From the Laboratory of Molecular and Cellular
Biochemistry, Faculty of Dental Science, and Station for Collaborative
Research and the
Laboratory of Pharmacology, Faculty of Medical
Science, Kyushu University, Fukuoka 812-8582 and the ¶ Department
of Pharmacology, Juntendo University School of Medicine, Tokyo
113-8421, Japan
Received for publication, October 23, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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The protein p130 was originally isolated
from rat brain as an inositol 1,4,5-trisphosphate-binding protein with
a domain organization similar to that of phospholipase C- D-myo-Inositol 1,4,5-trisphosphate
(Ins(1,4,5)P3),1
a product of receptor-induced hydrolysis of phosphatidylinositol
4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C (PLC),
plays an important role as an intracellular second messenger by
mobilizing Ca2+ from nonmitochondrial stores (1). We
previously isolated two Ins(1,4,5)P3-binding proteins with
molecular masses of 130 and 85 kDa from rat brain (2, 3) with the use
of an Ins(1,4,5)P3 affinity column (4, 5). Partial amino
acid sequencing revealed that the 85-kDa molecule was PLC- The Ins(1,4,5)P3-binding protein with a molecular mass of
130 kDa, termed p130, was a previously unidentified molecule (2, 3).
The predicted amino acid sequence of rat p130 shares 38.2% identity
with that of rat PLC- To investigate the physiological functions of PRIP family proteins, we
previously examined the possible role of the binding of inositol
compounds to the PH domain of p130 (10, 15-17). Our results suggested
that p130, which is localized predominantly in the cytoplasm,
contributes to Ins(1,4,5)P3-mediated Ca2+
signaling. The high affinity binding of Ins(1,4,5)P3 to the
PH domain of p130 might also serve to sequester
Ins(1,4,5)P3 and therefore prevent its interaction with
Ins(1,4,5)P3 receptors and metabolizing enzymes (18).
We have now applied the yeast two-hybrid system to identify proteins
that interact with p130. With the unique NH2-terminal region of p130 as the bait for screening a human brain cDNA
library, we isolated two positive clones, one of which was shown to
encode the catalytic subunit of protein phosphatase 1 Materials--
Cloning vectors pGBT9 and pACT2, a human brain
cDNA library, and yeast strains HF7c and SFY526 were obtained from
CLONTECH (Palo Alto, CA). All restriction
endonucleases and DNA-modifying enzymes were from Toyobo (Tokyo,
Japan). Dropout yeast selection medium and dropout base medium
were from BIO101 (Vista, CA). YPD medium for yeast and bacterial
medium were obtained from Becton Dickinson (Sparks, MD).
Polyvinylidene difluoride (PVDF) membranes were from Millipore
(Bedford, MA). [ Plasmid Construction--
For construction of p130 bait
plasmids, pcMT3 (9) was digested with XhoI, and the released
fragment (nucleotides (nt) 535 to 5233) was cloned into the
SalI site of pGBT9. The resulting plasmid, pGBT9-p130Full
(p130 plasmid 1; amino acid residues 24 to 1096) (see Fig.
1A), was digested with PstI, rendered blunt-ended with DNA polymerase, and then self-ligated at the
SmaI site (nt 1359), thereby generating p130 plasmid 2 (amino acids 24 to 298). In the same manner, pGBT9-p130Full was
self-ligated between the KpnI site (nt 1130) or the
BamHI site (nt 710) and the PstI site (all sites
were blunt-ended) to generate p130 plasmids 3 (amino acids 24 to 222)
and 4 (amino acids 24 to 82), respectively. The p130 plasmid 5 (amino
acids 222 to 298) was constructed by self-ligation between the
blunt-ended XhoI (nt 535) and KpnI (nt 1130)
sites of plasmid 2.
For construction of pGBT9-p130D (amino acids 848 to 1096), a
SpeI site (nt 3011) was introduced into pcMT3 by
site-directed mutagenesis, and the resulting plasmid was digested with
SpeI (nt 3011) and XhoI (nt 5233). The released
2.2-kilobase pair fragment was then ligated into the
SpeI-SalI sites of pGBT9. For construction of
pACT2-PP1c Yeast Two-hybrid Screening and GST Fusion Protein Precipitation and Protein Overlay
Analyses--
The recombinant GST-PP1c
For overlay analysis, samples were fractionated by SDS-PAGE, and the
separated proteins were transferred electrophoretically to a PVDF
membrane. After the blocking of nonspecific sites with 5% dried skim
milk, the membrane was incubated for 1 h at room temperature with
a protein probe (10 µg/ml). The membrane was then washed and
incubated with antibodies to the probe protein followed by alkaline
phosphatase-conjugated secondary antibodies, after which immune
complexes were detected by enzymatic reaction.
Immunoprecipitation--
COS-1 cells, COS-1p130
cells (COS-1 cells stably expressing p130) (18), and mouse brain
extract were subjected to immunoprecipitation with a specific
monoclonal antibody to p130 (2F9) or polyclonal antibodies to PP1c Analysis of Protein-Protein Interaction in Real
Time--
Protein-protein interaction was examined in real time with a
BIACORE 2000 surface plasmon resonance analyzer (Biacore International, Uppsala, Sweden). Recombinant GST-PP1c Assay of Glycogen Phosphorylase Activity--
COS-1 or
COS-1p130 cells (2 × 106) were lysed by
three freeze-thaw cycles in a solution containing 50 mM
NaCl, 10 mM MES-NaOH (pH 6.0), 1 mM EDTA, and
10 mM 2-mercaptoethanol. The lysate was subjected to
centrifugation at 15,000 × g for 30 min, and the
resulting supernatant was assayed for glycogen phosphorylase activity
as described (20).
Assay of Phosphatase Activity--
Phosphatase activity was
determined in a reaction mixture (40 µl) containing 139.2 mM KCl, 20 mM
4-morpholinepropanesulfonic acid-KOH (pH 7.0), 0.1 mM MnCl2, 0.5 mM dithiothreitol,
BSA (0.5 mg/ml), 2 µM phosphorylated myosin light chain
(from bovine stomach), 3.4 nM recombinant rabbit skeletal
muscle PP1c Two-hydrid Screening for Proteins That Interact with
p130--
Screening of a human brain cDNA library with a bait
plasmid (2) encoding the unique NH2-terminal region of rat
p130 (amino acids 24 to 298), including the PH domain and a portion of
the EF-hand motif (Fig. 1A),
yielded 51 positive clones of a total of 2 million clones examined; no
positive clones were obtained with a bait plasmid encoding the
COOH-terminal region (amino acids 848 to 1096) of p130. 10 of the 51 clones identified proved to be false positives, and the remaining 41 clones were divided into two groups on the basis of analysis of their
inserts by polymerase chain reaction amplification and restriction
enzyme digestion. Sequencing revealed that one of these 41 clones
encoded full-length PP1c Association of p130 with PP1c
The GST-PP1c
Analysis of the interaction of various regulatory subunits with PP1c
Given that p130 contains four consensus motifs for phosphorylation by
PKA (74Arg Arg Thr Ser77,
90Arg Lys Lys Thr93,
104Lys Ile Ser107, and
567Arg Arg Val Ser570 [underlining
refers to phosphorylatable residues] one of which (104Lys
Lys Ile Ser107) is present in p130PH, it was
possible that p130 associates with PP1c
The association between p130 and PP1c
We next investigated whether the activity of PP1c
The effects of Ins(1,4,5)P3 and water-soluble (short-chain)
PtdIns(4,5)P2 on the association of p130 with PP1c Association between p130 and PP1c
PP1c With the use of its specific NH2-terminal region as a
bait, we applied the yeast two-hybrid screen to identify human brain proteins that bind to p130, designated henceforth as PRIP-1. This approach identified PP1c Amino acid residues 95 to 97 of PRIP-1, located upstream of the PH
domain, appear to contribute to the binding site for PP1c Phosphorylation of PRIP-1 by PKA resulted in inhibition of the
association between PRIP-1 and PP1c PP1c In summary, we have shown that (i) p130, which belongs to the PRIP
family of proteins and is here renamed PRIP-1, associates with PP1c1
but which lacks phospholipase C activity. Yeast two-hybrid screening of
a human brain cDNA library for clones that encode proteins that
interact with p130 has now led to the identification of the catalytic
subunit of protein phosphatase 1
(PP1c
) as a p130-binding
protein. The association between p130 and PP1c
was also confirmed
in vitro by an overlay assay, a "pull-down" assay, and
surface plasmon resonance analysis. The interaction of p130 with
PP1c
resulted in inhibition of the catalytic activity of the latter
in a p130 concentration-dependent manner.
Immunoprecipitation and immunoblot analysis of COS-1 cells that stably
express p130 and of mouse brain extract with antibodies to p130 and to
PP1c
also detected the presence of a complex of p130 and PP1c
.
The activity of glycogen phosphorylase, which is negatively regulated
by dephosphorylation by PP1c
, was higher in COS-1 cells that stably
express p130 than in control COS-1 cells. These results suggest that,
in addition to its role in inositol 1,4,5-trisphosphate and
Ca2+ signaling, p130 might also contribute to regulation of
protein dephosphorylation through its interaction with PP1c
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (2).
Identification of the pleckstrin homology (PH) domain of PLC-
1 as
the site of Ins(1,4,5)P3 binding helped to define the PH
domain as an inositol compound binding module (6, 7).
1; the five identified domains of PLC-
1 (PH,
EF-hand, putative catalytic (X and Y), and C2 domains) are all
present in p130. The domain organization of p130 suggests that the
protein is likely to possess a fold similar to that of PLC-
1, a
notion that is supported by the results of limited proteolysis with
trypsin (8). However, p130 exhibits some distinct characteristics. It
is larger than the PLC-
isozymes, and it possesses unique regions
both at the NH2 terminus, preceding the PH domain, and at
the COOH terminus. Moreover, the residues within the catalytic domain
of PLC-
that are critical for enzyme activity (His356
and Glu390) are not conserved in p130 (9). The PH domain of
p130, like that of PLC-
1, is important for the binding of
Ins(1,4,5)P3 (10). Other molecules that show sequence
similarity to p130, including human PLC-L (11) and the K10F12.3 gene
product of Caenorhabditis elegans (12), have also been
described. Otsuki et al. (13) recently isolated a cDNA
from mouse brain that encodes a protein with 66% sequence identity to
PLC-L; they therefore termed this protein PLC-L2 and
renamed the original PLC-L as PLC-L1. Furthermore, the
gene for human type2 p130 (PLC-L2) has also been
cloned (14). All of these proteins exhibit characteristic
NH2- and COOH-terminal extensions and replacement of
critical catalytic residues. The identification of a p130-related
molecule in such a simple organism as C. elegans suggests
that this family of proteins diverged early from other PLC isozymes. We
propose that this distinct family of PLC-related proteins be designated
the PLC-related catalytically inactive protein (PRIP) family
(comprising PRIP-1 and -2 subfamilies).
(PP1c
). To
characterize the interaction between p130 and PP1c
, we studied the
association of these two proteins both in vitro and in
living cells, we delineated further the region of p130 that is
responsible for binding to PP1c
, and we examined the effect of such
binding on the enzymatic activity of PP1c
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (222 terabecquere/mmol) was obtained from DuPont-New England Nuclear.
A large scale plasmid preparation kit, QIAfilter Plasmid Giga kit, and
nitrilotriacetic acid-agarose beads for purification of
His6-tagged proteins were from Qiagen (Chatsworth, CA).
Protein G-Sepharose, glutathione-Sepharose 4B beads, and pGEX vectors were from Amersham Pharmacia Biotech. Ins(1,4,5)P3 was
synthesized as described (19). The catalytic subunit of
cAMP-dependent protein kinase (PKA) was obtained from
Promega (Madison, WI), and wild-type rabbit PP1c
was from
Calbiochem-Novabiochem (La Jolla, CA). A soluble form of
PtdIns(4,5)P2, diC8-PtdIns(4,5)P2,
was obtained from Echelon Research Laboratories (Salt Lake City, UT).
GM peptide (GRRVSFADNFGFN) and its random sequence
(GNFRGFRSADFVN) were synthesized using the Fmoc
(N-(9-fluorenyl)methoxycarbonyl) cleavage method on an
Advanced ChemTech 348MPS peptide synthesizer, and the purity was
checked by applying the sample to a µBondashere 5-µ C18 column mounted on a high-performance liquid chromatography column (more than 90%). Other reagents used were of the highest grade available.
(
29-163) a positive clone obtained from the yeast two-hybrid screening, pACT2-PP1c
, was digested with PvuII
(nt 116 and 815) and self-ligated. For expression of recombinant
PP1c
in Escherichia coli, the BamHI fragment
of pACT2-PP1c
was ligated into the BamHI site of pGEX-3X;
the resulting construct encodes a fusion protein of glutathione
S-transferase (GST) and PP1c
.
-Galactosidase
Assay--
Yeast two-hybrid screening of a human brain cDNA
library cloned in the pACT2 vector was performed in yeast strain HF7c
with the bait plasmids pGBT9-p130PH or pGBT9-p130D. Transformants
(total of 2 × 106) were plated and selected with a
combination of tryptophan, leucine, and histidine. The positive clones
identified by two-hybrid screening were sequenced with an ABI 373A
automated DNA sequencer. The domains required for the interaction
between p130 and PP1c
were investigated by expression of various
combinations of bait and target plasmids in yeast SFY526 cells
and measurement of
-galactosidase activity.
fusion protein was purified
from E. coli by affinity chromatography, and recombinant
full-length p130 (amino acids 24 to 1096) and the PH domain of p130
(p130PH; amino acids 95 to 232) were prepared as described previously
(8, 10). For "pull-down" assays, GST-PP1c
was incubated for
1 h at 4 °C with glutathione-Sepharose 4B beads in binding
buffer (50 mM Tris-HCl (pH 7.5), 5 mM
MgCl2, 100 mM NaCl, 10% glycerol, 0.5 mg/ml
bovine serum albumin (BSA), 5 mM 2-mercaptoethanol). The
beads were washed with 50 volumes of binding buffer and then incubated
(6 µg of GST-PP1c
) for 1 h at 4 °C, with gentle rotation, in a total volume of 150 µl with recombinant full-length p130 or
p130PH. After washing of the beads five times with 500 µl of binding
buffer, bound proteins were eluted with 50 µl of a solution containing 50 mM Tris-HCl (pH 8.0) and 10 mM
reduced glutathione and were then subjected to SDS polyacrylamide gel
electrophoresis (PAGE) and immunoblot analysis with antibodies to p130
(2F9) or to p130PH (3, 8).
(Santa Cruz Biotechnology, Santa Cruz, CA). Cells (5 × 106) or mouse brain (wet weight, 0.2 g) were
homogenized in 0.5 ml of a solution containing 20 mM
HEPES-NaOH (pH 7.4), 130 mM NaCl, 5 mM EDTA,
and a mixture of protease inhibitors. The homogenate was centrifuged
(14,000 × g, 20 min, 4 °C), and the resulting supernatant was incubated, with gentle rotation, for 1 h at
4 °C with 30 µg of antibodies to p130 or to PP1c
that had been premixed with 10 µl of a 50% slurry of protein G-Sepharose in phosphate-buffered saline containing 0.1% BSA. The beads were then
washed twice with a homogenizing solution (described above) containing 0.2% Triton X-100, boiled in SDS sample buffer, and subjected to SDS-PAGE and immunoblot analysis with antibodies to
PP1c
or to p130.
was immobilized on the surface of a CM5 sensor chip that had been activated with
N-hydroxysuccinimide and
N-ethyl-N'-(3-diethylaminopropyl) carbodiimide.
Recombinant full-length p130 (0.23, 2.3, 23, 230, or 2300 nM) was injected over the chip surface at a rate of 10 µl/min in a solution containing 10 mM HEPES-NaOH (pH
7.4), 0.15 M NaCl, 3.4 mM EDTA, and 0.005% Tween 20.
, and various concentrations of recombinant full-length
p130 or p130PH, in the absence or presence of 10 µM
Ins(1,4,5)P3. The mixture minus the substrate was incubated for 10 min at 25 °C, and the reaction was started by the addition of
phosphorylated myosin light chain and stopped after 20 min by the
addition of 0.2 ml of ice-cold 10% trichloroacetic acid. The
unphosphorylated and phosphorylated myosin light chains were separated
by two-dimensional electrophoresis, and the density of each spot was
determined as described (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a 37,510-Da protein composed of 330 amino
acids. To delineate more precisely the region of p130 required for
binding to PP1c
, we used plasmids encoding smaller portions of p130
as baits in two-hybrid analysis with the plasmid encoding full-length
PP1c
(Fig. 1A). Positive signals were obtained with a
bait plasmid (3) encoding amino acids 24 to 222, as well as with that
(1) encoding full-length p130. Neither a bait plasmid (4) encoding amino acids 24 to 82 nor one (5) encoding residues 222 to 298 yielded a
positive signal (Fig. 1B). A plasmid encoding a PP1c
mutant lacking amino acids 30 to 162 did not yield a positive signal
with any of the p130 bait plasmids examined. These results thus
suggested that the region of p130 composed of residues 83 to 222 interacts with that of PP1c
comprising residues 30 to 162.
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Fig. 1.
Yeast two-hybrid analysis of the
interaction between p130 and PP1c .
A, schematic representation of plasmids encoding various
regions of p130 (left) and PP1c
(right) that
were used for analysis of the domains required for binding. The initial
screening of the human brain cDNA library for p130-binding proteins
was performed with p130 bait plasmid 2 (pGBT9-p130PH(24-298)). Bait
plasmids 3 (XhoI-KpnI fragment of p130 cDNA),
4 (XhoI-BamHI), and 5 (KpnI-SmaI) encode amino acids 24 to 222, 24 to
82, and 222 to 298 of p130, respectively. These constructs were
introduced into yeast strain SFY526, together with pACT2-PP1c
or
pACT2-PP1c
(
30-162), the latter of which was prepared from the
former by digestion with PvuI and self-ligation.
B,
-Galactosidase assay of protein-protein interaction.
Two-hybrid analysis was performed with SFY256 cells transformed with
the indicated p130 (1 to 6) and PP1c
plasmids. The activity of
-galactosidase was determined with a filter assay.
in Vitro--
We next examined
the interaction of p130 and PP1c
in vitro by several
methods. The association was first analyzed with an overlay assay (Fig.
2A). Extracts of
nontransformed E. coli and of bacteria expressing a
GST-PP1c
fusion protein, as well as recombinant GST-PP1c
purified
from such a latter extract, were fractionated by SDS-PAGE, and the
separated proteins were transferred to a PVDF membrane and probed with
antibodies to PP1c
to confirm that the prominent band that migrated
at a position corresponding to a molecular size of 37 kDa was indeed
PP1c
. Duplicate membranes were incubated in the presence of a
recombinant p130 fragment containing the PH domain (p130PH; amino acids
95 to 232), recombinant full-length p130 (residues 24 to 1096), or BSA
(negative control). After washing, the membranes were exposed to the
corresponding antibodies to p130PH or to p130. Both the recombinant
GST-PP1c
present in the bacterial extract and the purified protein
interacted with both full-length p130 and p130PH. Together with the
results from the yeast two-hybrid analysis, these data indicate that
residues 95 to 222 of p130 (which include the entire PH domain and the 20 residues preceding it) mediate the interaction of this protein with
PP1c
.
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Fig. 2.
Association between p130 and
PP1c in vitro. A,
overlay assay. Extracts (10 µg of protein) of either nontransformed
E. coli (lane 3) or E. coli expressing
recombinant GST-PP1c
(lane 1), or 1 µg of purified
recombinant GST-PP1c
(lane 2), were subjected to
SDS-PAGE. The separated proteins were transferred to a PVDF membrane,
which was subsequently subjected to immunoblot analysis with antibodies
to PP1c
(panel a). Alternatively, the membranes were
first incubated in the presence of p130PH (panel b),
full-length p130 (panel c), or BSA (panel d),
each at a concentration of 10 µg/ml, and were then subjected to
immunoblot analysis with antibodies to p130PH or to p130 as indicated.
B, pull-down assay. Recombinant GST-PP1c
(or GST)
attached to glutathione-Sepharose 4B beads was incubated with various
protein samples, after which proteins that had bound to the beads were
eluted with reduced glutathione and subjected to SDS-PAGE and
immunoblot analysis with appropriate antibodies. Panel a,
beads were incubated with 1 µM p130PH in the absence or
presence of 0.01 or 0.1 µM full-length p130 or of 0.1 or
1 µM PLC-
1 (prepared as described in Ref. 7);
immunoblot analysis was performed with antibodies to p130PH.
Panel b, beads were incubated with 0.1 µM
full-length p130 in the absence or presence of 0.1, 1, or 10 µM p130PH; immunoblot analysis was performed with
antibodies to full-length p130. Panel c, beads were
incubated in the presence of 1 µM p130PH or full-length
p130, in the absence or presence of 50 µM GM
peptide (GRRVSFADNFGFN) or a peptide of the same amino acid composition
but of random sequence (GNFRGFRSADFVN); immunoblot analysis was
performed with antibodies to p130PH or to full-length p130. Panel
d, beads were incubated in the presence of 3 µM
wild-type (wt) or mutant (V95L or F97A) fragments of p130
spanning residues 82 to 232; immunoblot analysis was performed with
antibodies to p130PH(95-232) or to PP1c
. C,
phosphorylation of p130 by PKA and its effect on association with
PP1c
. Panel a, recombinant full-length p130 (150 pmol)
was incubated in a volume of 50 µl with 100 µM
[
-32P]ATP in the absence (lane 1) or
presence (lane 2) of 0.1 µg of the catalytic subunit of
PKA, after which the reaction mixtures were subjected to SDS-PAGE and
autoradiography. Panels b and c, recombinant p130
phosphorylated by PKA (lane 2) or treated with ATP alone
(lane 1) was incubated with recombinant GST-PP1c
immobilized on glutathione-Sepharose 4B beads, after which bead-bound
protein was eluted with reduced glutathione and subjected to SDS-PAGE
and immunoblot analysis with antibodies to p130 (b) or to
PP1c
(c). D, surface plasmon resonance
analysis. GST-PP1c
was immobilized on a sensor chip and exposed to
various concentrations of full-length p130 (0.23, 2.3, 23, 230, and
2300 nM) for 360 s before the application of buffer
alone; data are expressed in relative units (RU).
fusion protein was also subjected to a pull-down
assay with recombinant p130 or p130PH (Fig. 2B). Incubation of a GST-PP1c
resin with p130PH and subsequent immunoblot analysis of bead-bound proteins with antibodies to p130PH revealed that p130PH
was precipitated by GST-PP1c
and that this interaction was sensitive
to the presence of low concentrations of full-length p130 but not to
PLC-
1 (Fig. 2B, panel a). In contrast,
although full-length p130 also bound to GST-PP1c
(but not to GST
alone), this interaction was not sensitive to the presence of p130PH
(Fig. 2B, panel b). These results indicate that,
although the PH domain of p130 is primarily responsible for the binding
of this protein to PP1c
, other regions of p130 also contribute to
the interaction between these two proteins.
has led to the identification of a consensus sequence for binding,
(K/R)(K/R)(V/I)XF (22). The sequence VSF (residues 95 to 97) is present in the region of p130 shown to bind to PP1c
. To
determine whether this sequence participates in the interaction of p130
with PP1c
, we examined the effect of a peptide (GM
peptide, GRRVSFADNFGFN) that has been shown to inhibit the association between PP1c
and several regulatory subunits (22). This peptide inhibited the interaction of PP1c
with either full-length p130 or
p130PH, whereas a random peptide with the same amino acid composition had no such effect (Fig. 2B, panel c). To confirm
the role of the VSF sequence of p130 in the interaction of this protein
with PP1c
, we expressed in and purified from E. coli p130
fragments comprising amino acids 82 to 232. Whereas the wild-type
fragment bound to PP1c
, fragments containing either V95L or F97A
mutations bound to the lesser extent (Fig. 2B, panel
d).
because it is a substrate
for phosphatase activity of this enzyme. Indeed, p130 was
phosphorylated by PKA (Fig. 2C, lane 2), although
the precise site (or sites) phosphorylated remains to be determined.
However, this explanation for the interaction between p130 and PP1c
is unlikely, because phosphorylated p130 did not associate with
PP1c
, whereas p130 treated with ATP alone (without PKA) bound to
PP1c
(Fig. 2C, lane 1).
was further confirmed by
surface plasmon resonance analysis. Full-length p130 was introduced into the analysis chamber after immobilization of GST-PP1c
onto the
sensor chip. Positive signals indicative of protein-protein interaction
were generated in a p130 concentration-dependent manner and
were abolished by washing away of the applied p130 (Fig.
2D). The dissociation constant was calculated to be 1.2 ± 0.1 nM (mean ± S.E. of values from
five independent determinations). Replacement of the full-length p130
molecule with p130PH yielded a dissociation constant in the micromolar
range, consistent with the results obtained with pull-down assays (Fig.
2B, panels a and b).
is affected by the
association with p130. The dephosphorylation of phosphorylated smooth
muscle myosin light chain (21) by recombinant rabbit skeletal muscle
PP1c
was inhibited by full-length p130 in a
concentration-dependent manner (Fig.
3). Recombinant p130PH also inhibited the
activity of PP1c
, although higher concentrations of p130PH than of
full-length p130 were required for this effect.
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Fig. 3.
Effect of p130 on PP1c
activity. The phosphatase activity of recombinant PP1c
toward phosphorylated smooth muscle myosin light chain was assayed in
the presence of the indicated concentrations of p130
(circles) or p130PH (triangles) and in the
absence (open symbols) or presence (closed
circles) of 10 µM Ins(1,4,5)P3. Data are
means of triplicates from a representative experiment that was repeated
4 times with similar results.
, as
well as on the inhibition of PP1c
activity by p130, were also
examined, given that the site of p130 responsible for the association
with PP1c
was shown to be located immediately upstream of the PH
domain and that PH domains mediate binding to Ins(1,4,5)P3
or PtdIns(4,5)P2. The presence of Ins(1,4,5)P3
or short-chain PtdIns(4,5)P2 at a concentration of 10 µM in the reaction mixture for the pull-down assay had no
effect on the interaction of p130 with PP1c
(data not shown), and 10 µM Ins(1,4,5)P3 had no effect on p130-induced inhibition of PP1c
activity (Fig. 3).
in Intact Cells--
To
determine whether p130 and PP1c
interact in living cells, we first
examined COS-1 cells that stably express recombinant p130
(COS-1p130 cells) (18). Immunoblot analysis of extracts of
both control COS-1 cells (which lack endogenous p130) and
COS-1p130 cells with antibodies to PP1c
revealed that
both cell lines express similar amounts of PP1c
(Fig.
4A, a). Cell
extracts were then subjected to immunoprecipitation with antibodies to
either p130 (Fig. 4A, b) or PP1c
(Fig.
4A, c), and the resulting precipitates were
subjected to immunoblot analysis with the same two types of antibodies.
Stable association of p130 with PP1c
was apparent in
COS-1p130 cells but not in control COS-1 cells (Fig.
4A). We also examined whether these two proteins interact in
mouse brain, which contains both molecules (Fig. 4B).
PP1c
was detected in p130 immunoprecipitates (Fig. 4B,
b), and p130 was detected in PP1c
immunoprecipitates (Fig. 4B, c) prepared from mouse brain.
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Fig. 4.
Association between p130 and
PP1c in intact cells and mouse brain.
A, panel a, extracts prepared from COS-1 or
COS-1p130 cells were subjected to immunoblot analysis
(IB) with antibodies to p130 or to PP1c
, as indicated.
Panels b and c, extracts of COS-1 (lanes
1) and COS-1p130 (lanes 3) cells were
subjected to immunoprecipitation (IP) with antibodies to
p130 (b) or to PP1c
(c), and the resulting
precipitates were subjected to immunoblot analysis with the same two
types of antibodies. Lanes 2 correspond to
COS-1p130 cell extract subjected to immunoprecipitation
with protein G-Sepharose in the absence of antibodies. Upper
bands in lanes 1 and 3 in the blot analyzed
with anti-PP1c
in panel b are immunoglobulin heavy
chains. B, mouse brain extract was analyzed as described in
A; lanes 2 correspond to brain extract subjected
to immunoprecipitation with the indicated antibodies, whereas
lanes 1 correspond to extract subjected to precipitation
with protein G-Sepharose in the absence of antibodies.
is thought to catalyze protein dephosphorylation reactions that
underlie many aspects of cell function (23, 24). Glycogen
phosphorylase, which catalyzes the conversion of glycogen to glucose
1-phosphate, is a substrate for PP1c
in a wide variety of cell types
(25); its dephosphorylation by this phosphatase results in inhibition
of phosphorylase activity. Measurement of glycogen phosphorylase
activity in extracts of COS-1 and COS-1p130 cells yielded
values of 68 ± 6 and 130 ± 9 nmol per milligram of protein
per 5 min (means ± S.E. of six independent determinations), respectively. These results thus indicate that glycogen phosphorylase is phosphorylated to a greater extent in COS-1p130 cells
than in COS-1 cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
as one such protein. PP1 is a widely expressed serine-threonine protein phosphatase that exists in several
isoforms, including
,
2,
1,
2, and
(23, 24). Various
regulatory subunits have been shown to associate with PP1c
and
thereby to influence its catalytic activity (26). For example,
GM and GL subunits function to target PP1c
to glycogen granules; phosphorylation of these subunits by PKA induces
their dissociation from PP1c
, whereas that triggered by insulin
promotes their association with and activation of PP1c
, resulting in
inhibition of glycogen breakdown. The association of I-1
(inhibitor 1) or DARPP-32
(dopamine- and cAMP-regulated
phosphoprotein of 32 kDa) with
PP1c
appears not to affect phosphatase activity, whereas phosphorylation of I-1 or DARPP-32 by PKA induces marked inhibition of
such activity. Our results now suggest that PRIP-1 also functions as a
regulatory subunit of PP1c
that inhibits phosphatase activity. The
binding of Ins(1,4,5)P3 or PtdIns(4,5)P2 to
PRIP-1 had no effect on its association with or inhibition of PP1c
.
Our previous observations suggested that Ins(1,4,5)P3 may
be a physiological ligand for PRIP-1 and that this protein is localized
predominantly to the cytosol (18). PRIP-1 may therefore serve not only
to inhibit the activity of PP1c
but also to target this enzyme to the cytosol.
. The
fragment of PRIP-1 comprising residues 24 to 222 interacted with
PP1c
in the yeast two-hybrid assay, and p130PH (PRIP-1PH) (residues
95 to 232) as well as the full-length molecule, associated with PP1c
in vitro, as demonstrated with a variety of binding assays.
A GM peptide that disrupts the interaction of PP1c
with several regulatory subunits and that contains the VSF (residues 95 to
97) sequence of PRIP-1 also inhibited the association of PRIP-1 with
PP1c
. Furthermore, mutation of residues 95 or 97 of PRIP-1 prevented
the association of this protein with PP1c
. Other regions of the
PRIP-1 molecule may also interact with PP1c
, as suggested by the
observations that the full-length molecule bound to PP1c
was not
displaced by an excess amount of PRIP-1PH and that the dissociation
constant obtained by surface plasmon resonance analysis for the
interaction with PP1c
was smaller for the full-length molecule than
for PRIP-1PH. However, the observation that the GM peptide
was similarly effective in inhibiting the association of PP1c
with
full-length PRIP-1 and with PRIP-1PH suggests rather that other regions
of PRIP-1 promote the interaction of the region containing residues 95 to 97 with PP1c
.
. Although the phosphorylated residues of PRIP-1 that underlie this effect remain to be identified, T93, which is located immediately upstream of the putative binding site
for PP1c
, is a likely candidate.
contributes to the regulation of many aspects of cellular
metabolism, including glycogen metabolism (through dephosphorylation of
phosphorylase kinase, glycogen phosphorylase, and glycogen synthase)
and lipid metabolism (through dephosphorylation of acetyl-CoA carboxylase, hormone-dependent lipase, and
hydroxymethylglutaryl-CoA reductase). Furthermore, it participates in
the regulation of Ca2+ transport (through dephosphorylation
of phospholamban and Ca2+ channel proteins), smooth muscle
contraction (through dephosphorylation of myosin light chain), DNA
replication (through dephosphorylation of histones H2B and H1), and
protein synthesis (through dephosphorylation of initiation factor
eIF-2, RNA-dependent protein kinase, heat shock protein, S6
protein, and S6 kinase) (24, 25). It remains to be determined which of
these cellular activities are physiologically regulated by PRIP-1
through its interaction with PP1c
. Our data do suggest, however,
that the association between PRIP-1 and PP1c
occurs in living cells,
and we have shown that the activity of glycogen phosphorylase, which is
regulated exclusively by phosphorylation, was increased in COS-1 cells
by the expression of PRIP-1, probably as a result of the interaction of
PRIP-1 with, and the consequent inhibition of, PP1c
. Glycogen
phosphorylase may therefore be a physiological target for regulation by
the interaction of PRIP-1 with PP1c
.
through a GM peptide-like region located upstream of the PH
domain; (ii) association with PRIP-1 results in inhibition of the
catalytic activity of PP1c
as measured in vitro with
phosphorylated myosin light chain as substrate; and (iii) glycogen
phosphorylase activity was increased by expression of PRIP-1 in intact
cells, likely as a result of inhibition of PP1c
and accumulation of the phosphorylated, active form of glycogen phosphorylase. In addition
to its role in Ins(1,4,5)P3 and Ca2+ signaling
(18), PRIP-1 might therefore also contribute to the regulation of
protein dephosphorylation. Given that the binding of
Ins(1,4,5)P3 to the PH domain of PRIP-1 had no effect on
the association of PRIP-1 with PP1c
or on its inhibition of PP1c
activity, PRIP-1 may contribute to both Ca2+ signaling and
regulation of protein dephosphorylation simultaneously and, in some
instances, cooperatively.
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ACKNOWLEDGEMENTS |
---|
We thank M. Katan for helpful comments.
![]() |
FOOTNOTES |
---|
* This work was funded in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (to H. T., T. K., and M. H.), Kyushu University interdisciplinary programs in education and projects in research development (to M. H.), the Fujisawa Foundation (to M. H.), the Fugaku Trust for medicinal research (to M. H.), Kowa Life Science Foundation (to T. K.), and the Uehara Memorial Foundation (to H. T.).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.
§ Contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 81-92-642-6317. Fax: 81-92-642-6322; E-mail: hirata1@dent.kyushu-u.ac.jp.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M009677200
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ABBREVIATIONS |
---|
The abbreviations used are:
Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate;
PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate;
PLC, phospholipase C;
PH, pleckstrin homology;
PRIP, PLC-related
catalytically inactive protein;
PP1c, catalytic subunit of protein
phosphatase 1
;
PVDF, polyvinylidene difluoride;
PKA, cAMP-dependent protein kinase;
nt, nucleotide;
GST, glutathione S-transferase;
BSA, bovine serum albumin;
PAGE, polyacrylamide gel electrophoresis.
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