Phosphorylation-dependent Inhibition of Protein Phosphatase-1 by G-substrate
A PURKINJE CELL SUBSTRATE OF THE CYCLIC GMP-DEPENDENT PROTEIN KINASE*

Kelly Umstott HallDagger , Sean P. Collins§, David M. GammDagger , Enrique Massaparallel , Anna A. DePaoli-Roach**, and Michael D. UhlerDagger §Dagger Dagger

From the Dagger  Mental Health Research Institute, the § Department of Biological Chemistry, and the  Neuroscience Program, University of Michigan, Ann Arbor, Michigan 48109 and the ** Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202

    ABSTRACT
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Abstract
Introduction
References

G-substrate, a specific substrate of the cGMP-dependent protein kinase, has previously been localized to the Purkinje cells of the cerebellum. We report here the isolation from mouse brain of a cDNA encoding G-substrate. This cDNA was used to localize G-substrate mRNA expression, as well as to produce recombinant protein for the characterization of G-substrate phosphatase inhibitory activity. Brain and eye were the only tissues in which a G-substrate transcript was detected. Within the brain, G-substrate transcripts were restricted almost entirely to the Purkinje cells of the cerebellum, although transcripts were also detected at low levels in the paraventricular region of the hypothalamus and the pons/medulla. Like the native protein, the recombinant protein was preferentially phosphorylated by cGMP-dependent protein kinase (Km = 0.2 µM) over cAMP-dependent protein kinase (Km = 2.0 µM). Phospho-G-substrate inhibited the catalytic subunit of native protein phosphatase-1 with an IC50 of 131 ± 27 nM. Dephospho-G-substrate was not found to be inhibitory. Both dephospho- and phospho-G-substrate were weak inhibitors of native protein phosphatase-2A1, which dephosphorylated G-substrate 20 times faster than the catalytic subunit of protein phosphatase-1. G-substrate potentiated the action of cAMP-dependent protein kinase on a cAMP-regulated luciferase reporter construct, consistent with an inhibition of cellular phosphatases in vivo. These results provide the first demonstration that G-substrate inhibits protein phosphatase-1 and suggest a novel mechanism by which cGMP-dependent protein kinase I can regulate the activity of the type 1 protein phosphatases.

    INTRODUCTION
Top
Abstract
Introduction
References

Cyclic GMP acts as a second messenger in numerous physiological processes including smooth muscle relaxation, visual transduction, olfaction, neurotransmitter release, and long term depression of cerebellar Purkinje cells (1-3). cGMP mediates its effects by binding to and phosphorylating intracellular receptor proteins, such as ion channels, phosphodiesterases, and the cGMP-dependent protein kinases (cGKs).1 Three isoforms of the cGK protein have been well characterized, but elucidation of cGMP-cGK signaling cascades has been hampered by an overall similarity in the substrate recognition by cGK and cAMP-dependent protein kinase (cAK), as well as the existence of cross-activation of these kinases in vivo (3).

G-substrate was identified in the cytosol of rabbit cerebellum as the first soluble protein whose phosphorylation was stimulated specifically by cGMP and not cAMP (4). The subsequent purification and characterization of G-substrate confirmed its specificity as a substrate for cGK and found it to be a heat-stable, acid-soluble protein of low molecular weight (5). G-substrate is phosphorylated at two threonine residues located in nearly identical sites, as determined by sequencing of tryptic peptides (6). Antibodies raised against G-substrate localized G-substrate within the brain to the cerebellum by radioimmunoassay (7). Examination of mutant mice lacking certain neurons revealed the enrichment of G-substrate in cerebellar Purkinje cells, a cell type where cGK I is also found in great abundance (8).

The first suggestion as to the function of G-substrate arose from its similarity to the protein phosphatase inhibitor-1 (9). G-substrate shares a number of characteristics in common with inhibitor-1 and DARPP-32 (dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein-32,000), two specific inhibitors of the catalytic subunit of the type I protein phosphatases (PP1c) (10, 11). These common characteristics include low molecular weight, heat stability, acid solubility, a low content of hydrophobic residues, and a predicted elongated tertiary structure. In addition, inhibitor-1 and DARPP-32, both of which require phosphorylation by cAK to exhibit inhibitory activity, have phosphorylation sites with sequence homology to the phosphorylation sites of G-substrate (12, 13). Given these similarities, it has been suggested that G-substrate functions as an inhibitor of PP1c following its phosphorylation by cGK (6, 8, 10, 14).

More detailed studies of G-substrate phosphorylation and expression have been limited by the lack of complete amino acid sequence information. In this study, we report the isolation from mouse brain of a cDNA encoding murine G-substrate. Using this cDNA, we have examined G-substrate mRNA expression, as well as the interaction of recombinant G-substrate with several protein kinases and protein phosphatases.

    MATERIALS AND METHODS

Generation and Sequencing of Amplification Products-- Total RNA was isolated from mouse brain as described (15), and poly(A) RNA was purified by oligo(dT)-cellulose (16). First strand cDNA was synthesized from poly(A) RNA by reverse transcription in a mixture containing 50 mM Tris (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 0.5 mM each dATP, dCTP, dGTP, and dTTP, 50 µg/ml random hexamer primers, 40 µg/ml poly(A) RNA, and 400 units of Moloney murine leukemia virus reverse transcriptase. The reaction was incubated at 37 °C for 1 h and then diluted to 1 ml. RNase A was added to 20 µg/ml, and the mixture was incubated at 37 °C for 10 min, after which phenol/chloroform extraction and ethanol precipitation were performed. The first strand cDNA was then diluted to 100 ng/µl and used for amplification. The following oligonucleotide primers were synthesized at the University of Michigan Biomedical Research Core Facilities: GGAGATCT AAT GTG GAG TC(A/C/T) GA(C/T) CA(A/G) AA(A/G) AA(A/G) CC and CT(C/T) GT(C/T) TT(C/T) TT(C/T) GG(A/C) (G/T)C(A/C) GCC TTC CTCTTAAGGG. Amplification reactions (100 µl) contained 100 ng of first strand cDNA, 20 µM each oligonucleotide, 50 mM KCl, 10 mM Tris (pH 8.4), bovine serum albumin (10 µg/ml), 200 µM each dATP, dCTP, dGTP, and dTTP, and 5 units of Taq DNA polymerase (Life Technologies, Inc.). The amplification reactions were denatured for 30 s at 95 °C, annealed for 1 min at 45 °C, and extended for 3 min at 72 °C with 40 rounds of amplification in a model PTC-100 thermocycler (MJ Research, Inc.). Products of amplification were analyzed on a 3% NuSieve, 1% agarose gel and then isolated using phenol extraction as described (17). The isolated fragment was digested with BglII and EcoRI and ligated into BamHI/EcoRI-digested pGEM-3Zf(+). The resulting plasmid (pGSUBPCR) was denatured and sequenced with SP6 and T7 promoter primers using Sequenase DNA polymerase (Amersham Pharmacia Biotech).

Screening of a Mouse Brain cDNA Library and cDNA Sequence Analysis-- Approximately 1.2 × 106 recombinant lambda ZapII phage containing mouse brain cDNA fragments were screened by hybridization essentially as described (18). The HindIII/EcoRI fragment from pGSUBPCR was isolated and labeled by random primer extension with [alpha -32P]dATP (ICN Biomedicals). The resulting radiolabeled DNA fragment was hybridized to phage DNA immobilized on Hybond N at 37 °C in 50% formamide, 0.75 M NaCl, 20 mM NaPO4, 10 mM Hepes (pH 7.2), 5 mM EDTA, 100 µg/ml herring sperm DNA, and 0.1% each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll. Filters were washed at 60 °C in 0.5% SDS, 10 mM Tris (pH 7.4), and 1 mM EDTA (0.1× SET; where SET is SET, 5% SDS, 100 mM Tris (pH 7.4), 1 mM EDTA). The first screening of the library resulted in the isolation of a single clone (Fig. 1A, Clone 1), which was fully sequenced on both strands. The presence of a sequence of C-T repeats upstream of the open reading frame prompted a second library screening, which led to the isolation of three additional independent clones (Fig. 1A). Clones 2-4 were restriction mapped to confirm the identity of the open reading frame and sequenced at their 5' and 3' ends.

Identification of Homologous Sequences-- The nucleotide and the predicted amino acid sequences from G-substrate clone 1 were used to search the expressed sequence tag (EST) data base for homologous sequences using the basic local alignment search tool algorithm (19). An homologous sequence from human infant brain was identified. This integrated molecular analysis of genomes and their expression consortium (LLNL) cDNA clone (integrated molecular analysis of genomes and their expression consortium clone 46041/GenBankTM accession H09006) was obtained from Research Genetics, Inc., and sequenced in both directions. Alignment of predicted amino acid sequences was performed with DNASTAR software.

RNase Protection Analysis-- Total RNA was isolated from mouse brain and dissected mouse brain regions as described (15). The polymerase chain reaction (PCR) was performed with oligonucleotides 5' AGA TCT ACC CAG GAG GAA AGA CAC 3' and 5' AAG CTT GCA ACA AAG GGA GGC ATG 3' with G-substrate clone 1 (pGSUB-1) as a template, resulting in the amplification of a 201-bp fragment from the open reading frame of G-substrate flanked by a BglII site and a HindIII site. This amplified fragment was digested with BglII and HindIII, isolated, and ligated into the BglII/HindIII site of pSP73 to create pSP73.GRP, which was sequenced to confirm the coding region sequence and linearized with BglII. Linearized pSP73.GRP was used as a template to synthesize a 235-bp [alpha -32P]UTP-radiolabeled riboprobe using T7 RNA polymerase. Sense RNA was generated by SP6 polymerase using HindIII-digested pSP73.GRP as a template. RNase protection analysis was then performed as described (20).

Northern Blotting-- A plasmid containing the coding region of pGSUB-1 was generated by PCR (see below) and linearized with SacI. The linearized template was used with T7 RNA polymerase to generate a radiolabeled antisense probe as described (21). The resulting cRNA probe was hybridized with a mouse multiple tissue Northern blot (CLONTECH) at 60 °C for 16 h. Following hybridization, the blot was washed for 2 h at 70 °C in 0.5× SET containing 0.1% sodium pyrophosphate, dried, and autoradiographed.

In Situ Hybridization Histochemistry-- Mouse brains were removed, frozen, cut into 15-µm sections, and processed for in situ hybridization histochemistry as described (21). Antisense and sense cRNA probes to G-substrate were synthesized in the presence of [35S]UTP (Amersham Pharmacia Biotech) by SP6 or T7 RNA polymerase, using linearized pGSUB-1 as a template, according to the manufacturer's instructions. The cRNA probe was diluted to 2 × 106 dpm per 30 µl of hybridization buffer, and sections were hybridized at 54 °C for 16-20 h. Following hybridization, sections were treated with RNase A, washed in 75 mM NaCl, 7.5 mM sodium citrate, pH 7.0 (0.5× SSC; where 1× SSC is 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0), at 58 °C for 60 min, and dipped in Kodak NTB-2 nuclear emulsion (3-week exposure).

Construction of the G-substrate Mammalian Expression Vector, Transient Transfection of COS-1 Cells, and Protein Purification-- To simplify purification, an extension of six histidine residues was added to the amino terminus of G-substrate. pGSUB-1 plasmid DNA was used as a template, and the primers GGGAGATCTCCACC ATG CAC CAC CAT CAC CAT CAC ATG GCC ACT GAG ATG ATG AC and CTA CTA AAC CAG GTG GAA AG were used to amplify the coding region of the cDNA. The resulting PCR product was subcloned in pGEMT, and the plasmid pGEMT-GSUB was used for generation of radiolabeled RNA for Northern blotting, as well as construction of a CMV-driven expression vector. The addition of the hexahistidine sequence resulted in the creation of an NcoI site that could be used to distinguish the His6 G-substrate expression vector from the wild type G-substrate expression vector and that altered the Ser-2 codon to an alanine codon. The serine to alanine change and the addition of the hexahistidine tag changed the predicted molecular mass of the recombinant protein to 18,753 Da.

For mammalian expression, the plasmid pGEMT-GSUB was digested with BglII, and the fragment encoding histidine-tagged G-substrate was ligated into BglII-digested pCMV.neo (22), forming the vector pCMV.His6Gsub. For a typical G-substrate preparation, 20 15-cm plates of COS-1 cells at 30% confluency were transfected with 75 µg/plate of pCMV.His6Gsub DNA by calcium phosphate precipitation (23). Plates were incubated with DNA precipitate for 36 h and then washed three times with phosphate-buffered saline (150 mM NaCl, 20 mM NaPO4 (pH 7.4)) before being scraped into 20 ml of 10 mM Tris (pH 8), 200 mM NaCl, 0.1% Triton X-100, 5 mM beta -mercaptoethanol (Buffer A) containing 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 1 µg/ml pepstatin A. The resulting suspension was sonicated for 5 s and centrifuged at 9,000 × g for 30 min at 4 °C. Imidazole was added to the supernatant to a concentration of 10 mM, and the mixture was subjected to a second centrifugation, after which it was applied to a 1-ml column of nickel-affinity resin (Qiagen). The column was washed with 10 ml of Buffer A containing 10 mM imidazole, and the protein was eluted over a continuous gradient of 10-250 mM imidazole in Buffer A. Peak fractions were pooled analyzed by SDS-PAGE and silver staining (Bio-Rad Silver Stain Plus). Protein concentration was determined by bicinchoninic acid assay (Pierce Micro BCA). G-substrate so purified was typically 85% pure, as determined by densitometry, and was mainly used in kinetic analyses of phosphorylation.

Determination of Km and Vmax Values of cGK Ialpha , cGK II, and Calpha for G-substrate and Peptide Substrates-- The peptide Gln-Lys-Arg-Pro-Arg-Arg-Lys-Asp-Thr-Pro was synthesized at the University of Michigan Biomedical Core Facilities. The peptide, termed G-subtide, was purified by high pressure liquid chromatography, and its purity was confirmed by mass spectrometry. The peptide Arg-Lys-Arg-Ser-Arg-Ala-Glu (Glasstide) was purchased from Bachem. Phosphotransferase activities were assayed in 50-µl reactions of 20 mM Tris (pH 7.5), 10 mM MgAc, 200 µM ATP, 11 nM [gamma -32P]ATP (ICN) (specific activity, 200-300 cpm/pmol), 10 mM NaF, 10 mM dithiothreitol, and 0.2 mg/ml bovine serum albumin. Increasing concentrations of G-substrate, G-subtide, or Glasstide were assayed as indicated in Fig. 6. cGK Ialpha (24), cGK II (25), or Calpha (26) was added to a final concentration of 1.3, 5.8, or 5 nM, respectively, to start the reaction. Reactions were allowed to proceed at 30 °C for 30 min, after which they were terminated by spotting onto P81 phosphocellulose papers (Whatman), washed in 10 mM phosphoric acid, and counted. Km and Vmax values were calculated by Eadie-Hofstee analysis of three experiments (27, 28). All assays were linear with respect to enzyme concentration and time.

Construction of the G-substrate Baculovirus Transfer Vector, Sf9 Cell Infection, and Protein Purification-- An insert containing the histidine-tagged G-substrate cDNA was excised from pCMV.His6Gsub by BglII digestion and subcloned into the BglII site of the baculovirus transfer vector pBlueBac III (Invitrogen), creating the baculovirus transfer vector pBB.His6Gsub. Prior to protein production, recombinant baculovirus plaques were isolated and propagated as described (25).

To initiate protein expression, 500 ml of Sf9 culture was infected at a density of 6 × 106 cells/ml and a multiplicity of infection of 10. After 72 h, cells were collected by centrifugation and stored at -70 °C. For a typical G-substrate purification, pellets from three 500-ml cultures were thawed, combined, and resuspended in 120 ml of Buffer A, containing 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin A, and 5 mM benzamidine. Resuspended cells were sonicated for 15 s, heated at 100 °C for 5 min, and then centrifuged at 9,000 × g for 30 min at 4 °C. The supernatant was brought to 10 mM imidazole and kept on ice for 15 min, after which it was centrifuged as before. The secondary supernatant was loaded onto two 1-ml columns, connected in series, and packed with nickel-affinity resin (Qiagen). The columns were washed in series with 20 ml of Buffer A containing 10 mM imidazole. Protein was eluted from each column separately with a linear gradient of 10-250 mM imidazole in Buffer A.

DEAE chromatography was performed essentially as described (9). Peak fractions from the nickel affinity columns were pooled and dialyzed for 18 h against two changes of 10 mM KPO4 (pH 8), 0.2 mM EDTA, 15 mM beta -mercaptoethanol, 25 µM phenylmethylsulfonyl fluoride, 5 mM EGTA (Buffer B). After dialysis, the nickel affinity peaks were loaded onto a 1-ml column of Macro-Prep DEAE resin (Bio-Rad). The column was washed with 10 ml of Buffer B, and the protein was eluted in a linear gradient of 0-300 mM NaCl in Buffer B. Peak fractions were combined, concentrated by centrifugation (Amicon Centricon-3), and analyzed by SDS-PAGE and silver staining. Because the DEAE buffer interfered with the bicinchoninic acid assay, protein was determined with a modified Lowry assay (Pierce). G-substrate purified in this manner was judged to be greater than 95% pure by densitometry (Kodak Digital Science) and was used for phosphatase inhibition assays. Matrix-assisted laser desorption mass spectrometry was performed at the University of Michigan Biomedical Core Facilities on a VESTEC-2000 instrument.

Inhibition of PP1 and PP2A-- G-substrate purified from Sf9 cells was phosphorylated at a concentration of 15 µM in a reaction of 20 mM Tris (pH 7.5), 100 µM ATP, 10 mM MgAc, 100 µM EGTA, 20 µM cGMP, and 50 nM cGK Ialpha (Promega). Phosphorylation proceeded at 30 °C for 2.5 h, after which the kinase was denatured by heating at 100 °C for 10 min. To serve as a non-phosphorylated control in the phosphatase inhibition reactions, a second aliquot of purified G-substrate was subjected to a mock reaction excluding cGK Ialpha . After heating, both the phosphorylation and control reactions were diluted 10-fold with distilled water and concentrated back to their original volumes in centrifugal concentrators (Amicon Centricon-3) to dilute reaction components such at ATP which might themselves inhibit PP1. A second mock reaction excluding both cGK Ialpha and G-substrate was prepared, diluted, and concentrated for use as a buffer blank in the phosphatase reactions to control for any inhibitory activity the phosphorylation mix might retain. Following concentration, protein concentrations were determined by bicinchoninic acid assay (Pierce).

Incorporation of phosphate into G-substrate was quantified essentially as described (5). A small aliquot of G-substrate was phosphorylated as described above, except that [gamma -32P]ATP (Amersham Pharmacia Biotech) was added to a specific activity of 500 cpm/pmol. Phosphorylation was terminated by trichloroacetic acid precipitation, and following centrifugation, protein pellets were washed and counted by Cerenkov counting. Some aliquots of G-substrate were treated prior to phosphorylation with calf intestinal alkaline phosphatase (Boehringer Mannheim) at 37 °C for 1 h, after which they were heated at 100 °C for 10 min to denature the phosphatase.

Recombinant human DARPP-32 (Chemicon) was phosphorylated at a final concentration of 3.8 µM in a reaction of 20 mM Tris, 10 mM MgAc, 100 µM ATP and 42 nM Calpha (Promega) at 37 °C for 3 h, after which the reaction was heated, diluted, and concentrated as described for G-substrate. A control of non-phosphorylated DARPP-32 and a buffer blank were prepared as described for G-substrate.

Phosphatase activities were measured using a serine/threonine protein phosphatase assay kit (Life Technologies, Inc.). Radiolabeled phosphorylase a was prepared according to the instructions of the manufacturer and added as substrate to each phosphatase assay to a final concentration of 10 µM. Reactions of 30 µl were incubated at 30 °C for 20 min and terminated by trichloroacetic acid precipitation. Free phosphate in the reaction supernatants was measured by Cerenkov counting. Data were corrected using blanks in which phosphatase was excluded. The catalytic subunit of native rabbit skeletal muscle PP1 was purified as described (29) and was assayed at a final concentration of 0.7 nM in the presence of 8 mM caffeine. Uninhibited PP1c had a specific activity of 7.8 µmol/min·mg in the presence of 10 µM phosphorylase a. Native rabbit skeletal muscle PP2A1 was purchased from Upstate Biotechnologies and assayed at a final concentration of 15 nM in the presence of 10 mM MgAc and 1 mM dithiothreitol. Uninhibited PP2A1 had a specific activity of 0.24 µmol/min·mg in the presence of 10 µM phosphorylase a. PP1 and PP2A1 activities were assayed in the presence of increasing concentrations of phospho-G-substrate, dephospho-G-substrate, phospho-DARPP-32, dephospho-DARPP-32, and okadaic acid (Upstate Biotechnologies), as indicated. In PP1c assays, the G-substrate buffer alone was found to be inhibitory at only the two highest concentrations, and the data were corrected accordingly. In the PP2A1 assays, the G-substrate buffer alone was not inhibitory, even at the highest concentrations. All inhibition assays were performed in triplicate, and inhibition constants were calculated from curves fit to the data using Sigma Plot (Jandel Scientific), except for the IC50 of DARPP-32, which was extrapolated graphically. All phosphatase assays were linear with respect to enzyme concentration and time.

Dephosphorylation of G-substrate by PP1 and PP2A1-- G-substrate purified from Sf9 cells was phosphorylated as before, except that [gamma -32P]ATP (Amersham Pharmacia Biotech) was increased to a specific activity of 20,000 cpm/pmol. Phosphorylation was terminated by heating at 100 °C for 10 min. The reaction mix was then repeatedly diluted with distilled water and concentrated (Amicon Centricon-3) until contaminating free phosphate comprised less than 10% of the radioactivity counted in the pellet after a small aliquot was subjected to trichloroacetic acid precipitation. Native rabbit skeletal muscle PP1c was purchased from Upstate Biotechnologies and had a specific activity of 10 µmol/min·mg in the presence of 10 µM phosphorylase a. The ability of PP2A1 or PP1c to dephosphorylate phospho-G-substrate was measured as for phosphorylase a except that each phosphatase was assayed at a final concentration of 2 nM and phospho-G-substrate, at a final concentration of 190 nM, replaced phosphorylase a in the assay. Reactions were incubated at 30 °C for increasing periods, as indicated. Assays were performed in triplicate and curves fitted to the data using Sigma Plot (Jandel Scientific).

Transient Transfection and Regulation of Luciferase Activity by G-substrate-- Ten-cm plates of HEK-293 cells were transfected with 0.5 µg of human chorionic gonadotropin (HCG)-luciferase, 2 µg of SV2-beta -gal plasmid, and 20 µg of CMV-His6Gsub expression vector essentially as described previously (30). Twenty-four hours after transfection, the medium was changed to Dulbecco's modified Eagle's medium without serum and the cells incubated for 8 h. The media were then changed to Dulbecco's modified Eagle's medium with or without 300 µM chlorophenylthio-cAMP (CPT-cAMP), and the cells were incubated for an additional 24 h. Cells were then washed with phosphate-buffered saline and extracts assayed for luciferase activity as described previously (30). Transfection efficiencies were determined independently by assaying beta -galactosidase activity and normalizing luciferase activity relative to beta -galactosidase measurement for the same extracts. Normalization resulted in less than 25% corrections in luciferase activities. The mutant G-substrate expression vector, pCMV.His6Gsub(T72AT123A) was constructed by mutagenesis of the pGEMT-GSUB vector using the oligonucleotides GTG CAA GGC AGG CGC GTC TTT TCT, GTG CAC TGC TGG TGC GTC TTT CT, AG GAA GAC GCA CCA GCA GTG CAC, AGA AAA GAC GCG CCT GCC TTG CAC to alter the Thr codons of the G-substrate cDNA to Ala codons (underlined). The resulting G-substrate T72A, T1231 cDNA fragment was then sequenced and subcloned into pCMV.Neo at the BglII site.

    RESULTS

G-substrate Is Cloned from Mouse Brain-- For the present study, degenerate oligonucleotides based on the G-substrate phosphorylation site sequences (6) were designed with the goal of amplifying a cDNA fragment encoding the intervening residues. As described under "Materials and Methods," amplification of mRNA isolated from mouse brain produced a 200-bp amplification product, which was used to screen a mouse brain cDNA library. The initial library screening resulted in the isolation of clone 1, which was restriction-mapped (Fig. 1A) and fully sequenced on both strands (Fig. 1B). Clone 1 has 1275 bp and an open reading frame of 480 bp. Upstream of the open reading frame is a polypyrimidine stretch. A second library screening was conducted to determine whether the polypyrimidine stretch was a feature of the 5'-untranslated region or a cloning artifact.


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Fig. 1.   Cloning of murine G-substrate. A, schematic diagram of G-substrate cDNA clones. The four independent cDNA clones shown were isolated as described under "Materials and Methods" by hybridization of a mouse brain cDNA library with the product amplified using oligonucleotides based on the phosphorylation sites of G-substrate. The open reading frame is shown in gray, and the polypyrimidine stretch in the 5'-untranslated region is shown in black. Important restriction enzyme sites are indicated (E, EcoRI; Bm, BamHI; Bg, BglII). B, complete nucleotide sequence of G-substrate clone 1. The nucleotide sequence of both strands of DNA was determined. Below the nucleotide sequence, the predicted 159-amino acid sequence of the murine G-substrate protein is shown. Nucleotide numbers are indicated at the right of the sequence, and the amino acid numbers are indicated at the left of the sequence. The putative phosphorylation sites are underlined, and the phosphorylated threonine residues are indicated by boldface type.

The second library screening produced three additional independent clones. Clones 2-4 were restriction-mapped (Fig. 1A) and sequenced at their 5' and 3' ends. The 5' sequences of clones 2 and 4 are identical to the 5' sequence of clone 1, corroborating the presence of the 5'-polypyrimidine stretch. Clone 3 lacks any 5'-untranslated region, beginning 27 bp into the open reading frame. At the 3' end, clones 1 and 4 are identical. Clones 2 and 3 contain an additional 1 kilobase pair of 3'-untranslated region, giving clone 3, the longest clone, a size of 2.2 kilobase pairs. The 3'-untranslated regions of clones 2 and 3 were not fully sequenced, but the 3' ends of clones 2 and 3 are identical.

The open reading frame encodes a protein with a length of 159 residues (Fig. 1B) and a calculated molecular mass of 17.8 kDa. The differences between the phosphorylation site sequences reported here and those reported earlier for lapine G-substrate (6) are likely to arise from species differences between mouse and rabbit.

The amino acid composition of the predicted G-substrate protein is in overall agreement with that determined previously for lapine G-substrate (9). For example, mole percentages for the predicted protein and for lapine G-substrate are, respectively, 10.7 and 12.9 for aspartic acid, 10.1 and 9.1 for lysine, 10.7 and 9.1 for proline, 9.4 and 8.7 for leucine, 5.7 and 7.3 for arginine, 4.4 and 6.4 for glycine, 4.4 and 4.3 for serine, 1.9 and 2.7 for phenylalanine, and 0.63 and 0.95 for tryptophan. The only disparate residues are glutamic acid, at respective percentages of 6.9 and 14.8; methionine, at 3.8 and 1.2; and cysteine, at 1.3 and 0. The amino acid composition of the predicted protein, in addition to the putative phosphorylation site sequences, provided supportive evidence that the cDNA for murine G-substrate had been cloned, and the cDNA was used for further studies of G-substrate.

The Predicted Amino Acid Sequences of Human G-substrate and Murine G-Substrate Are 80% Identical-- A search of the expressed sequence tag (EST) data base for EST clones homologous to clone 1 identified two clones of interest. The first, a cDNA isolated from an infant brain library, appears to be a full-length cDNA encoding human G-substrate. This clone was obtained from Research Genetics (GenBankTM accession number H09006) and fully sequenced on both strands. The predicted amino acid sequence of human G-substrate, which contains 155 residues, is aligned with the predicted amino acid sequence of murine G-substrate in Fig. 2. The two proteins show 80% overall identity and almost complete conservation of the phosphopeptide sequence reported previously (6).


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Fig. 2.   Homology of mouse and human G-substrate amino acid sequences. A clone of human G-substrate was identified in the EST data base in a search for sequences homologous to the nucleotide and amino acid sequences of G-substrate clone 1. The human G-substrate clone was obtained and sequenced on both strands. The predicted protein sequences of murine and human G-substrate are compared in the above alignment, which was generated with DNASTAR software. Identical residues are shaded in gray, and boxes enclose the putative phosphorylation sites.

The second cDNA clone identified (GenBankTM accession number AA322502) was isolated from an adult brain cerebellum library and appears to encode a G-substrate splice variant lacking one of two phosphorylation sites of the protein. RNase protection analysis of mouse brain did not, however, provide evidence of any alternative G-substrate transcripts (see below), and characterization of this clone was not pursued.

G-substrate, DARPP-32, and Inhibitor-1 Have Homologous Phosphorylation Sites-- The inhibition of type I protein phosphatases by DARPP-32 and inhibitor-1 is dependent upon phosphorylation of a single threonine residue by cAK (10, 11). The phosphorylation sites of DARPP-32 (IRRRRP(p)TPAMLFR) (12) and of inhibitor-1 (IRRRRP(p)TPATLVL) (31) are very similar, sharing 10 out of 13 residues. Studies with DARPP-32 peptide analogs have shown that the phosphorylated threonyl residue is necessary for inhibitory activity and that substitution of a phosphorylated seryl residue nearly abolishes PP1c inhibition (32). The crystal structure of PP1c indicates an acidic groove on the surface of the enzyme containing negatively charged side chains, which are thought to participate in binding with the four arginyl residues NH2-terminal of the phosphothreonyl residue in inhibitor-1 and DARPP-32 (33).

Five of the 10 residues common to the phosphorylation sites of DARPP-32 and inhibitor-1 are also found in the phosphorylation sites of lapine G-substrate as follows: KPRRKD(p)TPAVHIP (site 1) and RPRRKD(p)TPALHMP (site 2) (6). The five common residues, which include the phosphothreonyl residue and two of the four NH2-terminal arginyl residues, are conserved in the predicted amino acid sequences of human and murine G-substrate. The fourth arginyl residue is replaced in the G-substrate sites by a lysyl residue, conserving the basic motif thought to interact with the acidic groove of PP1c.

G-substrate mRNA Localizes to the Purkinje Cells of the Cerebellum-- Within mammalian brain, the G-substrate protein has previously been localized by photoaffinity labeling and radioimmunoassay to the Purkinje cells of the cerebellum (4, 8), with trace levels of the protein also detected in cortex, hippocampus, and caudate (8). For the present study, the distribution of G-substrate mRNA was examined in mouse brain and mouse tissues by RNase protection analysis, Northern blotting, and in situ hybridization, as described under "Materials and Methods."

For RNase protection assays, a region of G-substrate clone 1 encompassing both phosphorylation sites was used to generate a 235-bp radiolabeled antisense RNA probe. A non-labeled sense transcript of 201 bp was synthesized from the same region for use as a standard during quantification. Autoradiograms from a representative assay are presented in Fig. 3A, and results of the signal quantification are presented in Fig. 3B. As expected, the predominance of signal was detected in the cerebellum. A 202-bp protected fragment in the cerebellum was clearly visible after a 1-day exposure (Fig. 3A, small autoradiogram). To develop weaker signals, a 1-week exposure was obtained (Fig. 3A, large autoradiogram). Within the brain, signals above background were observed for 202-bp protected fragments in hypothalamus and in pons/medulla. Signal above background was also detected for a 202-bp protected fragment in eye, although detection required higher amounts of total RNA.


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Fig. 3.   Localization of G-substrate mRNA in mouse brain, dissected brain regions, and tissues. A, RNase protection analysis of G-substrate mRNA distribution in mouse brain and dissected mouse brain regions. Total RNA from mouse brain (10 µg), brain regions (10 µg), mouse eye (30 µg), or sense standard RNA (0, 0.3, 1, 3, 10, or 30 pg) was hybridized to an antisense RNA probe generated from the coding region of G-substrate clone 1, RNase-treated, and separated by denaturing gel electrophoresis as described under "Materials and Methods." The position of the RNA probe (235 bp) and the sense standard RNA-protected fragments (207 bp) are indicated on the left with an arrow, and the position of the tissue RNA-protected fragments (202 bp) are indicated on the right with an arrow. The large autoradiogram pictured on the left was exposed for 1 week. The small autoradiogram showing only the cerebellum and pictured on the right was exposed for 1 day. B, PhosphorImager quantitation of RNase protection analysis of G-substrate mRNA distribution in mouse brain and dissected brain regions, using the 1-week exposure. Sense RNA standard assays were used to determine the number of picograms of protected fragment per µg of total RNA. WHL, whole brain; CER, cerebellum; CTX, cortex; HPC, hippocampus; HYPO, hypothalamus; MID, midbrain; P/M, pons/medulla; STR, striatum; THL, thalamus. C, RNA from various mouse tissues was analyzed by Northern blot using G-substrate clone 1. Hybridization with an antisense probe generated from the coding region was performed as described under "Materials and Methods." The sizes of RNA standards are indicated to the left of the figure in kilobases.

No protected fragments were detected to suggest the presence of splice variants in mouse. As discussed above, a search of the expressed sequence tag data base for homologous G-substrate sequences identified a cDNA clone that appeared to encode a human G-substrate isoform lacking one of the phosphorylation sites. If a transcript with a single phosphorylation site deletion had been present, a band would have been expected at approximately 150 bp. Such a band could be obscured by the high background seen for cerebellum after the week-long exposure, but its absence from the cerebellum after the day-long exposure indicates that, if present, this splice variant represents a minor fraction of the total G-substrate transcripts.

To compare the expression of G-substrate in different mouse tissues, poly(A)+ Northern blots were probed using a radiolabeled antisense RNA probe, as described under "Materials and Methods." A single transcript of 2.1 kilobase pairs was detected in brain but not in any other tissue (Fig. 3C). This transcript corresponds in size to clone 3, the longest G-substrate cDNA clone isolated. No other transcripts were detected on the Northern blot to provide evidence of other G-substrate isoforms or to indicate expression in the other tissues examined. Brain and eye were therefore the only tissues in which G-substrate mRNA was found.

The location of G-substrate within the brain was visualized by in situ hybridization. G-substrate mRNA was very abundant in cerebellum, where it localized to the Purkinje cells (Fig. 4). G-substrate mRNA was also detected at significantly lower levels in several regions including the paraventricular nucleus of the hypothalamus (Fig. 4).


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Fig. 4.   Comparative hybridization histochemical localization of G-substrate mRNA in mouse brain. Dark-field autoradiograms of sections of mouse brain cerebellum (A and B) or ventral hypothalamus (C and D) demonstrate regional distribution of neurons that hybridize with 35S-labeled cRNA probes for G-substrate. Sections were probed with antisense (A and C) or sense (B and D) cRNA probes, as described under "Materials and Methods." All slides were emulsion dipped for 3 weeks, with similar exposures. High levels of G-substrate mRNA are observed in the Purkinje cells of the cerebellum (A) and low levels are observed in the paraventricular nucleus of the hypothalamus (C).

Recombinant G-substrate Is Expressed and Purified-- Both mammalian and baculoviral expression systems were constructed. To facilitate purification, a six-residue histidine tag was added to the amino terminus of the protein using PCR, as described under "Materials and Methods." For expression in mammalian cells, transient transfection of 20 15-cm plates of COS-1 cells with pCMV.His6Gsub, followed by nickel affinity chromatography, typically resulted in the isolation of 1 mg of protein, judged to be approximately 85% pure by densitometry of silver-stained SDS-PAGE gels. Infection of 1.5 liters of Sf9 cell culture with pBB.His6Sub, followed first by nickel affinity chromatography and second by DEAE chromatography, typically resulted in the isolation of 1 mg of protein, judged to be greater than 95% pure by densitometry of silver-stained SDS-PAGE gels. The recombinant protein was analyzed by matrix-assisted laser desorption mass spectrometry and found to have a molecular mass of 18,752 Da, which is in good agreement with the predicted molecular mass of 18,753 Da for the protein (see "Materials and Methods"). When analyzed by SDS-PAGE, the recombinant, hexahistidine-tagged protein was found to have an anomalously high apparent Mr = 25,700, which is only slightly larger than the apparent Mr = 23,000 reported for the native protein.

Recombinant G-substrate Is Phosphorylated Preferentially by cGK Ialpha -- The ability of recombinant murine G-substrate to serve as a phosphorylation substrate was tested by incubation with cGK Ialpha in the presence of [gamma -32P]ATP in a phosphotransferase assay mix, as described under "Materials and Methods." Reactions excluding cGMP or enzyme were included to control for any kinase activity that might have co-purified with G-substrate. Reactions were subjected to SDS-PAGE, Coomassie staining, and autoradiography. A representative gel and its corresponding autoradiogram are presented in Fig. 5. For both pure and impure preparations of G-substrate, all of the 32P co-migrated with the recombinant protein at 25.7 kDa. Phosphorylation was induced by cGMP and caused a slight but reproducible reduction in the mobility of the protein on SDS-PAGE. No radiolabeling of the 27.5-kDa protein was detected on the autoradiogram in the absence of cGK Ialpha enzyme (data not shown).


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Fig. 5.   Phosphorylation of recombinant G-substrate by cGK Ialpha . Recombinant, hexahistidine-tagged G-substrate was expressed in COS-1 cells, purified, and phosphorylated by cGK Ialpha in a phosphotransferase mix containing [gamma -32P]ATP, as described under "Materials and Methods." Reactions were performed in the absence (left lane) or presence (right lane) of cGMP. Reactions were terminated by the addition of SDS sample buffer and subjected to SDS-PAGE and autoradiography. Representative lanes from a 12%, Coomassie-stained gel are shown on the left, and the corresponding autoradiogram is shown on the right. Numbers indicating molecular weight standards are shown in the center. The 68-kDa band visible in the Coomassie-stained gel is bovine serum albumin, added to the reactions to prevent loss of enzyme or substrate via nonspecific binding to the reaction tubes. Incorporation of radioactive phosphate into G-substrate is induced by the presence of cGMP and results in a slight reduction of the mobility of G-substrate on SDS-PAGE. Recombinant, hexahistidine-tagged G-substrate has a mass of 18.7 kDa, as confirmed by mass spectrometry but runs anomalously high at 27.5 kDa.

Once the ability of recombinant G-substrate to be phosphorylated by cGK Ialpha had been established qualitatively, assays were performed to determine the kinetic constants of its phosphorylation. Increasing concentrations of recombinant G-substrate were assayed with cGK Ialpha , cGK II, or the catalytic subunit of cAK (Calpha ), as described under "Materials and Methods." Results from a representative experiment are presented in Fig. 6A. cGK Ialpha was found to have a Km of 0.2 µM and a Vmax of 1.8 µmol/min·mg, and Calpha was found to have a Km of 2.0 µM and a Vmax of 2.6 µmol/min·mg. The kinetic constants obtained for recombinant G-substrate are in agreement with those determined for the native protein (5) and support the preferential phosphorylation of G-substrate by cGK Ialpha over cAK.


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Fig. 6.   Phosphorylation of recombinant G-substrate and the G-subtide peptide by cGK isoforms and the catalytic subunit of cAK. A, recombinant, hexahistidine-tagged G-substrate was expressed in COS-1 cells, purified, and phosphorylated at increasing concentrations by cGK Ialpha (open circles), cGK II (closed circles), or Calpha (open triangles) in a phosphotransferase mix containing [gamma -32P]ATP, as described under "Materials and Methods." Incorporation of phosphate was quantitated by spotting onto phosphocellulose papers and Cerenkov counting and is expressed as a percentage of the maximum phosphorylation obtained. B, increasing concentrations of the G-subtide peptide (circles) or the Glasstide peptide (triangles) were phosphorylated by cGK Ialpha (open symbols) or cGK II (closed symbols) in a phosphotransferase mix containing [gamma -32P]ATP, as described under "Materials and Methods." Incorporation of phosphate was quantitated as in A. The experiments were performed three times for both peptide substrates. Average Km and Vmax values and measurements of error for both peptide substrates are reported in Table I.

The kinetic constants of cGK II for G-substrate had not previously been determined. cGK II was found to have a Vmax of 1.3 µmol/min·mg and a Km of 1.1 µM, indicating that G-substrate is a poorer substrate for cGK II than for cGK Ialpha . A specificity index of a substrate for cGK Ialpha over cGK II can be calculated (25) by dividing the Vmax/Km ratio of cGK Ialpha by that of cGK II so that a value greater than 1 indicates a substrate phosphorylated preferentially by cGK Ialpha and a value less than 1 indicates a substrate phosphorylated preferentially by cGK II. By this calculation, G-substrate has a specificity index of 7.6, indicating a selectivity for cGK Ialpha over cGK II.

To study this selectivity further, a peptide substrate based on the phosphorylation sites of G-substrate was synthesized. This peptide has the sequence Gln-Lys-Arg-Pro-Arg-Arg-Lys-Asp-Thr-Pro and was termed G-subtide. Phosphorylation of G-subtide by cGK Ialpha and by cGK II was studied and compared with the phosphorylation of Glasstide, a commonly used peptide substrate of cGK I with the sequence Arg-Lys-Arg-Ser-Arg-Ala-Glu (34). Km and Vmax values were determined for both cGK Ialpha and cGK II with each peptide substrate, as shown in Table I. A representative experiment is shown in Fig. 6B. Glasstide, also known as H2Btide, has a cGK Ialpha /cGK II specificity index of 20, indicating a strong preference for cGK Ialpha as previously reported (25). G-subtide has a cGK Ialpha /cGK II specificity index of 0.3. This value less than 1 indicates the preference of G-subtide for cGK II and makes the peptide a useful substrate for detecting and characterizing cGK II activity. The difference in selectivity between G-subtide (0.3) and G-substrate (7.6) suggests there are specificity determinants present in the protein outside of sequences adjacent to the phosphorylation sites of G-subtide.

                              
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Table I
Summary of apparent kinetic constants for peptide substrates of cGK Ialpha and cGK II
Km and Vmax values are expressed as the average of three experiments ± S.D. for each substrate and cGK isoform. The cGK Ialpha /cGK II specificity index was obtained by dividing the Vmax/Km ratio of cGK Ialpha by the Vmax/Km ratio of cGK II for each peptide substrate. A specificity index greater than 1 is indicative of a cGK Ialpha -selective substrate, whereas a specificity index less than 1 is indicative of a cGK II-selective substrate. A representative experiment used to calculate these kinetic constants is shown in Fig. 7B.

Phosphorylated G-substrate Inhibits PP1c-- The common features of G-substrate, DARPP-32, and inhibitor-1 have led to speculation that G-substrate, like DARPP-32 and inhibitor-1, functions when phosphorylated as a type I protein phosphatase inhibitor (6, 12). Preliminary evidence supported this hypothesis (6, 8, 12, 14), but limited quantities of purified native protein prevented definitive characterization of G-substrate's interaction with PP1.

In order to test G-substrate for PP1c inhibitory activity, purified recombinant G-substrate was phosphorylated, as described under "Materials and Methods." The recombinant protein was found to incorporate a maximum of 1.34 mol of phosphate/mol of G-substrate. Treatment with alkaline phosphatase prior to phosphorylation did not increase phosphate incorporation, and phosphorylated G-substrate did not incorporate any phosphate upon back-phosphorylation (data not shown). Additionally, phosphorylation resulted in a slight but reproducible reduction in mobility of the entire 27.5-kDa protein band on SDS-PAGE (Fig. 5). For these reasons, it was determined that the predominance of G-substrate was phosphorylated after treatment with cGK Ialpha . The stoichiometry of phosphorylation lower than the theoretical value of 2 could be due to overestimation of protein concentration, as G-substrate protein determinations varied with the protein assay used (data not shown).

PP1c dephosphorylation of phosphorylase a was measured in the presence of increasing concentrations of phospho- and dephospho-G-substrate. Initial experiments with phosphorylated G-substrate and recombinant PP1c demonstrated no inhibition of protein phosphatase activity using phosphorylase a as substrate (data not shown). Since the recombinant PP1c has been reported to be less sensitive to inhibition by phosphorylated inhibitor-1 (29, 35), the inhibition of native PP1c by G-substrate was also tested.

Dephospho-G-substrate was found to have no inhibitory activity toward native PP1c, but phospho-G-substrate was inhibitory, with an IC50 of 131 ± 27 nM, determined as the mean of three independent experiments. A representative experiment is shown in Fig. 7A. As a positive control for inhibition, DARPP-32 was tested for PP1c inhibitory activity. Results of an experiment performed in triplicate are shown in Fig. 7B. Phospho-DARPP-32 was found to inhibit half of the PP1c activity at 1.8 nM, a value consistent with the range of reported IC50 values for phospho-DARPP-32 (36), whereas dephospho-DARPP-32 showed no inhibition at all.


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Fig. 7.   Inhibition of PP1c by G-substrate and DARPP-32. A, recombinant, hexahistidine-tagged G-substrate was expressed in Sf9 cells, purified, and phosphorylated as described under "Materials and Methods." Phospho-G-substrate was prepared in a phosphotransferase mix containing ATP and cGK Ialpha . Dephospho-G-substrate was prepared in a mock reaction excluding kinase. Increasing concentrations of phospho-G-substrate (closed circles) or dephospho-G-substrate (open circles) were added to PP1c activity assays in which phosphorylase a was the substrate. PP1c activity was measured as described under "Materials and Methods" and is expressed as a percentage of the activity obtained in the absence of G-substrate. Reactions were performed in triplicate, and standard deviation is represented by error bars in the figure. B, recombinant DARPP-32 (Chemicon) was phosphorylated as described under "Materials and Methods." Phospho-DARPP-32 was prepared in a phosphotransferase mix containing Calpha and ATP. Dephospho-DARPP-32 was prepared in a mock reaction excluding kinase. Increasing concentrations of phospho-DARPP-32 (closed circles) or dephospho-DARPP-32 (open circles) were added to PP1c activity assays in which phosphorylase a was the substrate. PP1c activity was measured as described in A. Reactions were performed in triplicate, and standard deviation is represented by error bars in the figure.

G-substrate Is a Weak Inhibitor of PP2A1-- To determine if G-substrate could inhibit type 2 protein phosphatases, PP2A1 dephosphorylation of phosphorylase a was measured in the presence of increasing concentrations of phospho- and dephospho-G-substrate, as described under "Materials and Methods." A representative experiment is shown in Fig. 8. Phospho-G-substrate had an IC50 of 382 nM and was a more effective inhibitor of PP2A1 than dephospho-G-substrate, which was not assayed at high enough concentrations to determine its IC50. The effect of okadaic acid on PP2A1 activity was tested as a positive control for inhibition. The IC50 of okadaic acid was determined to be 1.4 nM.


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Fig. 8.   Inhibition of PP2A1 by G-substrate and okadaic acid. Phospho-G-substrate and dephospho-G-substrate were prepared as described in the legend to Fig. 7. Increasing concentrations of phospho-G-substrate (closed circles), dephospho-G-substrate (open circles), or okadaic acid (open diamonds) were added to PP2A1 activity assays in which phosphorylase a was the substrate. PP2A1 activity was measured as described under "Materials and Methods" and is presented as a percentage of the activity obtained in the absence of inhibitor. Reactions were performed in triplicate, and standard deviation is represented by error bars in the figure.

PP2A1 Dephosphorylates G-substrate-- Native G-substrate, DARPP-32, and inhibitor-1 can all serve as substrates for type 2 protein phosphatases (37, 38). To determine if recombinant G-substrate could act as a substrate for either PP1c or PP2A1, purified recombinant G-substrate was phosphorylated in the presence of radiolabeled ATP. Radiolabeled phospho-G-substrate was then treated with 2 nM PP1c or 2 nM PP2A1 in a phosphatase assay mix for increasing periods, as described under "Materials and Methods." Over the course of 2 h, phospho-G-substrate was moderately dephosphorylated by PP1c, with an initial rate of 26 fmol of phosphate cleaved per min (Fig. 9). PP2A1, on the other hand, rapidly dephosphorylated phospho-G-substrate, with an initial rate of 555 fmol of phosphate released per min.


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Fig. 9.   Dephosphorylation of phospho-G-substrate by PP1c and PP2A1. Recombinant, hexahistidine-tagged G-substrate was expressed in Sf9 cells, purified, and phosphorylated in the presence of [gamma -32P]ATP and cGK Ialpha , as described under "Materials and Methods." Phospho-G-substrate was treated with PP1c (2 nM) (open circles) or PP2A1 (2 nM) (closed circles) for increasing periods. Reactions were terminated by trichloroacetic acid precipitation. Phosphate released from phospho-G-substrate was measured by Cerenkov counting of each supernatant after centrifugation. Reactions were performed in triplicate, and standard deviation is represented by error bars in the figure.

G Substrate Potentiates the Action of cAMP on Regulation of a cAMP-responsive Promoter-- In order to determine whether G substrate had the ability to inhibit phosphatase activity in vivo, cells were transfected with expression vectors for G-substrate was well as a cAMP-responsive reporter construct containing the promoter of the human chorionic gonadotropin gene directing expression of luciferase. This reporter has been used extensively to study the regulation of gene expression by the CREB transcription factor and the role of phosphorylation of CREB by cAMP-dependent protein kinase. As shown in Fig. 10, when cells were treated with 300 µM CPT-cAMP a 3.6-fold stimulation of luciferase activity was observed when only the endogenous cAMP-dependent protein kinase was activated. Transfection with a G-substrate expression vector alone resulted in a 1.5-fold increase in luciferase activity, but the combination of CPT-cAMP treatment and G-substrate co-expression resulted in a 30-fold increase in luciferase over basal levels. This increase in luciferase activity required Thr-72 and Thr-123 of the expressed G-substrate because when a mutant G substrate was expressed in which these residues were mutated to alanine, the luciferase activity was only induced 7.2-fold. These results are consistent with G-substrate inhibiting cellular phosphatases to potentiate the phosphorylation of the CREB transcription factor resulting in increased HCG-luciferase expression.


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Fig. 10.   Regulation of the HCG-luciferase expression by G-substrate. HEK-293 cells were transfected with HCG-luciferase and expression vectors for either a wild type G-substrate (Gsub) protein or a mutant (Gsub(MUT)) protein in which the codons for Thr-72 and Thr-123 were altered to Ala residues. Following transfection, cells were incubated in the presence or absence of 300 µM CPT-cAMP, and extracts were prepared for measurement of luciferase activities. Relative luciferase activities were determined by normalizing for transfection efficiencies using a co-transfected beta -galactosidase expression vector as described under "Materials and Methods." Similar results were obtained in two additional experiments.


    DISCUSSION

This report describes the cloning and characterization of G-substrate, one of the few cGK-specific substrates identified to date. Four cDNA clones encoding murine G-substrate have been isolated from mouse brain and have provided the first complete determination of the primary structure of G-substrate. The amino acid composition of the predicted protein and the putative phosphorylation site sequences are in close agreement with those published previously for lapine G-substrate (9), and the predominance of G-substrate mRNA localizes to cerebellar Purkinje cells, as determined for the G-substrate protein (8). Like the native protein, recombinant G-substrate is phosphorylated preferentially by cGK over cAK, and a single transcript corresponding in size to the largest cDNA isolated was detected in brain.

The predicted molecular mass of recombinant G-substrate (17.8 kDa) does not agree with the apparent molecular weight reported for the native protein (Mr = 23, 000) (9). However, the characteristics shared by G-substrate, inhibitor-1, and DARPP-32 include a high content of charged amino acid residues, which do not bind detergent well. It is therefore not surprising that G-substrate, like inhibitor-1 (31) and DARPP-32 (39), exhibits anomalously slow migration when subjected to SDS-PAGE.

The phosphorylation sites (RRPT) of both inhibitor-1 and DARPP-32 are nearly identical and contain the typical cAK consensus phosphorylation sequence (RRX(S/T), where X is any amino acid) (40). However, the selectivity of inhibitor-1 for cAK over cGK is twice that of DARPP-32, which is phosphorylated by both enzymes with nearly equal efficiency (41). This disparity of preference between proteins with such similar phosphorylation sites suggests the existence of substrate specificity determinants for the cyclic nucleotide-dependent protein kinases outside the phosphorylation sites of the substrates. This idea is supported by the interaction observed during this study of cGK with G-substrate and the G-subtide peptide.

The G-substrate phosphorylation sites (RKDT) do not have the typical cAK phosphorylation sequence, but neither do they have the basic p+1 residue (where the phosphorylated residue equals p) nor the p+4 phenylalanine reported to provide selectivity for cGK over cAK (42, 43). In this study, purified recombinant G-substrate was phosphorylated by cGK with the same efficiency reported previously for native G-substrate (5), exhibiting a preference for cGK over cAK, as well as a preference for cGK Ialpha over cGK II. The Km of cGK Ialpha for the peptide G-subtide, whose sequence is based on the phosphorylation sites of G-substrate, was 1,000-fold greater than the Km of cGK Ialpha for the full-length G-substrate. The peptide G-subtide was also phosphorylated preferentially by cGK II over cGK Ialpha , unlike the full-length G-substrate. The differing phosphorylation kinetics of the phosphorylation-site peptide and the full-length protein support the presence of specificity determinants for the kinases outside the substrate phosphorylation sites.

Both inhibitor-1 and DARPP-32, in their phosphorylated forms, inhibit PP1c with IC50 values in the range of 1-8 nM, depending on the preparation of PP1c (32, 44). The similarities among inhibitor-1, DARPP-32, and G-substrate have prompted speculation that G-substrate also functions as a PP1c inhibitor (6, 8, 11, 13), but this paper includes the first experimental demonstration of a G-substrate-PP1c interaction. The IC50 of 131 ± 27 nM determined for phospho-G-substrate is roughly 100-fold greater than the IC50 values for inhibitor-1 and DARPP-32 but still low enough for G-substrate to be a physiologically relevant inhibitor of PP1c, especially in Purkinje cells where concentrations of G-substrate have been estimated to be 10.8 ± 1.44 µM (8).

Radiolabeled phospho-G-substrate was not rapidly dephosphorylated by PP1c. Phospho-G-substrate inhibition of PP1c is therefore not likely to be the result of G-substrate serving as an alternate substrate in the assay. Conversely, phospho-G-substrate was dephosphorylated by PP2A1 at a rate 20 times faster than PP1c. This rapid dephosphorylation could have resulted in the observed PP2A1 inhibition via G-substrate replacement of phosphorylase a in the assay. The combination of dephosphorylation by type 2 phosphatases and inhibition of type 1 phosphatases is also observed for inhibitor-1 and DARPP-32 (37, 38), suggesting a possible mechanism for modulating the inhibitory activity of these proteins within the cell.

Studies have shown that phosphorylated peptides based on the conserved phosphorylation sequences of inhibitor-1 and DARPP-32 are up to 1,000-fold less inhibitory than the full-length proteins. These results suggest that there are determinants for inhibition outside the phosphorylation sequences (36, 45). The conserved sequence KIQF is located distally to the phosphorylation site in the amino terminus of both inhibitor-1 and DARPP-32 and has been implicated in the binding of the phospho- and dephospho-forms of each protein to PP1c (46, 47). This motif is functionally conserved in the binding sites of several PP1c targeting subunits, which bind to PP1c and regulate its subcellular distribution (48). Mutagenesis studies with the glycogen-targeting PP1c subunit have recently relaxed this binding motif to (R/K)(V/I)XF, where X is any amino acid (49). The closest sequence in G-substrate is an RYDV sequence located between the two phosphorylation sites, but what role it may play in binding to PP1c is unknown. The comparatively lower inhibitory potency of G-substrate could arise from its lack of this consensus PP1c binding motif.

The phosphorylation-dependent inhibition of PP1c by G-substrate represents a novel, neuron-specific pathway by which cGK can regulate the activity of the type 1 protein phosphatases. The main site of this regulation in the brain is likely to be the Purkinje cells, where G-substrate (8) and cGK I (36) are concentrated and where cGMP (50), PP1 (51), PP2A (52), and PP2B (53) have all been localized. Our results did not confirm the low levels of G-substrate detected previously in cortex, hippocampus, and caudate (8), but our results did provide evidence for low level expression of G-substrate in pons/medulla and hypothalamus. Photoaffinity labeling experiments have localized cGK I to the medulla at a concentration 10-fold less than what was found in cerebellum and to the pons and the hypothalamus at concentrations roughly 100-fold less than cerebellar concentrations (36).

Cyclic GMP stimulation of cGK I has been implicated in a number of neuron-specific phenomena, including long term depression in the cerebellum (1) and regulation of gonadotropin-releasing hormone expression in immortalized hypothalamic neurons (54). The potential physiological role of G-substrate is underscored by its localization with cGK I to these brain regions. G-substrate, as a phosphatase inhibitor controlled specifically by cGK, represents a potentially important component of cGMP signaling regulation. The G-substrate cDNA and recombinant protein thus provide unique tools for addressing the role of cGMP-activated phosphatase inhibition in these signaling cascades.

    ACKNOWLEDGEMENTS

We thank Dr. Audrey Seasholtz for assistance with the in situ hybridizations; Dr. Rafael Ballestero for skillful dissection of mouse brains; Dr. Timothy Angellotti for early baculovirus constructs; Linda Gates for tissue culture support; Junewai Reoma for assistance with sequencing; Cindy Overmyer and Stephanie McWethy for assistance with the figures; and members of the Uhler Lab for helpful discussions of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM50791 and DK36569.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF071562 (mouse) and AF071789 (human).

parallel Present address: Dept. of Biology, Texas A & M University, Kingsville, TX 78363.

Dagger Dagger To whom correspondence should be addressed: Mental Health Research Institute, Neuroscience Laboratory Bldg., 1103 East Huron St., University of Michigan, Ann Arbor, MI 48109-1687. Tel.: 734-647-3172; Fax: 734-936-2690; E-mail: muhler{at}umich.edu.

The abbreviations used are: cGK, cGMP-dependent protein kinase; cAK, cAMP-dependent protein kinase; DARPP-32, dopamine- and cAMP-regulated phosphoprotein of apparent Mr 32,000 determined by SDS-PAGE; PAGE, polyacrylamide gel electrophoresis; PP1c, protein phosphatase-1 catalytic subunit; PCR, polymerase chain reaction; CMV, human cytomegalovirus; Calpha , cAK catalytic subunit; PP2A1, protein phosphatase type-2A1; bp, base pairs; EST, expressed sequence tag; CPT-cAMP, chlorophenylthio-cAMP.
    REFERENCES
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Abstract
Introduction
References

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