From the Afdeling Biochemie, Faculteit Geneeskunde,
Katholieke Universiteit Leuven, B-3000 Leuven, Belgium and the
¶ Department of Pharmacology and Cancer Biology, Duke University
Medical Center, Durham, North Carolina 27710
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
NIPP-1 is a subunit of the major nuclear protein
phosphatase-1 (PP-1) in mammalian cells and potently inhibits PP-1
activity in vitro. Using yeast two-hybrid and
co-sedimentation assays, we mapped a PP-1-binding site and the
inhibition function to the central one-third domain of NIPP-1.
Full-length NIPP-1 (351 residues) and the central domain,
NIPP-1143-217, were equally potent PP-1 inhibitors
(IC50 = 0.3 nM). Synthetic peptides spanning
the central domain of NIPP-1 further narrowed the PP-1 inhibitory
function to residues 191-200. A second, noninhibitory PP-1-binding
site was identified by far-Western assays with digoxygenin-conjugated catalytic subunit (PP-1C) and included a consensus
RVXF motif (residues 200-203) found in many other
PP-1-binding proteins. The substitutions, V201A and/or F203A, in the
RVXF motif, or phosphorylation of Ser199 or
Ser204, which are established phosphorylation sites for
protein kinase A and protein kinase CK2, respectively, prevented
PP-1C-binding by NIPP-1191-210 in the
far-Western assay. NIPP-1191-210 competed for PP-1
inhibition by full-length NIPP-11-351, inhibitor-1 and
inhibitor-2, and dissociated PP-1C from inhibitor-1- and
NIPP-1143-217-Sepharose but not from full-length
NIPP-11-351-Sepharose. Together, these data identified
some of the key elements in the central domain of NIPP-1 that regulate
PP-1 activity and suggested that the flanking sequences stabilize the
association of NIPP-1 with PP-1C.
Reversible protein serine/threonine phosphorylation in higher
eukaryotes is catalyzed by protein kinases and phosphatases (1). The
functional diversity of protein phosphatases is increased by the
presence of isoforms of the catalytic subunits and numerous noncatalytic or regulatory subunits. For example, there are four mammalian isoforms of the catalytic subunit of type-1 protein phosphatases (2, 3). These catalytic subunits
(PP-1C)1 are
anchored at various subcellular locations by their association with
targeting subunits that also define the substrate specificity and
activity of the enzyme. Of the nearly 20 PP-1-targeting subunits thus
far identified in mammalian cells, the best characterized is the G
subunit which targets PP-1C to glycogen and enhances its
glycogen-synthase phosphatase activity (4-6). Interaction of the
skeletal muscle GM subunit with PP-1C is in
part mediated by an RVXF motif that binds in an extended
conformation within a hydrophobic channel near the C terminus of
PP-1C (5, 6). Phosphorylation of GM by protein
kinase A (PKA) at a threonine in the X position abolished
PP-1 binding. Variants of the RVXF motif are present in many
other PP-1 regulators, including the myosin-binding or M subunit, the
p53 binding-protein-2, inhibitor-1, DARPP-32, and inhibitor-2 (5-10).
Moreover, the regulation of PP-1C by these proteins was
impaired by mutations within the RVXF motif or by the
presence of competing RVXF-containing peptides. However,
these regulators also possessed other interaction sites with
PP-1C in addition to the RVXF motif (4, 5, 8).
Biochemical (11) and immunofluorescence (12) studies showed that a
large fraction of PP-1 is present in the nucleus. Nuclear PP-1 (PP-1N)
has been implicated in the regulation of mRNA splicing (13), in the
cell cycle-dependent control of the retinoblastoma protein
(14) and lamin B (15), and in the dephosphorylation of the
transcription factors CREB (16) and Sp1 (17). However, the PP-1
holoenzymes involved and their regulation remain poorly understood. The
two major nuclear targeting subunits of PP-1 have been identified (11)
as NIPP-1 and R111, which is also known as p99 (18) or PNUTS (19).
NIPP-1 and R111 are each associated with 30-50% of PP-1C
in mammalian nuclei and may target PP-1 to RNA (19-21). NIPP-1
potently inhibits PP-1C activity in vitro
(Ki = 1-10 pM), and the
NIPP-1·PP-1C complex (PP-1NNIPP-1) is
essentially inactive. However, PP-1NNIPP-1 can be
reactivated by phosphorylation with PKA and protein kinase CK2, without
disruption of the holoenzyme (21). Two phosphorylation sites,
Ser199 for PKA and Ser204 for CK2, flank an
RVXF motif that may contribute to PP-1 binding. Thus, the
phosphorylation of NIPP-1 may impair or disrupt the interaction of PP-1
with the RVXF motif and thereby enhance nuclear PP-1
activity. On the other hand, there must be yet other interactions between PP-1 and NIPP-1 that maintain the association of these two
proteins in the active complex. It is also highly unlikely that the
RVXF motif itself mediates PP-1 inhibition, because this sequence is present in many PP-1 regulators that do not inhibit enzyme
activity. Thus, the precise molecular basis for PP-1 inhibition by
NIPP-1 remains unknown.
In the studies described, we have analyzed the association of NIPP-1
and PP-1C by the yeast two-hybrid assay as well as by co-sedimentation. Further studies used numerous synthetic peptides based on NIPP-1 to identify two sequences in the central domain that
independently mediate PP-1 binding and inhibition. A synthetic peptide
encompassing these two sequences effectively competed for PP-1
inhibition by full-length NIPP-1 as well as other PP-1 inhibitors. Our
studies established that phosphorylation of the synthetic NIPP-1
peptide on the two serines flanking the RVXF motif resulted
in the dissociation of this motif from PP-1C and decreased
the potency of the peptide as a PP-1 inhibitor. Other experiments
suggested that sequences outside the central domain were also required
for the continued and stable association of PP-1C with
full-length NIPP-1 following its phosphorylation and inactivation by
PKA and CK2. Together, these studies provided the first insights into
the molecular determinants for the regulation of nuclear PP-1 by
NIPP-1.
Materials--
PP-1C (22) and phosphorylase
b (23) were purified from rabbit skeletal muscle.
Digoxygenin-labeled PP-1C was prepared according to
Beullens et al. (24) using the digoxygenin protein labeling
kit purchased from Roche Molecular Biochemicals. CNBr-activated Sepharose, glutathione-Sepharose, and Blue Sepharose were purchased from Amersham Pharmacia Biotech. Peptides were synthesized on Milligen
9050, using the N-(9-fluorenyl)methoxycarbonyl method. For
the PP-1-binding assays, the synthetic peptides were coupled to bovine
serum albumin using glutaraldehyde (25). Phosphorylase b was
phosphorylated in the presence of [ Preparation of Recombinant Proteins--
Bovine NIPP-1 was
expressed in Sf9 cells using the baculovirus system and purified
according to Vulsteke et al. (21). Oligonucleotides 5'-ATCGAACATATGGGTGGAGAGGATGATGAAC-3' (NdeI site
underlined) and 5'-CTGCACGGATCCTCAGTTCCGAAAGCGACCAACC-3'
(BamHI site underlined) were used as sense and antisense
primers in a PCR reaction using the NIPP-1 cDNA as template to
yield a product encoding NIPP-1143-217, which was verified
by double-stranded sequencing. The PCR product was digested with
NdeI/BamHI and subcloned into pET-16b (Novagen) for expression in bacteria as a polyhistidine-tagged protein. Escherichia coli BL21(DE3) cells, transformed with the
expression vector, were grown at 37 °C in LB medium supplemented
with 200 µg/ml ampicillin, to reach an A600 of
0.6-0.8. NIPP-1 protein was induced by the addition of 1 mM isopropyl-1-thio-
Human inhibitor-1 was expressed in E. coli as a fusion
protein with glutathione S-transferase and purified on
glutathione-Sepharose as described by Endo et al. (27).
Glutathione S-transferase inhibitor-1 was digested with
thrombin (1 units/ml for 60 min) and boiled for 5 min, and inhibitor-1
was separated from the denatured proteins by centrifugation (5 min at
10,000 × g). Purified inhibitor-1 was phosphorylated
with PKA catalytic subunit (27). Inhibitor-2 was purified from
heat-treated lysates from BL21(DE3) cells transformed with pET8d
inhibitor-2 plasmid (kindly provided by Prof. A. A. DePaoli-Roach)
using chromatography on Blue Sepharose (28).
Coupling of Inhibitor-1 and NIPP-1 to
Sepharose--
NIPP-11-351, NIPP-1143-217,
and phosphorylated inhibitor-1 (80 µg of protein) were each coupled
to 0.5 ml of CNBr-activated Sepharose as described by the manufacturer.
The gels were incubated with 10 pmol of PP-1C for 30 min at
4 °C on a rotating wheel. The unbound PP-1C was removed
by washing with buffer A (50 mM glycylglycine, pH 7.4, containing 0.5 mM dithiothreitol and 5 mM
Assays--
NIPP-1, its fragments, inhibitor-1, and inhibitor-2
were assayed as inhibitors of the phosphorylase phosphatase activity of PP-1C (29). The inhibitor-2-mediated inactivation of
PP-1C was monitored by the time-dependent
decrease in trypsin-revealed phosphorylase phosphatase activity (2).
The inhibitors were diluted in buffer A containing bovine serum albumin
(1 mg/ml) and 0.05% Triton X-100. All phosphatase assays were carried
out in buffer A, except for the inhibitor-2-induced inactivation, which
was undertaken in 50 mM imidazole, pH 7.4, containing 0.5 mM dithiothreitol, 5 mM
For the far-Western with digoxygenin-labeled PP-1C, the
albumin-coupled peptides were slot-blotted on polyvinylidene difluoride membrane (Bio-Rad), and the bound PP-1 was visualized using
anti-digoxygenin antibodies (24). Protein concentrations were measured
according to Bradford (30) with bovine serum albumin as standard.
Statistics are given as the means ± S.E. for the indicated number
(n) of experiments.
Yeast Two-hybrid Assays--
The plasmid pAS1-Glc7 (31) encodes
the yeast PP-1 catalytic subunit fused to the Gal4 DNA-binding
domain (residues 1-147). The cDNAs encoding full-length
NIPP-1 (351 residues) and various fragments
(NIPP-11-169, NIPP-1143-169,
NIPP-1143-224, NIPP-1143-351,
NIPP-1167-224, and NIPP-1225-351) were
subcloned into pACT2 (32), in-frame with the Gal4 activation domain
(residues 768-881) and the hemagglutinin (HA) epitope. Full-length
NIPP-1 cDNA was cloned into the pACT2 vector in two steps. First,
PCR was carried out using pBl-2175 as template (33) and
5'-ATCGAACCGCGGCCATGGAGGCCATGGCGGCAGCCGCGAACTC-3' (the SacII site is shown in bold and the SfiI
site is underlined) and 5'-AGAAAGTGCCGTGTGTAC-3' as primers. The PCR
product (300 base pairs) was digested with SacII and
ClaI, and the resulting 118-base pair fragment was exchanged
for the SacII-ClaI fragment of the original
pBl-2175 vector. The second step involved digestion of this vector with
SfiI and XhoI, which excised the entire NIPP-1 cDNA, which was then cloned into the
SfiI-XhoI sites of the pACT2 vector. The
NIPP-1143-351, NIPP-1225-351, and
NIPP-1143-224 deletion mutants were also constructed using
PCR. The sense oligonucleotides were:
5'-ATCGAACCGCGGCCATGGAGGCCATGGGTGGAGAGGATGATGAAC-3' for NIPP-1143-351 and NIPP-1143-224 and
5'-ATCGAACCGCGGCCATGGAGGCCATGGTGCAGACTGCAGTGGTC-3'
for NIPP-1225-351. The SfiI sites are
underlined. The antisense oligonucleotides were:
5'-CATTCCCTCGAGATCCCACCCTCCTC-3' for
NIPP-1143-351 and NIPP-1225-351 and
5'-CTGCACCTCGAGCCGAAAGCGACCAACC-3' for
NIPP-1143-224. The XhoI sites are underlined.
The PCR products were digested with SfiI and XhoI
and cloned into the SfiI-XhoI sites of pACT2. PCR
was performed using standard conditions and Pfu DNA
polymerase (Stratagene). NIPP-11-169 was obtained by
subcloning the SfiI-EcoRI fragment of
NIPP-11-351 into pACT2. NIPP-1143-169 was
obtained by subcloning the SfiI-EcoRI
fragment of NIPP-1143-224 into pACT2. To obtain
NIPP-1167-224, cDNA for NIPP-1143-224 was
digested with SfiI and EcoRI (to eliminate a
75-base pair 5'-fragment), gel-purified, filled in with Klenow, and
blunt-ended into pACT2. All constructs were verified by nucleotide sequencing.
In the yeast two-hybrid assay, Saccharomyces cerevisiae
strains Y190 and Y187 were transformed by the lithium acetate method of
Gietz et al. (34) with pACT2-NIPP-1-fragments and
pAS1-Glc7, respectively. Individual transformants were
purified on selection media (synthetic complete medium lacking leucine
for Y190 or tryptophan for Y187) and grown at 30 °C. After mating of
the appropriate yeast transformants, diploids were selected on a medium
deficient in both Trp and Leu. Expression of Co-sedimentation of NIPP-1 and PP-1C--
Overnight
cultures (20 ml) of Y190 yeast transformed with pACT2-NIPP-1 constructs
were inoculated into 200 ml of synthetic complete medium minus leucine.
Cells were grown to an A600 of 0.5-0.8,
harvested by centrifugation, washed with water, and resuspended in 500 µl of buffer B, containing 50 mM Tris/HCl, pH 7.5, 0.3 M NaCl, 0.1% Triton X-100, 1 mM
dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, 5 µM leupeptin, 50 µM
1-chloro-3-tosylamido-7-amino-2-heptanone, and 50 µM
L-1-tosylamido-2-phenylethyl chloromethyl ketone. Cells were lysed by vortexing with glass beads (six times for 30 s). The
homogenates were centrifuged for 10 min at 2000 × g to
remove cell debris. The pellets were extracted once with 500 µl of
buffer B, and the supernatants were combined with the extracts.
Immunoprecipitation of Gal4-HA-NIPP-1 fusion proteins from 250 µl of
yeast extract was performed with monoclonal antibodies (clone 12CA5)
against the HA tag, and protein A-TSK® (Affiland). The
immunoprecipitates were washed once with 0.25 M LiCl in TBS
(10 mM Tris/HCl, pH 7.5, 150 mM NaCl) and twice
with TBS and resuspended in 225 µl of buffer C containing 10 mM Tris/HCl, pH 7.5, 0.1 M NaCl, 0.05% Triton, 1 mM dithiothreitol, and the mixture of protease inhibitors
present in buffer B. Aliquots (50 µl) of the immunoprecipitates were
incubated for 10 min with either buffer or with 1 pmol
PP-1C on a rotating wheel, washed twice with buffer C,
resuspended in 45 µl buffer C, and assayed for spontaneous and
trypsin-activated phosphorylase phosphatase activity. The phosphatase
activities (derived from endogenous Glc7p and added muscle
PP-1C) were normalized for the amount of NIPP-1 fusion
proteins in the immunoprecipitates, as determined by Western blotting
with antibodies against the Gal4 activation domain
(CLONTECH).
Identification of PP-1 Binding Sites--
Initial studies of
NIPP-1 interactions with PP-1C were undertaken using the
yeast two-hybrid assay. For this purpose, the cDNAs encoding bovine
NIPP-1 and various fragments were subcloned in the pACT2 vector
containing the Gal4 transcriptional activation domain fused at its C
terminus to a HA tag (Fig.
1A). The cDNA encoding the
budding yeast PP-1 catalytic subunit (Glc7p) was subcloned in the pAS
vector containing the Gal4 DNA-binding domain. Additional experiments
showed that purified Glc7p was inhibited by bovine NIPP-1 with similar
potency to mammalian PP-1 catalytic subunit (data not shown). Mating
single transformants containing pAS-Glc7 with those containing
pACT2-NIPP-1 yielded double tranformants that contained the
In additional experiments, we used the monoclonal anti-HA antibodies to
immunoprecipitate Gal4-NIPP-1 fusion proteins from extracts of
single-transformed yeast. Subsequently, it was investigated whether
endogenous Glc7p had co-sedimented with the fusion proteins and whether
additional catalytic subunit could be bound by the immunoprecipitates,
when preincubated with an excess of PP-1C from rabbit
skeletal muscle. The "spontaneous" phosphatase activity in these
immunoprecipitates was assayed using phosphorylase a as
substrate. Because the complex of intact NIPP-1 and PP-1C
is inactive (21), we also assayed for "total" PP-1 activity
following the trypsin digestion of NIPP-1 present in the
immunoprecipitates. Because PP-1C is fully active after
trypsinization, the total activity can be taken as a measure of the
amount of catalytic subunit. Thus, the assay of spontaneous and total
activity enabled us to differentiate between fragments of NIPP-1 that
bound PP-1C and those that also inhibited enzyme
activity (2). In comparison with the endogenous Glc7p activity that was
co-immunoprecipitated, four to seven times more phosphorylase
phosphatase activity co-sedimented with the NIPP-1 fusion proteins when
the immunoprecipitates were preincubated with an excess of muscle
PP-1C (Fig. 1C). This suggests that the
immunoprecipitated NIPP-1 fusion proteins were only partially saturated
with endogenous Glc7p, either because the level of Glc7p was limiting
or because Glc7p was dissociated during the washing of the
immunoprecipitates with 0.25 M LiCl. Fig. 1C
compares the binding and inhibition of endogenous Glc7p or Glc7p plus
muscle PP-1C by equivalent molar amounts of NIPP-1 fusion
proteins, as measured by Western analysis with anti-Gal4 antibodies.
Compared with full-length NIPP-11-351, the
immunoprecipitates with the Gal4 fusions containing either the N
terminus, NIPP-11-169 or the C terminus,
NIPP-1225-351, showed no significant PP-1C
binding as assessed after trypsin digestion. On the other hand, the
Gal4-NIPP-1143-224 fusion protein bound PP-1C
and effectively inhibited its activity. Interestingly, a further
N-terminal deletion to produce NIPP-1167-224 yielded a
higher spontaneous PP-1 activity in the immunoprecipitates than
obtained with either NIPP-11-351 or
NIPP-1143-224. Moreover, this activity was further
increased by only 2-fold following trypsin treatment. This suggested
that residues 143-167, albeit not inhibitory by themselves (Fig.
1C), were also required for effective PP-1 inhibition.
Essentially the same results were obtained for the binding and
inhibition of Glc7p or muscle PP-1C by these NIPP-1 fusion
proteins (Fig. 1C), suggesting that similar structural
determinants mediate the interactions of NIPP-1 with the mammalian and
yeast catalytic subunits.
To more precisely delineate the PP-1 binding sites in NIPP-1, we
synthesized a series of overlapping 20-mer peptides that together
encompassed the central domain of NIPP-1 defined by the sequence
(residues 141-230). Of the eight peptides analyzed, only two,
NIPP-1181-200 and NIPP-1191-210, inhibited
PP-1C, with IC50 values of 32 and 11 µM, respectively (Fig. 2).
A decapeptide representing the overlapping sequence, NIPP-1191-200, was then synthesized and shown to inhibit
PP-1 activity with an IC50 of 8 µM (Table
I). This peptide lacked an
RVXF consensus sequence and thus clearly established that
enzyme inhibition was not mediated by this motif. Substitution of pairs
of adjacent basic residues with Ala increased the IC50 of
the decapeptide by 2-3-fold (Table I). On the other hand, substitution
of the three Lys residues for Arg did not significantly alter the
inhibitory potency of the decapeptide. Collectively, these data
suggested that the overall basic nature of the peptide, rather than
other features, such as the occurrence of alternating Lys and Arg
residues, mediates PP-1C inhibition.
We also investigated the direct association of the synthetic peptides
with PP-1C using a far-Western assay. The synthetic peptides directly immobilized on polyvinylidene difluoride membranes failed to bind the digoxygenin-conjugated PP-1C (data not
shown). However, when first coupled to albumin, the peptide
NIPP-1191-210 readily bound PP-1C (Fig. 2).
Surprisingly, NIPP-1181-200, already shown to inhibit
PP-1C activity, failed to bind PP-1C in the
far-Western assay. This suggested that the inhibitory site (residues
191-200) was not recognized in this assay and that the digoxygenin-conjugated PP-1C preferentially associated with
the region of NIPP-1 containing the RVXF motif. Support for
this hypothesis comes from the observation that the majority of
PP-1-binding proteins that can be visualized by far-Western analysis
contain this highly conserved PP-1-binding motif. To check whether the
binding of NIPP-1191-210 to PP-1C in the
far-Western assay was indeed mediated via the RVXF motif
(residues 200-203), we analyzed NIPP-1191-210 peptides
containing the substitutions V201A and/or F203A (Fig. 3B). These studies showed that
like NIPP-1181-200 and NIPP-1201-220 (which
do not contain the complete RVXF motif), the mutant peptides failed to bind PP-1C in the far-Western assay. This
suggested that three of the four residues in the RVXF motif,
Arg200, Val201, and Phe203, may be
required for PP-1C binding in the far-Western assay. On the
other hand, the mutations of both Val201 and
Phe203 did not diminish the inhibitory potency of the
NIPP-1191-210 peptide (Fig. 3A), further
emphasizing that the RVXF motif did not mediate PP-1
inhibition. These data also showed that the far-Western assay
visualized the interaction of NIPP-1 with PP-1C via the RVXF motif but could not be used as an indicator for the
binding to PP-1C via other NIPP1 site(s).
The above data identified at least two distinct interactions of
PP-1C with the central domain, NIPP-1143-217,
that inhibited PP-1C with essentially identical potency to
full-length NIPP-11-351 (IC50 = 0.3 nM). However, the synthetic peptide
NIPP-1191-210, which retained these two PP-1 binding
sites, was a much weaker inhibitor of PP-1 (IC50 = 11 µM). This could point to the existence of additional
interaction sites in the central domain that could not be identified by
two-hybrid analysis, far-Westerns, and phosphatase inhibition assays.
Alternately, the reduced potency of the NIPP-1191-210
peptide may arise from the inability of the PP-1 regulatory sequences to assume the conformation found in NIPP-1143-217. In this
regard, it is interesting to note that the deletion of residues
143-166, which did not directly mediate PP-1 binding, also increased
the spontaneous activity of the
PP-1C·NIPP-1167-224 complex (Fig.
1C).
Competition between NIPP-1191-210 and Endogenous PP-1
Inhibitors--
The inactive complex of NIPP-11-351 and
PP-1C, previously shown to be reactivated by
phosphorylation with PKA and/or CK2 (21), was also activated by the
addition of NIPP-1191-210 (Fig.
4A). This "deinhibition"
effect was observed at peptide concentrations that were themselves only
marginally inhibitory, indicating effective competition for PP-1
binding between NIPP-1 and the less potent NIPP-1191-210
peptide that alleviated PP-1 inhibition.
The deinhibition of PP-1NNIPP-1 by
NIPP-1191-210 was, however, only seen at physiological
salt concentration, i.e. 150 mM NaCl (data not
shown). A similar salt requirement was previously noted for the
activation of PP-1NNIPP-1 by PKA and CK2 (21). On the other hand, 150 mM NaCl was not required for the activation of a
complex formed with PP-1C and the central domain,
NIPP-1143-217, by either phosphorylation or addition of
the NIPP-1191-210 competitor (data not shown). This
indicated that despite their similar inhibitory potency, the central
domain forms a less stable complex with PP-1C than does
full-length NIPP-1. Our data also suggested that in the absence of
salt, there are additional phosphatase binding site(s) in the N- and/or
the C-terminal domains of NIPP-1.
Because the RVXF motif did not mediate PP-1 inhibition but
was a determinant of PP-1C interaction with
NIPP-1191-210, this raised the question of whether
the NIPP-1 peptide also competed with other PP-1 inhibitors that
contained a RVXF motif. Thus, we examined the
competition between NIPP-1191-210 and two other PP-1
inhibitors, inhibitor-1 and inhibitor-2, which contain variants of the
consensus RVXF motif (8, 10). Inhibitor-2 also mediates a
time-dependent inactivation of PP-1C that can be readily distinguished from enzyme inhibition by the inability to
recover total enzyme activity following the proteolytic degradation of
inhibitor-2 (2). Our data showed that PP-1 inhibition by inhibitor-1
and inhibitor-2 as well as the inhibitor-2-mediated inactivation of
PP-1C were reversed by NIPP-1191-210 (Fig. 4,
B and C), suggesting potential competition
between these inhibitors at the RVXF-binding site. This
hypothesis was strengthened by the finding that mutant
NIPP-1191-210 peptides with the substitutions V201A and/or
F203A within the RVXF motif, even at much higher
concentrations, failed to deinhibit PP-1 in the presence of NIPP-1,
inhibitor-1, or inhibitor-2 (data not shown).
NIPP-1191-210 Disrupts PP-1 Complexes--
To further
investigate the ability of NIPP-1191-210 to disrupt PP-1
binding to other inhibitors, the NIPP-1 peptide was added to phosphoinhibitor-1-Sepharose saturated with PP-1C. Fig.
5 shows that NIPP-1191-210
dissociated virtually all the inhibitor-1-bound PP-1 catalytic subunit.
Similarly, NIPP-1191-210 eluted most of the
PP-1C bound to NIPP-1143-217-Sepharose. On the
other hand, NIPP-1191-210 failed to release
PP-1C bound to full-length NIPP-1-Sepharose. These data are
consistent with our earlier findings that the phosphorylation of the
central one-third domain of NIPP-1 (35), but not full-length NIPP-1
(21), released the bound PP-1C and suggest once again that
the complex of the catalytic subunit with full-length NIPP-1 is more
stable.
Functional Effects of NIPP-1191-210
Phosphorylation--
Our previous studies had shown that the potency
of NIPP-1 as a PP-1 inhibitor was reduced by its phosphorylation by PKA
and/or CK2 and had established the phosphorylation sites for PKA
(Ser199) and CK2 (Ser204) that flanked the
RVXF motif (21). In the present study, we investigated the
functional impact of these phosphorylations on the
NIPP-1191-210 peptide. Synthesis of
NIPP-1191-210 with phosphoserine at either position 199 or
204 significantly reduced its activity as a PP-1 inhibitor (Fig.
6A). The doubly phosphorylated
peptide showed an even greater reduction in its PP-1 inhibitory
activity. We have also found that phosphoserine-199 and/or
phosphoserine-204 abolished PP-1C binding to
NIPP-1191-210 in far-Western assays (Fig. 6B).
Likewise, the phosphorylated NIPP-1191-210 was less
efficient in competing for inhibition of PP-1C by
inhibitor-1 and -2 (data not shown) and in reversing the
inhibitor-2-mediated inactivation of PP-1C (Fig.
7).
The above data suggested that phosphorylation of the flanking serines
impaired PP-1 binding at or near the RVXF motif and reduced
the activity of NIPP-1191-210 as a PP-1 inhibitor. We
speculate that the reduced inhibitory potency of phosphorylated
NIPP-1191-210 is accounted for by an electrostatic
interaction of the phosphate groups with basic residues in the upstream
inhibitory binding site, which we have shown to be positive
determinants of the inhibitory potency (Table I). This would also
explain why the substitutions V201A or F203A, albeit causing a
disruption of the RVXF motif, did not affect the potency of
NIPP-1191-210 as a PP-1 inhibitor (Fig. 3).
Conclusions--
The present study identified two interactions of
PP-1C with the central domain of NIPP-1. One, mediated by
the consensus RVXF motif found in many other
PP-1C-binding proteins, did not inhibit enzyme activity.
The second interaction occurred through a highly basic sequence
immediately N-terminal to the RVXF motif and inhibited enzyme activity. However, a peptide encompassing both sequences (NIPP-1191-210) did not inhibit PP-1 activity at
subnanomolar concentrations as seen with NIPP-1143-217.
This implied that other sequences within the central domain were also
required to enhance the potency of NIPP-1 as a PP-1 inhibitor. Because
no additional inhibitory fragments could be detected in the central
domain in our experiments (Fig. 2), we have postulated that
NIPP-1143-217 is a more potent inhibitor because of its
unique conformation.
NIPP-1191-210 also harbored the determinants for NIPP-1
regulation by PKA and CK2. Our data showed that the phosphorylation of
two serines in NIPP-1191-210 decreased its affinity for
PP-1 and concomitantly reduced its activity, both as a direct PP-1
inhibitor and as a competitor with other PP-1 inhibitors. Finally, we
have provided evidence suggesting that the N-terminal and/or C-terminal
thirds of NIPP-1 are required for its stable association with
PP-1C following phosphorylation by PKA and CK2.
The crystal structure of a complex between PP-1C and a
synthetic fragment of the muscle GM subunit has revealed
that the RVXF motif binds in a hydrophobic channel within
the C-terminal region of PP-1C, which is located some
distance from the catalytic-site channel (5, 6). The inhibitory
sequence (residues 191-200) in the central domain of NIPP-1 would
appear to be too close to the RVXF motif to bridge the
distance between the hydrophobic channel and the catalytic site. We
therefore propose that the inhibition of PP-1C by NIPP-1
does not result from its direct interaction with the catalytic site but
rather results from interactions of the basic inhibitory NIPP-1
site (residues 191-200) with acidic residues immediately adjacent to
the RVXF-binding site in PP-1C. This mechanism
would be different from that proposed for the inhibition of
PP-1C by the cytoplasmic proteins, inhibitor-1, and
DARPP-32, which only become potent PP-1 inhibitors after their
phosphorylation by PKA (5, 8, 9). It is worth noting that two PP-1
binding sites have also been mapped within the N-terminal 30 residues of inhibitor-1 and DARPP-32. One of these is the KIQF sequence, similar
to RVXF in other PP-1 regulators, which probably binds in
the same hydrophobic groove in the PP-1 catalytic subunit. The second
interaction most likely occurs through the PKA-phosphorylated threonine, which associates with the PP-1 catalytic site and functions as a pseudosubstrate to inhibit enzyme activity. Hence, multiple interactions between PP-1C and its endogenous inhibitors
may be a recurrent mechanism for PP-1 regulation.
In summary, we have defined a complex interaction of PP-1C
with a major nuclear regulatory subunit, NIPP-1, and identified a
unique polybasic sequence in NIPP-1 that is essential for PP-1 inhibition. We have also gained better insight into the phosphorylation mechanism that activates the nuclear phosphatase
PP-1NNIPP-1. Although the present studies hint at the
existence of additional regulatory sequences in NIPP-1 that greatly
enhance its activity as a PP-1 inhibitor, it raises the intriguing
possibility that mutations can be introduced into the PP-1 catalytic
subunit that will selectively impair its regulation by NIPP-1. Thus,
these structure-function studies open the way for addressing the
physiological role of NIPP-1 in the control of nuclear PP-1 activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP by
purified phosphorylase kinase (26).
-D-galactopyranoside. After 2 h at 30 °C, bacteria were collected by centrifugation (10 min at 5,000 × g) and resuspended in lysis buffer
(pH 7.9) containing 20 mM Tris/HCl, 5 mM
imidazole, 0.5 M NaCl, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM benzamidine, and 5 µM leupeptin. Cells were disrupted by three cycles of
freezing in liquid N2, followed by boiling for 15 min.
After centrifugation for 20 min at 20,000 × g, the
supernatant was applied to a 5-ml Ni2+-IDA-Sepharose column
(Sigma) equilibrated in lysis buffer. The column was washed with 25 ml
of lysis buffer containing in addition 100 mM imidazole,
and the protein was eluted with 50 ml of lysis buffer containing 400 mM imidazole. Fractions containing
NIPP-1143-217 were pooled, concentrated in a Vivaspin
ultrafiltration unit (VivaScience), and dialyzed against 20 mM Tris/HCl at pH 7.5.
-mercapthoethanol). The matrices were incubated with an equal volume
of buffer A containing 150 mM NaCl and 10 µM
NIPP-1191-210 for 10 min at 25 °C. Following
centrifugation (5 min at 15,000 × g), the gel was
washed twice with buffer A containing 150 mM NaCl and
resuspended in the same buffer. The pellet and supernatant were assayed
for phosphorylase phosphatase activity before and after incubation with
trypsin as described (29). One unit of phosphatase releases 1 nmol of
phosphate/min at 30 °C.
-mercapthoethanol,
and 1 mg/ml bovine serum albumin. Phosphatase assays at pH 5.0 and 6.0 were carried out in buffer containing 20 mM Tris, 20 mM MES, 20 mM CAPS, and 0.1% of Triton X-100,
adjusted to the desired pH at room temperature.
-galactosidase was
determined in duplicate after 8 h, using the
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside filter
assay described in the CLONTECH Yeast Protocols Handbook.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-galactosidase reporter gene under the control of the Gal4 promoter.
Analysis of numerous NIPP-1 constructs (Fig. 1B) showed that
only strains that expressed both Glc7p and NIPP-1 fragments containing
residues 167-224 were stained blue with 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (Fig. 1B).
View larger version (35K):
[in a new window]
Fig. 1.
Analysis of PP-1C interaction
with NIPP-1 by yeast two-hybrid and co-sedimentation assays.
A, a scheme of the Gal4-NIPP-1 constructs used in the yeast
two-hybrid assay. B, interaction of Glc7p and NIPP-1 in the
two-hybrid assay. The 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside filter assay was carried out in
duplicate for 8 h. Diploids containing pACT2-NIPP-1 and the empty
pAS1 vector were white in this assay (data not shown).
C, co-immunoprecipitation of PP-1C with Gal4-HA
(control) or Gal4-HA-NIPP-1 fusion proteins. The fusion proteins were
immunoprecipitated from yeast extracts with anti-HA antibodies and
protein A-TSK, and the immunoprecipitates were washed with 0.25 M LiCl (see "Experimental Procedures"). Subsequently,
aliquots of the immunoprecipitates were incubated with either buffer
(
exogenous PP1C) or muscle PP-1C (+ exogenous PP1C) and resedimented. The figure shows the
spontaneous (open bar fragments) and trypsin-revealed
(open plus closed bar fragments) phosphorylase phosphatase
activities that co-sedimented with the same amount of NIPP-1 fusion
proteins, as determined by densitometry of Western blots with
antibodies against the Gal4 tag. The figure shows a representative
profile of five different experiments.
View larger version (14K):
[in a new window]
Fig. 2.
Synthetic peptides identify
PP-1C-binding sites in the NIPP-1 central domain.
Full-length NIPP-1 is represented as a bar in which the
central domain is defined by the two methionines, Met143
and Met225. A series of overlapping 20-mer peptides
encompassing the entire central domain were tested for their ability to
inhibit PP-1C purified from rabbit skeletal muscle.
IC50 is the concentration of peptide (means ± S.E.,
n = 4) that inhibited the phosphorylase phosphatase
activity of PP-1C by 50%. Also shown is the ability of
albumin-coupled peptides to bind digoxygenin-labeled PP-1C
(DIG-PP-1C) in a far-Western assay.
Determinants of PP-1C inhibition by NIPP-1191-200
View larger version (18K):
[in a new window]
Fig. 3.
Importance of Val201 and
Phe203 for the interaction of NIPP-1191-210
with PP-1C. A shows the effects of
NIPP-1191-210 and the peptide with Val201
and/or Phe203 mutated to Ala on the phosphorylase
phosphatase activity of PP-1C (n = 3).
B shows the ability of the same peptides coupled to albumin
to bind digoxigenin-labeled PP-1C
(DIG-PP-1C) in the far-Western assay.
View larger version (33K):
[in a new window]
Fig. 4.
NIPP-1191-210 competes for
PP-1C inhibition by full-length NIPP-1, inhibitor-1, and
inhibitor-2. Inhibition of PP-1C by varying
concentrations of NIPP-1 (A), PKA-phosphorylated inhibitor-1
(B), and inhibitor-2 (C) was measured in the
presence or absence of NIPP-1191-210. The assays with
NIPP-1 were done in the presence of 150 mM NaCl. The
results (n = 3) are expressed as a percentage of
control activity without the inhibitors. The right panel of
C also shows the effect of NIPP-1191-210 on the
time-dependent inactivation of PP-1C by
inhibitor-2 (I2), as assessed by trypsin-activated
phosphorylase phosphatase activity (n = 3). The control
(100%) value was not significantly affected by up to 2.5 µM NIPP-1191-210. The 100% value with 3.1, 5, and 10 µM NIPP-1191-210 was 24, 34, and
40% lower than that without peptide.
View larger version (29K):
[in a new window]
Fig. 5.
NIPP-1191-210 competes for
PP-1C association with inhibitor-1 or NIPP-1.
Phosphoinhibitor-1, NIPP-1, and NIPP-1143-217 were
covalently bound to CNBr-activated Sepharose and saturated with rabbit
skeletal muscle PP-1C. The washed resins were incubated
either with buffer or 10 µM NIPP-1191-210.
The figure shows the trypsin-activated phosphorylase phosphatase
activity that was released from the matrix as a percentage of the total
activity associated with the column (n = 3).
View larger version (20K):
[in a new window]
Fig. 6.
Phosphorylation of NIPP-1191-210
reduces its affinity for PP-1C. A shows
PP-1C inhibition by indicated concentrations of
NIPP-1191-210 and the peptides phosphorylated on
Ser199 and/or Ser204 (n = 4).
B shows the binding of these peptides, after coupling to
albumin, to digoxigenin-labeled PP-1C in the far-Western
assay.
View larger version (25K):
[in a new window]
Fig. 7.
Phosphorylation of NIPP-1191-210
abrogates its ability to antagonize inhibitor-2-mediated inactivation
of PP-1C. PP-1C was incubated at 25 °C
with 0.3 nM inhibitor-2 (control) and with 1 µM NIPP-1191-210 or
NIPP-1191-210 phosphorylated on Ser199 and/or
Ser204. At indicated times, aliquots were taken and assayed
for trypsin-activated phosphorylase phosphatase activity
(n = 3).
![]() |
ACKNOWLEDGEMENTS |
---|
Stefaan Wera and Juan Colomer are acknowledged for contributions to the identification of inhibitory NIPP-1 peptides. Karl Beckers, AnneMie Hoogmartens, Nicole Sente, Peter Vermaelen, and Cindy Verwichte provided expert technical assistance. Valère Feytons is acknowledged for synthesis of peptides and oligonucleotides.
![]() |
FOOTNOTES |
---|
* This work was supported by the Fund for Scientific Research Grant G.0179.97, funds from ASLK, funds from Flemisch Concerted Research Action, Prime Minister's office Grant IUAP P4/23, and National Institutes of Health Grant DK52044 (to S. S.).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.
§ Postdoctoral fellow of the National Fund for Scientific Research-Flanders.
To whom correspondence should be addressed: Afdeling
Biochemie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-34-57-01; Fax: 32-16-34-59-95, E-mail:
Mathieu.Bollen{at}med.kuleuven.ac.be.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PP-1, protein phosphatase-1; PP-1C, catalytic subunit of PP-1; PP-1N, nuclear PP-1; NIPP-1, nuclear inhibitor of PP-1; PKA, protein kinase A; CK2, protein kinase CK2, previously called casein kinase-2; HA, hemagglutinin; PCR, polymerase chain reaction; MES, 4-morpholineethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|