also suggested a single gene.Studies in progress, with mutant protein in which the hydrophobic region has been deleted, should prove useful in addressing the question of how the same polypeptide might be directed to different cellular compartments. Analysis of the tissue distribution of the RG,subunit mRNA (Fig. 4) supports previous observations by Western immunoblot (28); which indicated that thepolypeptide is specifically expressed in skeletal and cardiac muscle, but not in other tissues examined. This also suggests that the polypeptide responsible for targeting the phosphatase to glycogen in liver is not homologous to the muscle form, although their molecular weights appear to be very similar and both interactwith a highly conservedcatalytic subunit (62). The rabbit skeletal muscle glycogen-associatedphosphatase undergoes in vivo and in vitro phosphorylation at several sites all of which are located near the NH2 terminus. The CAMPdependent protein kinase sites are at residues 48 and 67, respectively, and the glycogen synthase kinase-3 sites at positions 40 and 44. Other potential recognition sites for CAMPdependent protein kinase are threonine 498 and 978 and serine 636. Since purification of intact R c l subunit has been difficult and some ofthe site identifications have beencarried out on proteolyzed species (50),it is possible that additional phosphorylation sites might have been missed.Threonine 978, in the motif -Arg-Arg-Val-Thr-, is an especially strong candidate. Phosphorylation of this residue, similarly to serine 48, could form a recognition site for glycogen synthase kinase-3 (31). Search of the Swiss protein (release 13) and the EMBL (release 21) data bases with PCGENE utilizing the FASTN and FASTP programs (63) revealed no significant homology between the Rc, subunit and other known sequences. However, a search of the protein data base assembled by Dr. Mark Goebl, at Indiana University, indicated significant homology with the product of the yeast gene, GACl, isolated by Dr. Kelly Tatchell at North Carolina State Uni~ersity.~ Over a segment of 144 residues,the identity is 27% and thehomology
'K. M. Swiderek and A. A. DePaoli-Roach, unpublished results. K. Tatchell, personal communication.
38% if conservative replacements are taken into a c c ~ u n t . ~ Utilizing the algorithm of Lipman and Pearson (63), the optimal alignment score between R G 1 and GACl amino acid sequence is 13 standard deviations over the mean of the optimal score of 100 random shufflings of the GAG1 sequence. It is of significance that thesimilarity lies within the 40-kDa NHz-terminal portion of the protein, whichisable to interact with glycogen and with the catalytic subunit of type 1phosphatase (61). The GACl protein appears to be involved in activation of glycogen synthase and glycogen accumulation. Both of these functions are consistent with the GACl gene product being the yeast homologue of the R G subunit. ~ Gene replacement in yeast should allow us to address this question. In addition site specific and deletion mutagenesis will provide a powerful tool to elucidate the physiological role and regulation of the glycogen-associated protein phosphatase. Acknoufedgments-We are grateful to Dr. P. Cohen for making available amino acid sequences from RGI subunitpeptides, to Dr. F. W. Studier for the generous gift of the T7 RNA polymerase expression system, to Drs. R. C. Hardison and T. Maniatis for the rabbit genomic library, to Dr. K. Tatchell for allowing comparative analysis with the GACl gene product, and toDr. M. Goeblfor carrying out the analysis. We also thank Dr. P. J. Roach for his advice and criticism in t,he preparation of the manuscript.
REFERENCES I. Edelman, A. M.,Blumenthal, D. K., and Krebs, E. G. (1987) Annu. Rev. Biochem. 56,567-613 2. Hunter, T. (1987) Cell 50,823-829 3. Stull, J. T., Nunnally, M. H., and Michnoff, C. H. (1986)in The Enzymes (Boyer, P. D.) 3rd Ed., Voi. 17, pp. 114-166, Academic Press, Orlando, FL 4. Taylor, S. S. (1989) J. Biol. Chem. 264,8443-8446 5. Soderling, T. R. (1990) J. Biol. Chem. 265,1823-1826 6. Kemp, B. E., and Pearson, R. B. (1990) Trends Biochem. Sei. 177,342-346 7. Ingebritsen, T. S., and Cohen, P. (1983) Science 221,331-338 8. Cohen, P. (1989) Annu. Rev. Biochem. 58,453-508 9. Cohen, P., and Cohen, P. T. W. (1989) J . Biol. Chem. 2 6 4 , 21435-21438 10. Berndt, N., Campbell, D. G., Caudwell,F. B., Cohen, P., da Cruz e Silva, E. F., da Cruz e Silva, 0. B., and Cohen, P. T. W. (1987) FEBS Lett. 223,340-346 11. Bai, G., Zhang, Z., Amin, J., Deans-Zirattu, S. A., and Lee, E. Y. C. (1988) FASEB J. 2,3010-3016 12. Arino, J., Woon, C. W., Brautigan, D. L., Miller, Y. B. Jr., and Johnson, G. L. (1988) Proc. Natl. Acad.Sci. U. S. A. 85,42524256 13. Green, D. D., Yang, %-I., and Mumby, M. C. (1987) Proc. Natl. Acad. Sci. U. S. A . 84,4880-4884 14. Da Cruz e Silva, 0. B., Alemany, S., Campbell, D. G., and Cohen, P. T. W. (1987) FEBS Lett. 221,415-422 15. Kincaid, R. L., Nightingale, M. S., and Martin, B. M. (1988) Proc. Natl. Acad. Sci. U.S. A. 85,8983-8987 16. Tamura, S., Lynch, K. R., Larner, J., Fox, J., Yasui, A., Kikuchi, K., Suzuki, Y., and Tsuiki, S. (1989) Proc.Natl.Acad.Sci. U. S. A . 86, 1796-1800 17. Yang, S. D., Vandenheede, J. R., Goris, J., and Merievede, W. (1980) J . Bwl. Chem. 2 5 5 , 11759-11767 18. Ballou, L. M., Brautigan, D. L., andFisher, E. H. (1983) Biochemistry 22,3393-3399 19. DePaoli-Roach, A. A. (1985) Adu. Prot. Phosphatases 1,59- 72 20. Stralfors, P.. Hirana. A.,. and Cohen, P. (1985) Eur. J. Biochem. 149,295-303 21. Villa-Moruzzi. E.. and Heilmever, - . L. M. G..~.Jr. (1987) Eur. J . Bwchem. 169,659-667 22. Hubbard, M. J., Dent, P., Smythe, C., and Cohen, P. (1990) Eur. J. Biochem. 189,243-249 23. Chisholm, A. A. K., and Cohen, P. (1988) Biochim. Biophys. Acta 968,392-400 I
.
The following groupings of amino acids were considered homologous: Val-Ile-Leu; Phe-Tyr;Ala-Ser-Thr; Asp-Glu;Asn-Gin;ArgLys.
Phosphatase
Protein
24. Schlender, K. K., Wang, W., and Wilson, S. E. (1989) Biochem. Biophys. Res. Commun. 159, 72-78 25. Kuret, J., Bell, H., and Cohen, P. (1986) FEBS Lett. 203, 197202 26. Jakes, S., Merlgren, R. L., and Schlender, K. K. (1986) Biochim. Biophys. Acta 888, 135-142 27. Hiraga, A,, and Cohen, P. (1986) Eur. J. Biochm. 161, 763-769 28. DePaoli-Roach, A. A. (1989) Adu. Protein Phosphatases 5, 479500 29. Hubbard, M. J., and Cohen, P. (1989) Eur. J. Biochem. 180, 457-465 30. Hubbard,M. J., and Cohen, P. (1989) Eur. J. Biochem. 186, 701-709 31. Fiol, C. J., Haseman, J. H., Wang, Y., Roach, P. J., Roeske, R. W., Kowalczuk, M., and DePaoli-Roach, A. A. (1988) Arch. Biochem. Biophys. 267, 797-802 32. Dent, P., Campbell, D. G., Hubbard, M. J., and Cohen, P. (1989) FEBS Lett. 248,67-72 33. Dent, P., Campbell, D. G., Caudwell, F. B., and Cohen, P. (1990) FEBS Lett. 259,281-285 34. Dent, P., Lavoinne, A., Nakielny, S., Caudwell, F. B., Watt, P., and Cohen, P. (1990) Nature 348, 302-307 35. Zhang, W., Browner, M. F., Fletterick, R. J., DePaoli-Roach, A. A., and Roach, P. J. (1989) FASEB J. 3, 2532-2536 36. Maruyama, T., Gojobori, T., Aota, A.-I., and Ikemura, T. (1986) Nucleic Acids Res. 14, (suppl.) 151-197 37. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning:A laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 38. Messing, J. (1983) Methods Enzymol. 101, 20-78 39. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem 132, 61. 6-13 40. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463-5467 41. Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O’Connel, C., and Quon, D. (1978) Cell 15, 687-701 42. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517
15789 43. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 44. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 52015205 45. Rigby, P. W., Dieckmann, M., Rhodes, C., and Berg, P. (1977) J . Mol. Biol. 113, 237-251 46. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113130 47. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89 48. Laemmli, U. K. (1970) Nature 227, 680-685 49. Burnette, W. N. (1981) Anal. Biochem. 112, 195-203 50. Caudwell, F. B., Hiraga, P.Cohen, A,, and (1986) FEBS Lett. 194,85-90 51. Nevins, J. R. (1983) Annu. Reu. Biochem. 52,441-466 52. Kozak, M. (1987) J . Mol. Biol. 196, 947-950 53. Rao, M. J. K., and Argos, P. (1986) Biochim. Biophys. Acta 869, 197-214 54. Gubler, U., and Hoffman, B. J. (1983) Gene (Amst.)25, 263-269 55. Nimmo, G. A., and Cohen, P. (1978) Eur. J. Biochem. 87, 341351 56. Aitken, A., Bilham, T., and Cohen, P. (1982) Eur. J . Biochem. 126,235-246 57. Holmes, C. F. B., Campbell, D. G., Caudwell, F. B., Aitken, A., and Cohen, P. (1986) Eur. J. Biochem. 155, 173-182 58. Foulkes, J. G., and Cohen, P. (1980) Eur. J . Biochem. 105, 195203 59. Goeddel, D. V., Heyneker, H. L., Hozumi, T., Arentzen, R., Itakura, K., Yansura, D. G., Ross, M. J., Mizzari, G., Crea, R., and Seeburg, P. H. (1979) Nature 281, 544-548 60. Goeddel, D. V. (1990) Methods Enzymol. 185, 3-7 Hiraga, A., Kemp, B. E., and Cohen, P. (1987) Eur. J. Biochem. 163,253-258 62. Wera, S., Bollen, M., and Stalmans, W. (1991) J. Biol. Chem. 266,339-345 63. Lipman, D. J., and Pearson, W. R. (1985) Science 227, 14351441
