From the 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
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ABSTRACT |
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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.
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.
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
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 [ 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 Determination of Km and Vmax Values of cGK
I 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
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 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 I
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 [
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 C
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 [ 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- 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.
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).
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.
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).
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
I
Once the ability of recombinant G-substrate to be phosphorylated by cGK
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 I
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 I 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
I
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.
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.
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.
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.
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 I 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.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
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 [
-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.
-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).
-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.
, cGK II, and C
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 [
-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 I
(24), cGK II (25), or C
(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.
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.
-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.
(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 I
. 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 I
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).
-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.
(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.
-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).
-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
-galactosidase activity and normalizing luciferase activity relative to
-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
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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.
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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.
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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.
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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).
--
The ability of recombinant murine G-substrate to serve as a
phosphorylation substrate was tested by incubation with cGK I
in the
presence of [
-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 I
enzyme
(data not shown).
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Fig. 5.
Phosphorylation of recombinant G-substrate by
cGK I . Recombinant, hexahistidine-tagged G-substrate was
expressed in COS-1 cells, purified, and phosphorylated by cGK I
in a
phosphotransferase mix containing [
-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.
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 I
,
cGK II, or the catalytic subunit of cAK (C
), as described under
"Materials and Methods." Results from a representative experiment
are presented in Fig. 6A. cGK
I
was found to have a Km of 0.2 µM
and a Vmax of 1.8 µmol/min·mg, and C
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 I
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 I (open circles), cGK II
(closed circles), or C
(open triangles) in a
phosphotransferase mix containing [
-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 I
(open
symbols) or cGK II (closed symbols) in a
phosphotransferase mix containing [
-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.
. A specificity index of a substrate for cGK
I
over cGK II can be calculated (25) by dividing the
Vmax/Km ratio of cGK I
by
that of cGK II so that a value greater than 1 indicates a substrate phosphorylated preferentially by cGK I
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 I
over cGK II.
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 I
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 I
/cGK II specificity index of 20, indicating a strong preference for
cGK I
as previously reported (25). G-subtide has a cGK I
/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.
Summary of apparent kinetic constants for peptide substrates of cGK
I and cGK II
/cGK II specificity index was obtained by
dividing the Vmax/Km ratio of cGK
I
by the Vmax/Km ratio of cGK
II for each peptide substrate. A specificity index greater than 1 is
indicative of a cGK I
-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.
. 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).
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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 I . 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 C
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.
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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.
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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 [ -32P]ATP and cGK
I
, 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.
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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 -galactosidase expression vector as described under
"Materials and Methods." Similar results were obtained in two
additional experiments.
DISCUSSION
over cGK II. The Km of cGK
I
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 I
for the full-length G-substrate. The
peptide G-subtide was also phosphorylated preferentially by cGK II over
cGK I
, 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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).
Present address: Dept. of Biology, Texas A & M University,
Kingsville, TX 78363.
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; C, cAK
catalytic subunit; PP2A1, protein phosphatase
type-2A1; bp, base pairs; EST, expressed sequence tag; CPT-cAMP, chlorophenylthio-cAMP.
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REFERENCES |
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