From the Department of Biochemistry, College of
Natural Sciences and
Department of Molecular and Cellular
Biochemistry, School of Medicine, Kangwon National University,
Chunchon, Kangwon-Do 200-701, Korea and § Laboratory of
Molecular and Cellular Neuroscience, The Rockefeller University,
New York, New York 10021
Received for publication, September 19, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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PNUTS, Phosphatase 1 NUclear Targeting Subunit, is a
recently described protein that targets protein phosphatase 1 (PP1) to the nucleus. In the present study, we characterized the biochemical properties of PNUTS. A variety of truncation and site-directed mutants
of PNUTS was prepared and expressed either as glutathione S-transferase fusion proteins in Escherichia
coli or as FLAG-tagged proteins in 293T cells. A 50-amino acid
domain in the center of PNUTS mediated both high affinity PP1 binding
and inhibition of PP1 activity. The PP1-binding domain is related to a
motif found in several other PP1-binding proteins but is distinct in
that Trp replaces Phe. Mutation of the Trp residue essentially
abolished the ability of PNUTS to bind to and inhibit PP1. The central
PP1-binding domain of PNUTS was an effective substrate for protein
kinase A in vitro, and phosphorylation substantially
reduced the ability of PNUTS to bind to PP1 in vitro and
following stimulation of protein kinase A in intact cells. In
vitro RNA binding experiments showed that a C-terminal region
including several RGG motifs and a novel repeat domain rich in His and
Gly interacted with mRNA and single-stranded DNA. PNUTS exhibited
selective binding for poly(A) and poly(G) compared with poly(U) or
poly(C) ribonucleotide homopolymers, with specificity being mediated by
distinct regions within the domain rich in His and Gly and the domain
containing the RGG motifs. Finally, a PNUTS-PP1 complex was isolated
from mammalian cell lysates using RNA-conjugated beads. Together, these studies support a role for PNUTS in protein kinase A-regulated targeting of PP1 to specific RNA-associated complexes in the nucleus.
Protein phosphatase 1 (PP1)1 is a multifunctional
serine/threonine phosphatase that plays a key role in regulation of
diverse cellular processes, including gene expression, muscle
contraction, cell cycle progression, glycogen metabolism, and
neurotransmission (1-3). The catalytic subunit of PP1, which exists as
four different isoforms (PP1 A variety of studies have suggested an important role for PP1 in the
nucleus (9). PP1 interacts with the retinoblastoma protein p110Rb (10)
and is believed to act as a positive regulator of the interaction of
p110Rb with the transcription factor, E2F (10, 11). PP1 is likely to
play an important role in dephosphorylation of the transcription
factors CREB and Sp-1 (12-14). PP1 interacts with a protein termed
NIPP1 that functions as a splicing factor at a late stage of spiceosome
assembly (15, 16). Other studies (17, 18) have suggested a role for PP1
in modulation of mammalian splicesome assembly and in the subcellular
distribution of pre-mRNA splicing factors. During the cell cycle,
biochemical and genetic studies have shown that PP1 activity is
regulated by phosphorylation (19, 20) and that the enzyme plays a key
role in the mitotic transition by dephosphorylating various nuclear
phosphoproteins that are essential for driving structural
reorganization of the nuclear envelope, spindle apparatus, and
chromosomal DNA (21-25). PP1 also interacts with other nuclear
proteins including the p53-binding protein, p53BP (26), Hox11 (27), and
with sds22, a protein implicated in chromosome stability (28,
29).
We and others have reported recently (30, 31) the cloning and initial
characterization of a novel nuclear PP1-binding protein named PNUTS
(Phosphatase 1 NUclear Targeting
Subunit) or p99. PNUTS exhibits a discrete nuclear
compartmentalization and is found in a stable complex with PP1 in
mammalian cell lysates. Recombinant PNUTS potently inhibits the
catalytic activity of PP1 toward exogenous substrate in
vitro. Primary sequence analysis indicates that the C terminus of
PNUTS contains several closely spaced RGG sequences, motifs that are
often found in RNA-binding proteins (32). PNUTS also contains a novel
region of repetitive amino acid sequence that is rich in His and Gly,
and a putative Zn2+ finger domain with the signature
CX8CX5CX3H.
In the present study, we have characterized further the biochemical
properties of PNUTS. PNUTS contains a short ~50-amino acid central
region that contains closely associated PP1-binding domains and
inhibitory domains. Moreover, the interaction of PNUTS with PP1 is
regulated by phosphorylation within the binding domain. We have also
found that PNUTS binds to homopolymeric RNA with high selectivity for
poly(A) and poly(G) via the RGG motifs and the novel region rich in His
and Gly. These studies support the conclusion that PNUTS may mediate
the reversible association of PP1 with specific RNAs in the nucleus of
mammalian cells.
Materials--
Ribonucleotide homopolymer-agarose beads were
obtained from Sigma. An in vitro transcription and
translation kit was purchased from Promega.
[35S]Methionine, ssDNA-agarose (1-3 mg of denatured calf
thymus DNA/ml gel), secondary antibodies, and enhanced
chemiluminescence (ECL) reagents were obtained from Amersham
Biosciences. Synthetic peptides (DARPP-32-(1-39) (with a Cys residue
included at the C terminus), PNUTS-(392-408), PNUTS-(392-415),
PNUTS-(392-408;W401A), and PNUTS-(392-415;W401Y)) were prepared by
the W. M. Keck Biotechnology Resource Center, Yale University. The
catalytic subunit of protein kinase A (PKA) was purified from bovine
heart as described (33). [32P]Phosphorylase a
(1-3 × 106 cpm/nmol) was prepared from phosphorylase
b as described (34). Rabbit polyclonal PNUTS, PP1 pcDNA1/Neo-FLAG Plasmid Construction and
Co-immunoprecipitation--
DNA fragments of PNUTS were amplified by
Pfu polymerase; PCR products were digested by
SalI/NotI restriction enzymes and subcloned into
pcDNA1Neo (Invitrogen) (encoding a FLAG epitope with a 5' SalI site in-frame with the FLAG sequence). Internal
deletion mutants were prepared from the ligation of two individual PCR fragments. HEK293T cells grown in Dulbecco's modified Eagle's culture
medium (10% fetal bovine serum) were transiently transfected with
different plasmids (10 µg) using the calcium phosphate method. Cells
were washed with fresh medium 5 h after transfection and were
cultured for 16 h. Cells were washed with PBS, harvested, and
resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl,
0.6 mM PMSF, 20 µg/ml leupeptin and antipain, 10 µg/ml
pepstatin A and chymostatin, 0.5% Nonidet P-40). Lysates were briefly
sonicated and then centrifuged at 15,000 × g for 20 min. Protein concentration of supernatants was determined using the BCA
assay (Pierce). Lysates (1 mg) were incubated with anti-FLAG affinity
beads (Sigma) for 2 h at 4 °C. Immunocomplexes were washed with
lysis buffer and eluted from beads by boiling in SDS sample buffer.
Samples were analyzed by SDS-PAGE (12% polyacrylamide), and proteins
were transferred to polyvinylidene difluoride membrane (Immobilon-P,
Millipore) by electroblotting (200 mA, overnight). Blots were incubated
with either an anti-FLAG antibody or an anti-PP1 Preparation of GST Fusion Proteins in Bacteria--
The
cDNAs encoding various deletion mutants of PNUTS were amplified by
PCR with primers containing 5' EcoRI and 3' NotI
restriction sites. PCR fragments were digested with EcoRI
and NotI and subcloned into the pGEX-5x-1 expression vector.
Plasmids were transformed into Escherichia coli
(BL21 DE3), and bacteria were cultured in LB media in the presence of
50 mg/ml ampicillin to an A600 value of 0.6-0.8
at 37 °C. Expression of GST fusion proteins was induced by addition
of 0.3 mM
isopropyl-1-thio- Site-directed Mutagenesis of GST-PNUTS-(382-433) and
-(382-459)--
Point mutations were produced by Quick Change
Mutation Kit using the manufacturer's protocol (Stratagene). The
W401A, V399A, K397A, and R396A mutant cDNAs were prepared by
Pfu-mediated PCR using pGEX-5x-1 vectors containing
wild-type PNUTS-(382-433) and PNUTS-(382-450). The primers used are
as follow: W401A,
5'-GAAAAACTGTGACGGCGCCTGAGGAGGGC-3'; V399A,
5'-GAAAGAGAAAAACTGCGACGTGGCCTGAGG-3'; K397A,
5'-GGGCAGAAAGAGAGCAACTGTGACGTGGCC-3'; and R396A,
5'-CGAAAGGGCAGAAAGGCAAAAACTGTGACGTGG-3'.
Mutations were confirmed by DNA sequencing.
Protein Phosphatase Assay--
Purified rabbit muscle PP1 was
kindly provided by Dr. Hsien-bin Huang. PP1 was assayed using
[32P]phosphorylase a as substrate essentially as
described (34). Briefly, assays (final volume 30 µl) contained 50 mM Tris-HCl, 0.15 mM EGTA, 15 mM
2-mercaptoethanol, 0.01% (w/w) Brij 35, 0.3 mg/ml bovine serum
albumin, 5 mM caffeine, 10 µM
[32P]phosphorylase a, various concentrations
of GST fusion proteins, and <0.1 unit/ml of PP1 Pull-down of PP1 Using GST-PNUTS Fusion Proteins--
Various
GST-PNUTS mutants (7 µg) were incubated with glutathione-agarose
beads and then mixed with PC12 cell lysates (1 mg of protein with lysis
buffer containing 50 mM Tris-HCl, pH 7.4, 1 mM
EDTA, 1 mM EGTA, 150 mM NaCl, 0.6 mM PMSF, 20 µg/ml leupeptin and antipain, 10 µg/ml
pepstatin A, and chymostatin, 0.5% Nonidet P-40) for 2 h. After
centrifugation, beads were washed with lysis buffer, eluted with SDS
sample buffer, and analyzed by SDS-PAGE (10% acrylamide). PP1 was
detected by immunoblotting using an anti-PP1 PP1 Overlay Assay--
PP1 overlay assays were carried out
essentially as described (37). Briefly, proteins were separated by
SDS-PAGE and transferred to nitrocellulose filters. Filters were
incubated with a buffer containing 10 mM Tris-HCl (pH 7.4),
2% (w/v) dried milk, and 0.1% Tween 20. Filters were washed with PBS
containing 0.2% Nonidet P-40 and then incubated with PBS/Nonidet P-40
containing 0.1 µg/ml recombinant PP1 and 100 nM
microcystin (to inhibit potential dephosphorylation of PNUTS) for
2 h at 4 °C. Filters were washed with PBS/Nonidet P-40, and
bound PP1 was detected by immunoblotting using PP1 Metabolic Labeling--
PC12 cells were incubated in 200 µCi/ml of [32P]inorganic phosphate (PerkinElmer Life
Sciences) and phosphate-free, serum-free Dulbecco's modified Eagle's
medium for 2 h. After metabolic labeling, cells were washed three
times with PBS, harvested by lysis in an immunoprecipitation buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1 mM
Na3VO4, 1 mM PMSF, 0.5% Nonidet
P-40, 20 mM NaF), and clarified by centrifugation at
14,000 × g for 10 min. Unlabeled HEK293 cells,
incubated in the absence or presence of 8-Br-cAMP, were lysed in the
same way. Lysates were incubated with control IgG or anti-PNUTS
antibody in the absence or presence of antigen peptide, followed by the
addition of protein A-agarose beads. Immunoprecipitated proteins were
eluted from the protein A-agarose with SDS sample buffer and separated
by SDS-PAGE (10% acrylamide). Gels were dried, and proteins were
visualized by either autoradiography (32P-labeled samples)
or by immunoblotting with PNUTS or PP1 Phosphorylation of GST-PNUTS Fusion Proteins by
PKA--
Phosphorylation reactions were performed using the protein of
interest and the catalytic subunit of PKA (40 µg/ml) in an incubation mixture using 50 mM HEPES, pH 7.4, 10 mM
MgCl2, 1 mM EGTA at 30 °C. Reactions were
initiated by the addition of ATP (50 µM) in the absence
or presence of [ Phosphopeptide Mapping and Phosphoamino Acid Analysis--
After
autoradiography, gel pieces containing 32P-labeled
GST-PNUTS-(382-433), GST-PNUTS-(382-486), or PNUTS were re-swollen in
destain (50% methanol, 10% acetic acid in water), washed twice with
50% methanol, and dried. Gel pieces were then incubated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (50 µg/ml, Worthington) in 50 mM
NH4HCO3, pH 8.0 (1 ml), for 18 h at
37 °C. The supernatants containing the soluble phosphopeptides were
recovered after centrifugation. The extraction efficiency (~85%) was
quantified by Cerenkov counting of the gel pieces and supernatants
before and after digestion. Two-dimensional phosphopeptide mapping was
performed as described previously (38). For phosphopeptide mapping,
electrophoretic separation was at pH 3.5 for 90 min at 400 V, and
ascending chromatography was in pyridine/n-butyl
alcohol/acetic acid/water (15:10:3:12). The pattern of tryptic
phosphopeptides was detected by autoradiography. For phosphoamino acid
analysis, ~10% of the digested sample was hydrolyzed in HCl for
1 h at 110 °C. Lysates were lyophilized and resolved in pH 1.9 buffer containing standard phosphoamino acids
(o-phospho-DL-serine,
o-phospho-DL-threonine, and
o-phospho-DL-tyrosine from Sigma). Phosphoamino
acids were separated by one-dimensional thin layer electrophoresis.
Standard amino acids were visualized by ninhydrin staining, and
autoradiography was used to detect phosphorylated amino acids.
RNA Gel Retardation Assay--
GST fusion proteins (10-40 ng)
were incubated with ~5 ng of 32P-radiolabeled In Vitro Transcription and Translation--
cDNA encoding
full-length PNUTS was subcloned into the pGEM T vector (Promega); the
plasmid was linearized by digestion with ScaI and used as a
template for RNA synthesis with T7 polymerase. The resulting RNAs were
translated in rabbit reticulocyte lysate in the presence of
[35S]methionine according to the manufacturer's
suggested conditions (Amersham Biosciences). Translated protein was
analyzed SDS-PAGE (10% acrylamide) and autoradiography.
Ribonucleotide Homopolymer and ssDNA Binding Assays--
Assays
were initiated by addition of 25 µl of ribonucleotide
homopolymer-agarose or ssDNA-agarose into binding buffer with various
GST fusion proteins (total volume of 125 µl of 10 mM
HEPES, pH 7.4, 2 mM MgCl2, 0.1% Triton X-100,
3 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 0.05 mM EDTA, 0.1 M NaCl, or other salt at the
indicated concentrations, 1 mM PMSF) at 4 °C. After 30 min of incubation, beads were centrifuged and washed 6 times with 0.5 ml of binding buffer, and proteins were eluted with SDS sample buffer.
Samples were analyzed by SDS-PAGE (10% polyacrylamide) and
immunoblotting using an anti-GST antibody.
Binding of in vitro translated protein to ribonucleotide
homopolymer-agarose was performed essentially as described (39). An
equivalent of 105 cpm of 35S-labeled in
vitro translation product and 25 µl of homopolymer RNA beads
were incubated at 4 °C for 10 min in a total volume of 0.25 ml of
binding buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM
MgCl2, 0.5% Triton X-100, at the salt concentrations
indicated). The beads were pelleted by a brief centrifugation and
washed 5 times with 0.5 ml of binding buffer, and protein was eluted
with SDS sample buffer. Samples were analyzed by SDS-PAGE (10%
acrylamide) and autoradiography.
Ribonucleotide Homopolymer Pull-down Assay and Poly(G)-Agarose
Column Chromatography--
HEK293T cells were lysed by brief
sonication in buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl,
0.6 mM PMSF, 20 µg/ml leupeptin and antipain, 10 µg/ml
pepstatin A and chymostatin, 0.5% Nonidet P-40). Lysates were
centrifuged at 15,000 × g for 20 min. The supernatant
(400 µl, 500 µg of protein) was mixed with 50 µl of homopolymer
RNA beads and incubated at 4 °C for 1 h. The beads were
centrifuged and washed 5 times with 0.5 ml of lysis buffer containing
0.25 M NaCl, and proteins were eluted in 50 µl of SDS
sample buffer. Samples were analyzed by SDS-PAGE (10% acrylamide) and
transferred to polyvinylidene difluoride membrane (Immobilon-P,
Millipore). Proteins were detected by immunoblotting using antibodies
against PNUTS, PP1
For poly(G) column chromatography, 2 ml of the supernatants (2 mg of
protein) were mixed with 200 µl of poly(G) and incubated at 4 °C
for 1 h. Beads were washed once with 1 ml of lysis buffer and
loaded onto a column, which was then washed extensively with lysis
buffer. Bound proteins were eluted with a linear 0.1-2.0 M
NaCl gradient in lysis buffer. Fractions (0.5 ml) were collected, and
proteins in each fraction were analyzed by SDS-PAGE and immunoblotting with antibodies against PNUTS, PP1 Characterization of Binding of PP1 to PNUTS and Effect on Cell
Viability--
Previous studies (30) have shown that full-length PNUTS
and PNUTS-(309-872) (the protein product of clone 14 originally isolated in the yeast two-hybrid screen) were able to interact with
PP1
The viability of the 293T cells was significantly affected by
transfection with many of the PNUTS plasmids. Notably, all of the PNUTS
fragments that bound strongly to PP1 caused cell death (Fig. 1).
Neither a nuclear localization signal (a KKKRK motif at residues
157-161) nor the C-terminal region (residues 590-872) was required
for cell toxicity. Expression of PNUTS-(357-486) caused cell toxicity
more effectively than PNUTS-(357-433) or PNUTS-(309-433), suggesting
that residues 434-486 contributed to regulation of cell viability but
were not required for PP1 binding.
PNUTS Contains Closely Associated PP1 Binding and Inhibitory
Domains--
Our previous studies showed that a GST fusion protein
containing residues 309-872 potently inhibited the phosphorylase
phosphatase activity of PP1 in vitro (30). The results shown
in Fig. 1 and other preliminary studies indicated that both PP1 binding
and inhibition appeared to be contained within residues 382-537 of PNUTS. Various GST fusion proteins containing residues 382-537 of
PNUTS were expressed in bacteria, purified, and incubated with a 293T
cell extract. PP1 co-precipitated with GST-PNUTS fusion proteins
containing residues 382-486, 382-450, 382-444, 382-438, 382-433,
and 382-417 (Fig. 2). Further C-terminal
deletion of amino acids 410-417 (GST-PNUTS-(382-409)) significantly
reduced PP1 binding, and PP1 did not bind to GST-PNUTS-(404-537).
Analysis of residues 382-409 of PNUTS identified a sequence,
Arg-Lys-Thr-Val-Thr-Trp, that is similar to the
(Arg/Lys)-(Arg/Lys)-X-(Val/Ile)-X-Phe docking motif found in many PP1-binding proteins. The interaction of PP1 with
GST-PNUTS-(382-433) was completely abolished by mutation of
Trp401, reduced by mutation of Val399, but not
significantly affected by mutation of Lys397 or
Arg396 (all mutations to alanine). These results indicate
that Trp401 appears to functionally correspond to the
phenylalanine in the (Arg/Lys)-(Arg/Lys)-X-(Val/Ile)-X-Phe motif and
is essential for binding of PP1 to PNUTS.
Notably, of the GST fusion proteins examined, only GST-PNUTS-(382-486)
and PNUTS-(382-450) inhibited PP1 activity, both with very potent
IC50 values of ~0.1 nM (Figs.
2 and 3A and Table
I, and data not shown). Deletion of the
PP1 docking motif (GST-PNUTS-(404-537)) resulted in loss of PP1
inhibition. Surprisingly, deletion of only 6 residues from the C
terminus of GST-PNUTS-(382-450) almost completely abolished PP1
inhibitory activity (GST-PNUTS-(382-444) at 1 µM reduced
PP1 activity by less than 5%, see Fig. 3A). Consistent with
the binding studies, mutation of Trp401 resulted in an
almost complete loss of PP1 inhibition (an increase in IC50
of more than 4 orders of magnitude), whereas mutation of
Val399 increased the IC50 value by almost 2 orders of magnitude (Table I). Mutation of Lys397 increased
the IC50 value by ~5-fold, whereas mutation of
Arg396 had no effect. Together, these results suggest that
inhibition of PP1 by PNUTS is mediated by two small regions between
residues 382 and 450. Residues 399-402 include Val399 and
Trp401 that are necessary for binding to PP1, and residues
445-450 are necessary for inhibition of PP1.
Regulation of PP1 Activity by Peptides Encompassing the PP1-binding
Site of PNUTS--
We examined in more detail the features of PNUTS
surrounding the PP1 docking motif. GST-PNUTS-(382-433) (at only 30 nM) antagonized the ability of thiophospho-DARPP-32-(1-39)
to inhibit PP1 activity as demonstrated by an increase in the
IC50 by more than 2 orders of magnitude (Fig. 3B
and Table I). However, mutation of Trp401
(GST-PNUTS-(382-433;W401A)) resulted in a PNUTS fragment that was
unable to antagonize the inhibitory action of
thiophospho-DARPP-32-(1-39) (Fig. 3B). A shorter synthetic
peptide encompassing residues 392-415 of PNUTS was also very effective
at antagonizing the inhibitory action of phospho-DARPP-32 (Fig.
3C and Table I) (note here that full-length
thiophospho-DARPP-32 and higher concentrations of competing peptide
were used). However, a shorter peptide PNUTS-(392-408) was much less
effective in antagonizing the actions of thiophospho-DARPP-32, suggesting that residues 409-415 (Glu-Tyr-Phe-Tyr-Phe-Glu-Leu) contribute to the binding of PNUTS to PP1. Similar results were obtained when PP1 was inhibited using various concentrations of spinophilin or inhibitor-2 (data not shown). As expected, mutation of
Trp401 in PNUTS-(392-408) rendered the peptide completely
ineffective in competing with thiophospho-DARPP-32 (Fig.
3C). However, surprisingly, PNUTS-(392-415;W401Y) was very
effective in antagonizing the actions of thiophospho-DARPP-32.
In the studies of the various PNUTS peptides as antagonists of the
actions of PP1 inhibitors, we noted an unusual property of
PNUTS-(392-415). PNUTS-(392-415) alone was able to activate consistently PP1 activity by ~30-40% (Fig. 3D).
PNUTS-(392-408) was much less effective as an activator, and
PNUTS-(392-408;W401A) had no effect on PP1 activity. Consistent with
the ability of the PNUTS peptide to activate PP1 via the C-terminal
docking site where Trp401 binds, PNUTS-(392-415) had no
effect on a PP1/PP2A chimeric enzyme in which the C terminus of PP1
(residues 274-330) was replaced by the equivalent residues of PP2A
(data not shown) (see also Ref 40).
Phosphorylation of PNUTS Regulates Its Interaction with
PP1--
Examination of the amino acid sequence of residues 382-450
of PNUTS revealed the presence of several consensus sites for
phosphorylation by PKA. In addition, in preliminary studies using PC12
cells metabolically labeled with [32P]phosphate,
full-length PNUTS was found to be phosphorylated (Fig.
4A). In vitro,
GST-PNUTS-(382-433), -PNUTS-(382-450), and -PNUTS-(382-486) were all
found to be excellent substrates for PKA (Fig. 4B).
GST-PNUTS-(382-486) was more efficiently phosphorylated, and to a
higher stoichiometry, than GST-PNUTS-(382-450). Moreover, tryptic
phosphopeptide mapping studies indicated that GST-PNUTS-(382-486) contained two major phosphorylation sites, whereas GST-PNUTS-(382-433) contained only one major phosphorylation site (Fig. 4C, note
the two phosphopeptides are likely derived from alternative tryptic digestion). Notably, GST-PNUTS-(382-486) contains two potential PKA
consensus sites,
Arg-Lys-Arg-Lys-Thr-Val-Thr-Trp (residues 394-401, either Thr residue might be phosphorylated) and
Arg-Arg-Leu-Ser-His (residues 448-452), suggesting the
possibility that phosphorylation of Ser451 might explain
the increased level of phosphorylation of GST-PNUTS-(382-486). However, phosphoamino acid analysis of the 32P-labeled
GST-PNUTS proteins indicated that phosphorylation occurred almost
exclusively on threonine (Fig. 4D). These results indicate that Ser451 is not phosphorylated but that one or more
threonine residues close to the PP1 docking motif are phosphorylated by
PKA.
To determine whether phosphorylation of PNUTS by PKA might affect the
interaction with PP1, GST-PNUTS-(382-433) was phosphorylated by PKA
and [32P]ATP for various times (Fig.
5A, top panel). Maximal
phosphorylation was reached between 40 and 70 min (a maximal
stoichiometry of ~1 mol/mol was determined). The phosphorylated
samples were separated by SDS-PAGE, transferred to Immobilon-P
membrane, and incubated with PP1 The C Terminus of PNUTS Binds to mRNA and Single-stranded
DNA--
PNUTS contains multiple closely spaced repeats of the amino
acid sequence, RGG, a motif often found in RNA-binding proteins (32)
(Fig. 6A). The RGG motifs are
followed by a region with several imperfect repeats of a sequence rich
in histidine and glycine. The extreme C-terminal region then contains a
putative zinc finger domain. These features together with its nuclear
localization suggested that PNUTS might interact with nucleic acids. To
initially examine the interaction with RNA, a gel retardation assay was performed with various GST fusion proteins and 32P-labeled
Selective Binding of a PNUTS-PP1 Complex to Different
Ribonucleotide Homopolymers--
The entire rat PNUTS cDNA was
transcribed in vitro using T7 polymerase and translated in a
rabbit reticulocyte system in the presence of
[35S]methionine. Three radiolabeled bands were detected
using SDS-PAGE, the largest corresponding to full-length PNUTS
(apparent molecular mass ~110 kDa) (Fig.
7A). The other bands are
likely to be proteolytic fragments or incomplete translation products.
Full-length PNUTS bound to poly(A) and poly(G) but not to poly(U) or
poly(C) (Fig. 7A). As a control, in vitro
translated hnRNP K, which is a known poly(C)-specific RNA-binding
protein, bound selectively to poly(C) (Fig. 7A).
Because PNUTS is able to interact directly with PP1 as well as with
RNA, we hypothesized that the PNUTS-PP1 complex may bind to RNA. To
examine this possibility, 293T cell lysates were incubated with
poly(A)-, poly(C)-, poly(G)-, and poly(U)-agarose beads, and bound
proteins were eluted and analyzed by immunoblotting. Consistent with
the properties of the recombinant GST-PNUTS fusion proteins, endogenous
PNUTS was efficiently precipitated by poly(A) and poly(G) but not by
poly(U) and poly(C) (Fig. 7B). Moreover, PP1
To characterize further the PNUTS/PP1/RNA interaction, 293T cell
lysates were loaded onto a poly(G)-agarose column (equilibrated in a
buffer containing 100 mM NaCl), and the bound proteins were eluted with a linear salt gradient (Fig.
8). PNUTS and both PP1 Different Subdomains of PNUTS Bind to Poly(A) and Poly(U)--
In
order to characterize further the ribonucleotide-binding properties of
PNUTS, GST-PNUTS fragments were incubated with poly(A), poly(C),
poly(G), and poly(U) at 100 mM NaCl. GST-PNUTS-(617-872) and GST-PNUTS-(617-837) bound with very similar efficiency to both
poly(A) and poly(U) (Fig. 9). However,
further deletion of the histidine/glycine-rich region
(GST-PNUTS-(617-762)) resulted in a preferential decrease in binding
to poly(A). Partial or complete deletion of the RGG motifs led to
complete loss in binding to the poly(G) ribonucleic acid homopolymer.
Increasing the bead volumes of poly(C) and poly(U) up to 4 times
compared with poly(A) did not alter the inability of PNUTS to bind to
poly(U) and poly(C) (data not shown).
In the present study, we have characterized the properties of the
interactions between PNUTS and both PP1 and RNA. The results obtained
suggest that PNUTS may bind to specific types of RNA implicating PP1 in
specific functions within the nucleus. Studies of the interaction with
PP1 revealed that PNUTS contains two closely associated subdomains in
the center of the protein within ~50 amino acids (residues 400-450).
These include a high affinity PP1-binding domain located within
residues 397-401 of PNUTS and a distinct inhibitory region located
within residues 445-450. The binding domain,
Lys397-Thr-Val-Thr-Trp401, resembles the
consensus motif (Arg/Lys-Arg/Lys-Val/Ile-X-Phe) found in
many other PP1 regulatory subunits except for the replacement of Phe
with Trp. Consistent with residues 397-401 of PNUTS binding to a
common docking site in PP1, competition studies indicated that PNUTS
peptides as short as residues 392-408 were potent antagonists of the
inhibitory actions of thiophospho-DARPP-32. Site-directed mutagenesis
studies of PNUTS indicated that the order of importance of amino acids
contributing to the association with PP1 is Trp401 > Val399 A notable feature of the PP1 docking motif in PNUTS is the presence of
Trp instead of Phe as the most important binding residue (30). In an
analysis of a random peptide library that bound to PP1, Phe and Trp
were identified with equal frequency in the interacting peptides (42).
However, PNUTS appears to be the first well characterized PP1-binding
protein that contains Trp within the PP1-docking motif. The affinity of
PNUTS for PP1 is suggested from the PP1 inhibition and competition
assays to be very high, presumably being in the low nM
range. It is possible that the presence of Trp401
contributes to the high affinity, but it is clear that parts of PNUTS
outside of the minimal PP1-docking motif also play a role.
PNUTS-(392-408) was very effective in competing against the inhibitory
action of thiophospho-DARPP-32, but addition of residues 409-415 made
PNUTS-(392-415) a much more effective antagonist. In addition,
mutation of Trp401 to Tyr had only a small effect on the
antagonist properties of PNUTS-(392-415), and mutation of
Phe11 in DARPP-32 to Trp did not affect its inhibitory
potency significantly (41), consistent with the idea that the presence
of Phe or Trp as the key docking residue is not critical. The results
with PNUTS also support the idea that like DARPP-32, inhibitor-1 and
inhibitor-2, targeting proteins are likely to bind to PP1 via multiple
subdomains (8, 36, 41, 43, 44).
PNUTS is a highly potent inhibitor of PP1, with an IC50
value of ~10 The PP1-binding domain of PNUTS contains a number of potential
consensus sites for phosphorylation. These include a site at Thr398 within the PP1-docking motif and at
Ser451 close to the inhibitory subdomain. Our results show
that a polypeptide encompassing residues 382-433 of PNUTS was
efficiently phosphorylated by PKA in vitro, and the
resulting phosphorylation decreased its affinity for PP1 in
vitro and also in intact cells following stimulation of PKA. Based
on analysis of phosphorylation of different PNUTS fragments, and
phospho-amino acid analysis, Thr398 is a likely candidate
for phosphorylation by PKA; notably Ser451 was not
phosphorylated in vitro. In the human homologue of PNUTS, Thr398 is replaced by serine (31). Therefore, these results
suggest that the interaction of PNUTS with PP1 may be negatively
regulated by phosphorylation in an analogous manner to that observed in studies of a few other PP1-binding proteins. For example,
phosphorylation of Ser67 in the glycogen-binding
GM subunit results in the dissociation of
PP1-GM complex (5, 45). Similarly, NIPP1 has
phosphorylation sites for PKA (Ser199) and CK2
(Ser204) that flank the PP1-binding motif, and the
phosphorylation of either serine impairs PP1 binding and reduces the
activity of NIPP1 as a PP1 inhibitor (46, 47). We have also shown that phosphorylation of the brain-specific actin-binding protein, neurabin, at Ser461 by PKA significantly reduces its binding to PP1
(48). These observations together with our present data suggest that
reversible phosphorylation of a site at or near the
Arg-Lys-Arg/Lys-Val/Ile-X-Phe/Trp motif of PP1 regulatory
proteins may be a common control mechanism that adds to the complexity
of PP1 regulation in the nucleus and other cellular compartments.
The present study shows that the C-terminal region, including the seven
closely repeated RGG boxes and particularly the histidine/glycine-rich domain, mediates the interaction of PNUTS with RNA and ssDNA. However,
the zinc finger region did not appear to play any role in the
interaction with RNA or ssDNA. The RGG box was first described as an
RNA-binding domain in hnRNP U (32, 49). Typically, multiple RGG boxes
are found, with as few as 6 and as many as 18 being present in an
RNA-binding protein. The RGG boxes are also frequently found adjacent
to or interspersed with other RNA-binding motifs such as KH and RBD
domains (32). In some cases such as the fragile X mental retardation
protein (FMRP) or the TLS protein, the RGG boxes have intrinsic
RNA-binding properties (50-52), whereas in other cases such as
nucleolin, the RGG boxes complement the specificity of additional
RNA-binding domains (53).
Together, the histidine/glycine region, and to a lesser extent the RGG
boxes, appear to be responsible for the high affinity interaction of
PNUTS with RNA and may be responsible for specifying the interaction of
PNUTS with specific mRNAs in the nucleus. Our studies with RNA
homopolymers indicated that PNUTS has high selectivity for binding to
poly(A) or poly(G) but not for poly(U) or poly(C). The RGG boxes of
PNUTS appeared to be involved in the preferential binding to poly(G).
In other cases, the RGG boxes of hnRNP U exhibited highest binding to
poly(G) and intermediate binding to poly(U) (49); Nopp44/46 has been
shown to bind preferentially to poly(U) (54), and FMRP showed selective
binding to poly(G) and poly(U) (54). Interestingly, in the splicing
factor, TLS, an N-terminal group of RGG boxes showed selective binding
to poly(U), whereas a C-terminal group of RGG boxes showed selective
binding to poly(G) (52). The amino acid sequences surrounding the RGG
boxes are often rich in aromatic amino acids. However, in PNUTS, the
amino acid composition of the RGG domain differs from that of other proteins by the abundance of proline. Therefore, it is likely that the
specific amino acid residues surrounding the RGG boxes influence the
specificity and avidity of RNA interaction (54). The histidine/glycine
region of PNUTS was also necessary for the interaction with native
mRNA and ssDNA and perhaps is involved in the preferential binding
to poly(A). The histidine/glycine-rich domain is unique to PNUTS, and
presumably the repetitive feature of the domain plays some role in the
specific interaction with RNA and ssDNA.
The results from our studies indicate that PP1 associates indirectly
with RNA through its interaction with PNUTS and suggests a role for
PNUTS in anchoring PP1 to RNA-associated complexes. Several lines of
evidence indicate that PP1 plays a number of roles in mRNA splicing
by reversing the actions of multiple protein kinases (9, 55). PP1 can
regulate initial steps in splicing by dephosphorylating factors
necessary for spliceosome assembly (17, 18). PP1 interacts with NIPP1
in nuclear speckles, and NIPP1 appears to play a role in a late stage
of spliceosome assembly. However, the spliceosome function of NIPP1
seems not to be related to PP1 binding (16, 56), suggesting the
possibility that NIPP1 plays an additional role to target PP1 to the
splicing machinery. The PP1 It is also possible that PNUTS serves to target PP1 to the nucleus for
functions of the phosphatase in addition to, or instead of, regulation
of RNA processing. Recent studies (59) have highlighted the close
physical association of the transcriptional and splicing machinery.
Moreover, although a subpopulation of PP1 is localized in the nucleus,
recent studies (60) indicate that this localization is dynamic, and PP1
can rapidly move between subnuclear compartments. Within the nucleus
PP1 plays an important role in the dephosphorylation of transcription
factors such as CREB and Sp1 (9, 12, 61). PP1 dephosphorylates
Ser133 of CREB which is phosphorylated by a number of
kinases, including PKA and multiple
Ca2+-dependent kinases (12). During
de-differentiation associated with liver regeneration, PP1 was also
identified as the enzyme that dephosphorylated Sp1, reversing the
action of CK2 (61). PP1 is also an important phosphatase involved in
regulation of the retinoblastoma protein, pRb (62). PP1 can bind to pRb
and selectively dephosphorylate specific sites of pRb and appears to be
the phosphatase responsible for dephosphorylation of pRb at the time of
mitotic exit. Moreover, biochemical studies have identified a high
molecular complex that dephosphorylates pRb (63) and that contains PP1
and a 110-kDa protein that appears to be identical to PNUTS (62). Thus,
PNUTS may not only serve to target PP1 to the nucleus but to influence
its specificity toward nuclear substrates.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, -
1, -
2, and -
), is widely
distributed in various subcellular compartments (1, 4) where it is
regulated by association with a growing number of identified regulatory
proteins. These regulatory proteins include several heat-stable
inhibitors, such as inhibitor-1, its neuronal homologue DARPP-32, and
inhibitor-2, which are controlled by phosphorylation (1, 2). PP1 is
also regulated by a family of proteins, termed targeting subunits, that
direct the catalytic subunit of PP1 to specific subcellular locations
and also influence the specificity of the enzyme at these sites (1, 2,
5). For example, the glycogen-binding proteins, GM and
GL, target PP1 to glycogen and enhance the activity of PP1
toward glycogen synthase. Similarly, the myofibril-binding protein,
M110, mediates the association of PP1 with the myofibrils of skeletal muscle and smooth muscle and stimulates the activity of PP1
toward phosphorylated myosin light chain (6). Despite little overall
amino acid sequence homology, several studies have identified a common
docking motif in many of the targeting proteins that binds to a defined
region of PP1 removed from the active site of the enzyme (7, 8).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and
PP1
antibodies were prepared as described (30, 35). Anti-glutathione
S-transferase (GST) monoclonal antibody and anti-hnRNP C
monoclonal antibody were generous gifts from Drs. E.-Y. Choi and M.-Y.
Choi, respectively. Full-length DARPP-32 was expressed in bacteria,
purified, and phosphorylated at Thr34 with PKA and ATP
S
essentially as described (36).
antibody, followed
by horseradish peroxidase-conjugated secondary antibody. Proteins
were visualized using ECL.
-D-galactopyranoside at 30 °C for
3 h. Cells were collected by centrifugation, resuspended in lysis
buffer (20% sucrose, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF,
150 mM NaCl, and 1% Triton X-100), and lysed by sonication
on ice. Lysates were centrifuged at 12,000 × g for 20 min, and supernatants were loaded onto a column containing glutathione-agarose beads (Sigma) and washed extensively with PBS.
Fusion proteins were eluted with 5 mM glutathione, 50 mM Tris-HCl, pH 8.0. Protein purity was analyzed by
SDS-PAGE and stained with Coomassie Brilliant Blue, and protein
concentration was determined using the BCA assay.
(~50
pM). All components except [32P]phosphorylase
a were preincubated at 30 °C for 2 min. Assays were
initiated by addition [32P]phosphorylase a;
incubations were performed at 30 °C for 10 min and then terminated
by the addition of 100 µl of 10% (w/v) trichloroacetic acid. Samples
were centrifuged for 3 min, and 32P in the
supernatant was measured by Cerenkov counting.
antibody.
antibody.
antibodies.
-32P]ATP. Reactions were terminated at
various time points by dilution of the reaction mixture into SDS-PAGE
sample buffer, and the stoichiometry of phosphorylation was assessed
after SDS-PAGE and autoradiography.
-globin
mRNA in binding buffer (total volume 40 µl) containing 10 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM MgCl2, and 40 ng of RNasin (Roche Molecular
Biochemicals). Samples were incubated for 10 min on ice. After addition
of 5 µl of electrophoresis buffer containing 10% glycerol and 0.01%
bromphenol blue, reaction mixtures were separated on a 4% native
polyacrylamide gel for 2-3 h at 20 mA (about 6 V/cm) at room
temperature. After electrophoresis, gels were dried and exposed to
Hyperfilm MP.
, and hnRNP C, and detection was by the ECL method.
, PP1
, and hnRNP C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. To characterize further the interaction of PP1 and PNUTS
protein, various FLAG-tagged PNUTS fragments were expressed in HEK293T
cells, proteins were immunoprecipitated with anti-FLAG antibody, and
PP1
was detected in the immunoprecipitates (Fig. 1). All the PNUTS fragments were
expressed at equivalent levels (data not shown). PP1 was bound to
fragments containing residues 309-872, 309-589, 309-433, 357-537,
357-486, 357-433, 143-433, and a fragment between residues 143 and
872 with an internal deletion of residues 434-589. A low level of PP1
was found to bind to PNUTS-(309-401), but no binding was detected for
PNUTS-(404-537), PNUTS-(590-872), and PNUTS-(724-872) or for two
fragments with an internal deletion of residues 254-589. Together,
these results indicate that PP1 binds to PNUTS between residues 357 and
433, with the C-terminal boundary of the binding site close to residue
401.
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Fig. 1.
Characterization of binding of PP1 to PNUTS
and effect on cell viability. A, the domain
organization of PNUTS is shown at the top of the figure. The
PP1-binding domain is localized approximately in the middle
of the molecule (black shading), and a putative motif
(KKKRK) for nuclear localization is at residues 157-161 (gray
shading). The C terminus of PNUTS contains three distinct domains
potentially involved in RNA binding (detailed in Fig. 6A).
293T cells were transiently transfected with various FLAG-tagged PNUTS
mutants (N- and C-terminal amino acid number is indicated within each
rectangle). Cell toxicity was estimated by counting viable
cell numbers 24 h after transfection. PP1 binding was measured as
shown in B. For cell toxicity, indicates no effect; + and
++ indicate slight and potent toxicity, respectively.
B, cells were lysed and anti-FLAG antibody was used to
immunoprecipitate each PNUTS mutant. Immunoprecipitated (IP)
samples were analyzed by immunoblotting (WB) using a PP1
antibody. Lane 1 shows untransfected cells; lanes
2-15 correspond to the mutants shown in A. Qualitative
analysis of the amount of co-precipitated PP1 is shown in the 1st
column in A. Results are representative of three
experiments.
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Fig. 2.
PNUTS contains closely associated PP1 binding
and inhibitory domains. A, the amino acid sequence
between residues 390 and 455 of PNUTS is shown, with important residues
shown in boldface. Various GST fusion proteins were prepared
as indicated. The N- and C-terminal amino acid number for mutants 2-4
is shown within each rectangle. Fusion proteins 5-8 all
started at residue 382. Fusion proteins 10-14 all contained residues
382-433, together with the point mutations as indicated.
B, the GST fusion proteins were bound to
glutathione-Sepharose and then incubated with PC12 cell lysates for
1 h. Bound proteins were eluted and analyzed by immunoblotting
using a PP1 antibody. Lane 1 contained GST as a control.
Inhibition of PP1 activity was assayed using
[32P]phosphorylase a as a substrate, in the
presence of various concentrations of each GST-PNUTS protein. Some
representative data are shown in Fig. 3. Qualitative analysis of the
amount of PP1 bound to each mutant, and of the level of PP1 inhibition
is shown in the columns in A. For inhibition, + indicates an
IC50 <0.1 nM;
indicates an IC50
>500 nM. Results are representative of three
experiments.
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Fig. 3.
Regulation of PP1 activity by PNUTS.
A, the effect of GST-PNUTS fusion proteins on PP1
activity was determined using 10 µM
[32P]phosphorylase a as substrate.
GST-PNUTS-(382-450) (open square), -(382-450;W401A)
(filled square), -(382-450;V399A) (open
diamond), -(382-450;K397A) (filled circle),
-(382-450;R396A) (open circle), and GST-PNUTS-(382-444)
(open triangle). B, the activity of PP1 was
measured using [32P]phosphorylase a as
substrate with the indicated concentrations of
thiophospho-DARPP-32-(1-39) (P-D32-(1-39)), in
the absence (CON, filled square) or presence of
30 nM GST-PNUTS-(382-433) (open square) or 30 nM GST-PNUTS-(382-433;W401A) (open circle).
Results are representative of at least three separate experiments.
C, the activity of PP1 was measured using
[32P]phosphorylase a as substrate with the
indicated concentrations of thiophospho-DARPP-32 (S-DARPP),
in the absence (CON, filled circle) or presence
of PNUTS-(392-415) (10 µM) (filled square),
PNUTS-(392-408) (10 µM) (filled diamond),
PNUTS-(392-408;W401A) (10 µM) (open diamond),
or PNUTS-(392-415;W401Y) (20 µM) (open
square). Results are representative of at least three separate
experiments, and the error bars show S.D. Activity was
normalized to that measured in the presence of 10 µM
PNUTS. D, the activity of PP1 was measured using
[32P]phosphorylase a as substrate with the
indicated concentrations of PNUTS-(392-415) (filled
square), PNUTS-(392-408) (filled diamond), or
PNUTS-(392-408;W401A) (open diamond).
Regulation of PP1 activity by GST-PNUTS fusion proteins and peptides
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Fig. 4.
Phosphorylation of PNUTS within its
PP1-binding domain. A, phosphorylation of PNUTS in
intact cells. PC12 cells were incubated with 200 µCi/ml
[32P]inorganic phosphate in phosphate-free medium for
2 h. PNUTS was immunoprecipitated using control (Con
IgG) or PNUTS antibody (Ab), in the absence or presence
of a PNUTS peptide antigen, as indicated. 32P
incorporation was analyzed by SDS-PAGE and autoradiography.
B, GST-PNUTS-(382-433), GST-PNUTS-(382-450), or
GST-PNUTS-(382-486) was phosphorylated by PKA for indicated times by
using [ -32P]ATP. Samples were separated by SDS-PAGE,
and 32P-labeled GST-PNUTS proteins were detected by
autoradiography. C, two-dimensional phosphopeptide maps
of 32P-labeled GST-PNUTS-(382-433) (right
panel) or full-length PNUTS immunoprecipitated from
32P-labeled cells (left panel). Proteins were
excised from gels as shown in lane 2 of A and
lane 5 of B and were digested with trypsin. The
resultant phosphopeptides were separated by two-dimensional
phosphopeptide mapping and visualized by autoradiography; O
shows the origin. D, phosphoamino acid analysis of
32P-labeled GST-PNUTS-(382-433), GST-PNUTS-(382-486), and
full-length PNUTS immunoprecipitated from 32P-labeled
cells. Proteins from gels as shown in lane 2 of A
and lanes 5 and 15 of B were
hydrolyzed and subjected to phosphoamino acid analysis. The positions
of ninhydrin-stained phosphoamino acid standards (pSer,
pThr, and pTyr) are indicated with
arrows.
. The amount of PP1 bound to
GST-PNUTS-(382-433) decreased in parallel to the increase in
phosphorylation (Fig. 5A, middle panel). These results
indicate that phosphorylation of PNUTS by PKA within the site of PP1
binding blocks the association of PNUTS with PP1. We further
investigated whether PKA regulates interaction of PNUTS with PP1 in
intact cells. In intact HEK293 cells incubated in the presence of
forskolin, PNUTS was phosphorylated largely on threonine (Fig.
4D). In addition, phosphopeptide mapping studies indicated
that PNUTS was phosphorylated at the same site as that phosphorylated
by PKA within GST-PNUTS-(382-433) (Fig. 4C). In parallel
studies, HEK293 cells were incubated in the absence or presence of
8-Br-cAMP (500 µM for 10 min), and the interaction between PP1
and PNUTS was examined following co-immunoprecipitation (Fig. 5B). By using antibodies specific for either PP1
or
PNUTS, the interaction between PNUTS and PP1 was shown to be
significantly reduced by activation of PKA with 8-Br-cAMP. Together
these results suggest that phosphorylation of PNUTS by PKA negatively
regulates the interaction of PNUTS with PP1.
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Fig. 5.
PKA-mediated phosphorylation of PNUTS
modulates its interaction with PP1. A, the
interaction of PP1 is abrogated by PKA-mediated phosphorylation of
PNUTS-(382-433). Top panel, GST-PNUTS-(382-433) was
phosphorylated by PKA for the indicated times using
[ -32P]ATP. Samples were separated by SDS-PAGE, and
32P-labeled GST-PNUTS-(382-433) was detected by
autoradiography. Middle panel, GST-PNUTS-(382-433) was
phosphorylated as described in the top panel, and samples
were separated by SDS-PAGE and transferred to Immobilon-P membrane. The
membrane was incubated with 100 ng/ml PP1
and 100 nM
microcystin; the membrane was washed, and then bound PP1 was detected
using PP1 antibody. Bottom panel, the membrane was stripped
and re-blotted using anti-PNUTS antibody to determine the amount of
GST-PNUTS-(382-433) in each lane. B, activation of PKA
reduces the interaction of PP1 with PNUTS. HEK293 cells were incubated
without (
) or with (+) 8-Br-cAMP (500 µM) for 10 min.
Either PP1
or PNUTS was immunoprecipitated using specific antibodies
as indicated, and samples were analyzed by SDS-PAGE and immunoblotting
as indicated.
-globin mRNA. GST-PNUTS-(617-872) retarded the mobility of
-globin mRNA, but deletion of the putative zinc finger domain and most of the region rich in histidine and glycine
(GST-PNUTS-(617-762)) resulted in a marked decrease in RNA binding
(Fig. 6B). Moreover, partial or complete deletion of the RGG
motif (GST-PNUTS-(617-726) or GST-PNUTS-(404-662)) led to an almost
complete loss of RNA binding. We next examined the abilities of PNUTS
fragments to bind to single-stranded DNA (ssDNA). Both
GST-PNUTS-(617-872) and GST-PNUTS-(617-837) bound effectively to
ssDNA (Fig. 6C). However, GST-PNUTS-(617-762),
GST-PNUTS-(617-726), or GST-PNUTS-(404-662) did not bind to ssDNA.
These results suggest that the C-terminal region of PNUTS, including
the histidine/glycine-rich motifs, is necessary for binding to both RNA
and ssDNA and that the RGG motifs may contribute to a lesser extent to
the binding to RNA.
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Fig. 6.
mRNA and single-stranded DNA binding
properties of GST-PNUTS C-terminal fusion proteins.
A, the C-terminal amino acid sequence of PNUTS. The
domain containing 7 RGG repeats is shown with the RGG sequences in
boldface and marked with an asterisk. Residues
751-834 (boxed) are rich in histidine and glycine and
contain two perfect and one imperfect repeats of 14 residues. Following
this, three additional pentamer repeats are found. Residues 844-863
(underlined) contain a cysteine/histidine-rich putative
Zn2+ finger motif with the signature
CX8CX5CX3H.
B, the interaction of various GST-PNUTS fusion proteins
(including those indicated) with -globin mRNA was analyzed using
a gel retardation assay. Each GST-PNUTS fusion protein (40 µg) was
incubated with 32P-labeled
-globin mRNA for 90 min
at 37 °C. Samples were analyzed using non-denaturing gel
electrophoresis and autoradiography. C, the interaction
of various GST-PNUTS fusion proteins with ssDNA was measured. Each
GST-PNUTS fusion protein (200 ng) was incubated with 25 µl of
ssDNA-agarose. Bound protein was analyzed by SDS-PAGE and immunoblotted
by using anti-GST monoclonal antibody.
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Fig. 7.
Selective binding of a PNUTS-PP1 complex to
different ribonucleotide homopolymers. A,
full-length PNUTS mRNA and hnRNP K were translated in
vitro using [35S]methionine and a rabbit
reticulocyte lysate. [35S]PNUTS was bound to the
indicated ribonucleotide, and bound protein was analyzed by SDS-PAGE
and autoradiography (left panel). For analysis of RNA
binding to hnRNP K, the binding buffer contained 500 mM
NaCl. Samples were analyzed by SDS-PAGE and hnRNP K was detected by
autoradiography (right panel). S indicates that
in vitro translated 35S-labeled protein that was
loaded directly on the gel as a control. B, protein
extract from 293T cells (~500 µg) was incubated with 50 µl of the
indicated ribonucleotide homopolymer (poly(U),
-(A), -(G), or -(C)) immobilized to
agarose beads at 4 °C for 1 h. Bound proteins were analyzed by
SDS-PAGE, and various proteins were detected by immunoblotting using
the indicated PNUTS, PP1, and hnRNP C antibodies. Cell lysate (25 µg,
CL) was loaded directly on the gel as a reference.
was found
only in the precipitates from poly(A)- or poly(G)-agarose beads. In
addition, depletion of PNUTS from 293T cell extracts by
immunoprecipitation using anti-PNUTS antibody significantly reduced the
amount of PP1 bound to poly(G)-beads (data not shown). As a
control, hnRNP C, one of the most abundant heterogeneous nuclear
ribonucleoproteins (hnRNPs) and known to bind to pre-mRNA, was
detected in all the precipitates.
and PP1
were
detected in the same fractions at salt concentrations ranging from 0.5 to 0.7 M. hnRNP C was also detected in fractions that
contained PNUTS and PP1 but was also found in fractions eluting at
higher ionic strength that did not contain PNUTS/PP1.
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Fig. 8.
Fractionation of PNUTS-PP1-RNA complexes
using poly(G)-agarose column chromatography. Protein extract from
293T cells (~5 mg) was incubated with 200 µl of poly(G)-agarose
beads at 4 °C for 1 h. The beads were loaded into a column and
extensively washed with lysis buffer. Bound proteins were eluted with a
linear gradient of 0.1-2.0 M NaCl. Fractions (0.4 ml) were
collected, and proteins were analyzed by SDS-PAGE and immunoblotting
using the indicated PNUTS, PP1, and hnRNP C antibodies. Cell lysate (25 µg) was loaded directly on the gel as a reference (extreme left
lanes in each panel).
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Fig. 9.
Characterization of the RNA binding
specificity of PNUTS. Various GST-PNUTS fusion proteins or GST
(200 ng) (S indicates the input for each fusion protein)
were incubated with 25 µl of the indicated ribonucleotide
homopolymers (poly(U), -(A), -(G), or
-(C)) immobilized to agarose beads. Bound protein was
analyzed by SDS-PAGE and immunoblotting using anti-GST monoclonal
antibody.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Lys397, a pattern consistent with
our previous studies of DARPP-32, where Phe11 and
Ile9 play critical roles, with Lys7 playing a
lesser role in the interaction with PP1 (8, 41). Therefore, it is
likely that the conserved docking motif of PNUTS interacts with the
exposed hydrophobic docking site in PP1 in a similar manner to DARPP-32
and other PP1-binding proteins.
10 nM using phosphorylase
a as substrate. Deletion mutagenesis studies identified a
short sequence, ETARRL (residues 445-450), that was responsible for a
greater than 104-fold factor in inhibitory potency. Like
DARPP-32 and inhibitor-1, the PP1 docking and inhibitory subdomains are
contained in a short stretch of amino acids, but unlike
phospho-DARPP-32 (phosphorylated at Thr34 by PKA), PNUTS
does not have to be phosphorylated in this subdomain to be an effective
inhibitor. Possibly, residues 445-450 of PNUTS interact with the
active site of PP1 in the manner of a pseudosubstrate, or these
residues may act to block interactions of phosphorylase at a
substrate-binding site that is situated close to but not within the
active site of the phosphatase. Surprisingly, in contrast to the highly
potent inhibitory actions of residues 382-450 of PNUTS, addition of
peptides encompassing just the docking motif resulted in activation of
PP1. This effect required the presence of Trp401 but was
more robust when residues 409-415 were included. The molecular basis
for this is not currently known, but may reflect an allosteric effect
that stabilizes the substrate-binding site or alters the active site.
isoform has also been found to interact
with the polypyrimidine tract-binding protein-associated splicing
factor, although the function of this interaction is not known (57).
Finally, in a recent study PP1 has been implicated in the control of
alternative splicing of caspase 9 and bcl-x genes in
lung adenocarcinoma cells (58). Initial studies of the localization of
PNUTS indicate that the protein exhibits a discrete punctate
nucleoplasmic staining pattern with some accumulation in the nucleolus
(30, 31). This pattern of localization and the fact that PNUTS
interacts with RNA is consistent with a specific function for the
protein in the nucleus. Most likely this would involve some aspect of RNA processing or transport of RNA within the nucleus and probably would involve the action of PP1 that is bound to PNUTS at these sites.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. M.-Y. Choi for providing anti-hnRNP C antibody and the cDNA for hnRNP C and Dr. M. M. Konarska for advice on the RNA gel retardation assay.
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FOOTNOTES |
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* This work was supported by Grant D00359 from the Korea Research Foundation and Vascular System Research Center (to Y.-G. K.), Grant 981-0508-038-2 from the Korea Science and Engineering Foundation (to Y.-G. K.), and by United States Public Health Grant MH40899 (to A. C. N. and P. G.).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.
¶ Present address: Dept. of Biochemistry and Molecular Vascular Biology, Kanazawa University Graduate School of Medical Science, 13-1, Takara-machi, Kanazawa 920-8640, Japan.
** Present address and to whom correspondence may be addressed: Dept. of Psychiatry, Yale University School of Medicine, New Haven, CT 06508. Tel.: 203-974-7725; Fax: 203-974-7724; E-mail: angus.nairn@yale.edu.
To whom correspondence may be addressed: Dept. of Biochemistry,
College of Natural Sciences, Kangwon National University, Chunchon,
Kangwon-Do 200-701, Korea. Tel.: 82-33-250-8517; Fax: 82-33-242-0459;
E-mail: ygkwon@kangwon.ac.kr.
Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M209621200
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ABBREVIATIONS |
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The abbreviations used are:
PP1, protein
phosphatase-1;
PNUTS, protein phosphatase-1 nuclear targeting subunit;
DARPP-32, dopamine- and cAMP-regulated phosphoprotein,
Mr 32,000;
PBS, phosphate-buffered saline;
GST, glutathione S-transferase;
ssDNA, single-stranded DNA;
FLAG, epitope tag of sequence DYKDDDDK;
PKA, protein kinase A;
hnRNP, human
nuclear ribonucleoprotein;
ECL, enhanced chemiluminescence;
PMSF, phenylmethylsulfonyl fluoride;
ATPS, adenosine
5'-O-(thiotriphosphate);
pRb, retinoblastoma protein;
CREB, cAMP-response element-binding protein.
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