(Received for publication, March 10, 1995; and in revised form, May 8, 1995)
From the
The activity of protein phosphatase-1 in rat liver nuclei
(PP-1N) was decreased by up to 97% by associated inhibitory
polypeptides, depending on the assay and extraction conditions. These
inhibitors were rapidly degraded by endogenous proteases, resulting in
the accumulation of active heat-stable intermediates. Two major species
of PP-1N could be differentiated by fractionation of a nuclear extract.
PP-1N
Most processes in eukaryotic cells are controlled through
phosphorylation and dephosphorylation of key proteins by protein
kinases and protein phosphatases, respectively. The serine/threonine
protein phosphatases of type 1 (PP-1)
The noncatalytic subunits of
PP-1 also have a targeting function, enabling the phosphatase to
associate with a particular cellular
structure(1, 2, 3, 4) . This
targeting role explains the broad subcellular distribution of PP-1 and
forms the basis for the nomenclature of PP-1 holoenzymes. It is now
firmly established that unique species of PP-1 are associated with
glycogen (PP-1G), myofibrils (PP-1M), and in the liver also with the
endoplasmic reticulum (PP-1E). A minor fraction of PP-1 is soluble
(PP-1S). By far the highest concentration of PP-1, however, is found in
the nucleus (PP-1N), where it can be found both in the nucleoplasm and
associated with particulate structures like chromatin(4) .
Although there is convincing genetic and biochemical evidence for a
role of PP-1N in nuclear processes like (alternative) pre-mRNA
splicing, chromatin condensation, and gene expression, the exact
substrates remain largely to be
identified(4, 5, 6) . One of the underlying
problems is a complete lack of understanding of the structure and
properties of native PP-1N. Although some groups reported that PP-1 was
extracted from nuclei as free catalytic
subunit(7, 8) , others provided evidence for an
oligomeric structure of PP-1N(4, 9) . We have purified
from bovine thymus nuclei small (16-18 kDa) nuclear inhibitory
polypeptides of PP-1
We have started to analyze the
properties and regulation of PP-1N in rat liver nuclei and report here
on the existence of at least two species of PP-1N with regulatory
subunits of 41 and 111 kDa, respectively. The 41-kDa polypeptide is
shown to represent the native form of hepatic NIPP-1. We also provide
evidence for a physiological regulation of NIPP-1 by reversible
phosphorylation.
Phosphorylase b was phosphorylated in the presence of
[
For the preparation of
digoxygenin-labeled PP-1
To
check whether phosphorylation of NIPP-1 by PKA also occurs in vivo (see Fig. 7), the animals were first injected
intraperitoneally with propranolol (1 mg in 0.2 ml of saline) plus
glucose (1 g in 3 ml of saline). 30 min later, the activity of liver
PKA was enhanced by the intraperitoneal injection of glucagon (0.3 mg
in 0.2 ml of saline). 15 min before excision of the liver, the animals
were anesthetized by an intraperitoneal injection of sodium
pentobarbital (12 mg in 0.2 ml of saline). In this experiment, nuclei
were prepared in the presence of 40 mM NaF and 0.2 µM microcystin to prevent dephosphorylation of NIPP-1.
Figure 7:
Reduced binding of DIG-PP-1
The nuclear pellet was homogenized
with a Dounce homogenizer (B pestle) in a hypotonic solution (buffer C)
containing 50 mM glycylglycine/NaOH at pH 7.4, 5 mM 2-mercaptoethanol, and 0.5 mM dithiothreitol, and the
lysed nuclei were submitted to centrifugation during 5 min at 13,000
For Western blotting, incubation with the antisera
was done for 2 h at room temperature at a final dilution of 1:2000, and
the peroxidase-labeled secondary antibodies were detected by enhanced
chemiluminescence. For immunoprecipitation of PP-1N
Figure 1:
Proteolytic activation of
PP-1N during incubation of nuclei. Nuclei were prepared in the presence
of either 0.5 mM phenylmethanesulfonyl fluoride as sole
protease inhibitor (
Since we
had previously identified heat-stable inhibitory polypeptides of PP-1
(NIPP-1) in thymus nuclei(10) , we wondered whether the
presence of such inhibitor(s) could account for the low spontaneous
activity of hepatic PP-1N. To our surprise, however, we found that
freshly prepared liver nuclei contained very little or no heat-stable
inhibitory activity of PP-1
Figure 2:
Proteolytic generation of heat-stable
inhibitor(s) of PP-1
Figure 3:
Fractionation of PP-1N on Mono Q. A
freshly prepared nuclear extract from the livers of two rats was
dialyzed against buffer C and was applied to a Mono Q column,
equilibrated in the same buffer. Bound proteins were eluted with a
linear salt gradient (0-0.4 M NaCl). The fractions were
assayed for spontaneous (
That the 41- and 111-kDa polypeptides,
further denoted as R41 and R111, were physically associated with the
catalytic subunit is substantiated by their co-immunoprecipitation with
PP-1
Figure 4:
Immunoprecipitation of PP-1N
Figure 5:
Gel filtration of PP-1N. 200 µl of the
Mono Q pool of PP-1N
Figure 6:
Identification of the
Although phosphorylation of native
PP-1N
We have also used the
DIG-PP-1
We did not find any evidence for a
regulation of the activity of PP-1N
A widely used approach for the initial
identification of the protein phosphatase(s) that dephosphorylate a
particular substrate is based on the screening of subcellular fractions
in the presence or absence of specific phosphatase
inhibitors/activators(1) . Such a strategy has for example
recently been employed for the identification of the nuclear
phosphatase(s) acting on p34
Due to the extreme sensitivity of the noncatalytic
polypeptides of PP-1 to proteolysis (this study and (1) ), the
PP-1 holoenzymes are very resistant to purification. We have therefore
adopted an alternative strategy for the identification of the
noncatalytic subunits in crude fractions that is based upon their high
affinity binding of DIG-labeled PP-1
The copurification and
coimmunoprecipitation of R111 and PP-1N
Western
analysis suggested that both PP-1N
We thank Dr. A. A. DePaoli-Roach for the generous gift
of polyclonal antibodies against PP-1
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
contained, besides the
-isoform of the
catalytic subunit, an inhibitory polypeptide of 111 kDa. PP-1N
was found to be an inactive heterodimer between the
-isoform
of the catalytic subunit and NIPP-1, a nuclear inhibitor of PP-1, which
in its undegraded form is heat labile and migrates during
SDS-polyacrylamide gel electrophoresis as a polypeptide of 41 kDa.
Native hepatic NIPP-1 displayed a reduced affinity for the catalytic
subunit after phosphorylation by protein kinase A in vitro and
after glucagon-induced phosphorylation in vivo.
(
)comprise
an abundant and widely distributed group of enzymes that
dephosphorylate proteins involved in such diverse cellular processes as
metabolism, intracellular transport, protein synthesis, and cell cycle
control(1, 2, 3) . They all possess an
isoform of a phylogenetically very conserved catalytic subunit
(PP-1
), which displays some unique properties that readily
allow differentiation from other protein phosphatases. For example,
only type 1 protein phosphatases are inhibited by specific cytoplasmic
(inhibitor-1, inhibitor-2, DARPP-32) and nuclear (NIPP-1) polypeptides.
Also, with phosphorylase as substrate, the known PP-1 holoenzymes are
activated by limited trypsinolysis. This can be explained by the
destruction of inhibitory noncatalytic polypeptide(s), resulting in the
release of the catalytic subunit. Trypsin also hydrolyzes the carboxyl
terminus of PP-1
, but this has little effect on the
phosphorylase phosphatase activity.
, termed NIPP-1(10) .
Phosphorylation of NIPP-1 by protein kinase A and/or casein kinase 2
results in a drastic decrease in affinity for
PP-1
(11, 12) . In Schizosaccharomyces
pombe, a polypeptide (sds22) has been identified that is partially
nuclear and acts as a positive regulator of
PP-1(13, 14) . It has also been demonstrated that
inhibitor-2 is partially nuclear during the S-phase of the cell cycle (15) and that some PP-1
is bound to the
retinoblastoma protein during mitosis and early
G
(16) .
Materials
PP-1(17) ,
PP-2A
(18) , inhibitor-2(19) , and
phosphorylase b(20) were purified from rabbit
skeletal muscle. Protein kinase p34
was
purified from Xenopus oocytes by affinity chromatography on
p13-Sepharose(21) . The recombinant
-,
-,
-, and
-isoforms of rat PP-1
(22) were a kind gift of Prof. E. Y. C. Lee (University
of Miami, Miami, FL). Casein was prepared according to the procedure of
Mercier et al.(23) . Histone IIA, myelin
basic protein, catalytic subunit of PKA from beef heart and PP-2B from
bovine brain were purchased from Sigma. Histone H1 and the digoxygenin
(DIG) protein labeling and detection kit were obtained from Boehringer.
Polyvinylidene difluoride membranes (Immobilon) were purchased from
Millipore. Protein A coupled to TSK® beads, and Affi-T-agarose,
used for the purification of immunoglobulins, were obtained from
Affiland (Liège, Belgium). A kit for the development of Western
blots by enhanced chemiluminescence was purchased from Amersham Corp.
P]ATP by purified phosphorylase
kinase(24) . Casein, myelin basic protein, and histone IIA were
phosphorylated by PKA(10) , and histone H1 was phosphorylated
by p34
(21) .
, 200 µl of purified catalytic
subunit (0.5 mg/ml) was dialyzed against 50 mM sodium
tetraborate at pH 8.5 plus 1 mM dithiothreitol, and incubated
with 15 µl of
digoxygenin-carboxymethyl-N-hydroxysuccinimide ester for 1 h
at room temperature. Subsequently, the mixture was extensively dialyzed
against a solution containing 20 mM Tris-HCl at pH 7.5, 1
mM dithiothreitol, and 60% glycerol.
Treatment of the Animals
All experiments were
performed with normally fed female Wistar rats of about 250 g. Unless
indicated otherwise, the animals were sacrificed by decapitation.
after phosphorylation of hepatic NIPP-1 by PKA. An aliquot of the
Mono Q peak fraction of PP-1N
was incubated during 60 min
at 30 °C in buffer C plus 0.2 mM ATP, 2 mM
magnesium acetate, 1 mg/ml bovine serum albumin in the absence or
presence of 80 nM PKA. Following electrophoresis and blotting,
the membranes were screened for binding of DIG-PP-1
(upperleftpanel). Other samples of
PP-1N
were first subjected to denaturing electrophoresis
and blotting. Subsequently, the latter blots were incubated under
phosphorylation conditions in the absence or presence of PKA, which was
followed by an incubation during 120 min at room temperature in buffer
C, 1 mg/ml bovine serum albumin in the absence or presence of
PP-2A
(2 units/ml) and PP-2B (25 units/ml). Finally, the
blots were labeled with DIG-PP-1
(upperrightpanel). The lowerleftpanel shows the binding of DIG-PP-1
to NIPP-1 in hepatic
nuclear extracts prepared from rats that were treated for the indicated
times with glucagon, as detailed under ``Experimental
Procedures.'' The lowerrightpanel shows DIG-PP-1
binding to the same samples after
preincubation of the blots with PP-2A
plus
PP-2B.
Purification of Nuclei and Subnuclear
Fractionation
Unless indicated otherwise, all purification and
fractionation buffers were supplemented with a mixture of protease
inhibitors including 0.5 mM phenylmethanesulfonyl fluoride, 50
µM 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), 50
µML-1-tosylamido-2-phenylethyl chloromethyl
ketone (TPCK), and 5 µM leupeptin. Liver nuclei were
prepared according to the method of Blobel and Potter(25) ,
with slight modifications. Briefly, livers were homogenized in 2
volumes of buffer A containing 15 mM PIPES/NaOH at pH 7.2, 80
mM KCl, 15 mM NaCl, 0.5 mM dithiothreitol,
and 0.25 M sucrose. The homogenate was mixed with 2 volumes of
buffer A containing 2.3 M instead of 0.25 M sucrose,
and put on top of 1 volume of the same buffer. After high speed
centrifugation (120,000 g for 30 min), the pelleted
nuclei were resuspended with a Dounce homogenizer (A pestle) in buffer
B, containing 20 mM Tris-HCl at pH 7.4, 1 mM
MgCl
, 0.25 M sucrose, and 0.5% Triton X-100. After
standing on ice for 10 min, enabling the complete solubilization of the
outer nuclear membrane, the nuclear suspension was centrifuged for 5
min at 3,500
g.
g. The pellet was resuspended in buffer C plus 0.3 M NaCl, incubated for 30 min at 4 °C, and centrifuged
during 5 min at 13,000
g. The latter supernatant is
further referred to as ``nuclear extract.''
Affinity Labeling with Digoxygenin-bound
PP-1
The protocol was based on the procedure
described by Alessi et al.(26) . Briefly, following
SDS-PAGE (10%), the separated polypeptides were blotted onto Immobilon
membranes and incubated for 3 h at room temperature with a buffer
containing 50 mM Tris-HCl at pH 7.5, 0.5 M NaCl, and
5% skim milk powder. Subsequently, the blots were incubated for 3 h in
10 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1 mg/ml bovine
serum albumin, and a 1:1000 dilution of DIG-labeled PP-1.
Finally, the free DIG-PP-1
was washed away, and the bound
phosphatase was detected with DIG-specific antibodies according to the
manufacturer's protocol.
Western Blotting and Immunoprecipitation
Rabbit
polyclonal antibodies against a synthetic peptide encompassing the
carboxyl-terminal domain (residues 302-322) of the -isoform
of PP-1
were kindly donated by Prof. A. A. DePaoli-Roach
(Indiana University, Indianapolis, IN). Antibodies against a synthetic
peptide encompassing the 11 carboxyl-terminal residues of NIPP-1 from
bovine thymus were raised in a rabbit and purified by chromatography on
Affi-T-agarose.
and
PP-1N
, 300 µl of the Mono Q peak fractions (see
``Results'') were first incubated with the PP-1
antiserum (final dilution of 1:60) or with Affi-T-purified NIPP-1
antibodies (final concentration of 30 µg/ml) during 45 min at 4
°C. Subsequently, 30 µl of protein-A-TSK® was added, and
the mixture was rotated during 30 min at 4 °C. Following
centrifugation (1 min at 10,000
g), the pellet was
first washed in 0.5 ml of a buffer containing 10 mM Tris-HCl
at pH 7.5, 1 mM dithiothreitol, and 0.25 M LiCl and
then washed twice in 1 ml of the same buffer without LiCl. The
supernatant and resuspended pellet were assayed for spontaneous and
trypsin-revealed phosphorylase phosphatase activities. An aliquot of
these fractions was boiled in SDS-sample buffer and centrifuged (1 min
at 10,000
g), and the supernatant was checked for
binding of DIG-PP-1
following denaturing electrophoresis
and blotting.
Assays
The spontaneous and trypsin-revealed
protein phosphatase activities were measured as described
previously(10) . One unit of phosphatase releases 1 nmol of
phosphate/min at 30 °C. Heat-stable inhibitory polypeptides of
PP-1 were assayed using
P-labeled
phosphorylase a as substrate. Following a heat treatment (3
min at 90 °C), the inhibitory activity was assayed before and after
preincubation of the fractions with trypsin (0.1 mg/ml during 5 min).
The difference between both assays was taken as protein-derived
inhibitory activity.
Hepatic PP-1N Is a Largely Latent Enzyme
Since
all known species of PP-1 can dephosphorylate phosphorylase a,
we have chosen this substrate for the initial characterization of
PP-1N. Although the phosphorylase phosphatase activity was very low in
freshly prepared liver nuclei, the activity was increased up to 40-fold
by a preincubation of the nuclear fraction with trypsin (Fig. 1), which is known to release the catalytic subunit (see
the Introduction). Both, the spontaneous and the trypsin-revealed
activities could be nearly completely blocked by the addition of
inhibitor-2 (not illustrated), showing that the activity stemmed
virtually exclusively from PP-1. A large activation (5-25-fold)
of PP-1N by trypsinolysis was also obtained after prior extraction of
PP-1N with 0.3 M NaCl (see below).
,
) or in the presence of 0.5 mM phenylmethanesulfonyl fluoride, 50 µM TLCK, 50
µM TPCK, and 5 µM leupeptin (
). The
nuclei (10
10
/ml) were incubated at 37 °C in 20
mM Tris-HCl at pH 7.5, 0.3 M sucrose, 5 mM
MgCl
, 2 mM EDTA, 2.5 mM CaCl
and the same mixture of protease inhibitors. At the indicated
times, samples were taken for the assay of spontaneous (opensymbols) and trypsin-revealed (
) phosphorylase
phosphatase activities. For the assay, the samples were 30-fold diluted
in a hypotonic medium. The results represent the means ± S.E.
for three different preparations.
The spontaneous
phosphorylase phosphatase activity gradually increased during
incubation of the nuclei at 37 °C in the presence of
phenylmethanesulfonyl fluoride as sole protease inhibitor (Fig. 1). This increase was completely blocked by the addition
of the protease inhibitors leupeptin, TPCK, and TLCK. In separate
experiments, it was found that the addition of leupeptin alone was able
to prevent completely the time-dependent increase in the spontaneous
activity of PP-1N, while the addition of TPCK or TLCK was only
partially effective (not illustrated). On the other hand, the
trypsin-revealed phosphorylase phosphatase activity, which is a measure
for the concentration of the catalytic subunit, remained constant
during incubation with phenylmethanesulfonyl fluoride only.
, although such an inhibitory
activity could be generated by simple incubation of the nuclei at 37
°C (Fig. 2). Again, the generation of this heat-stable
inhibitory activity was completely blocked by the addition of the
protease inhibitors leupeptin, TLCK, and TPCK. Taken together, the
above data suggest that the activity of PP-1
in the nucleus
is suppressed by polypeptide(s) that are destroyed by trypsin. These
inhibitory polypeptides seem also to be degraded by endogenous
protease(s), which explains the gradual increase of the spontaneous
phosphorylase phosphatase activity (Fig. 1). However, the
endogenous proteases also generate some active heatstable inhibitor
fragments (Fig. 2).
. From the same nuclear suspensions
that were used for the assay of phosphorylase phosphatase (Fig. 1), samples were taken for the assay of heat-stable
inhibitory protein, as indicated under ``Experimental
Procedures.'' The nuclear suspension had been prepared and
incubated in the absence (
) or presence (
) of TPCK, TLCK,
and leupeptin. The results represent the means ± S.E. for three
different preparations.
Identification of Two Species of PP-1N
Upon
chromatography of a nuclear salt extract on Mono Q, the phosphorylase
phosphatase activity eluted in two major peaks (Fig. 3A). The first peak, termed PP-1N,
was nearly completely latent, but the activity could be revealed by a
preincubation with trypsin, which results in the release of free
catalytic subunit (see the Introduction). The second peak
(PP-1N
) was always partially active but could be
additionally activated up to 5-fold by trypsin. The extent of
activation of PP-1N
by trypsin varied among preparations
and moreover decreased rapidly during storage of the fractions on ice
or at -20 °C.
) and trypsin-revealed (
)
phosphorylase phosphatase activities (A) and for binding of
DIG-PP-1
after SDS-PAGE (10%) and blotting onto Immobilon
membranes (B). The peak fractions of PP-1N
and
PP-1N
were also used for immunoprecipitation with
antibodies against PP-1
. PanelC shows the labeling of the immunoprecipitates with
DIG-PP-1
, performed as in panelB.
Although the addition of leupeptin, TPCK,
and TLCK blocked the degradation of PP-1N during purification and
incubation of liver nuclei (Fig. 1), these inhibitors failed to
block proteolysis during further purification of PP-1N. Thus, efforts
to purify PP-1N and PP-1N
until
homogeneity failed and invariably resulted in the generation of free
catalytic subunit. Problems with proteolysis were particularly
pronounced for PP-1N
(see also below). For the
identification of the noncatalytic subunits of PP-1N, we have therefore
used an indirect method that is based upon the high affinity binding of
DIG-labeled PP-1
to specific polypeptides, following
SDS-PAGE (10%), and blotting of a nuclear extract. Using this approach,
we could identify two major PP-1
-binding polypeptides of 41
± 1 (n = 12) and 111 ± 2 kDa (n = 7), that coeluted during chromatraphy on Mono Q with
PP-1N
and PP-1N
, respectively (Fig. 3B).
, using polyclonal antibodies against the C-terminal
domain of
-isoform of the catalytic subunit (Fig. 3C). In agreement with the extreme sensitivity of
R111 to proteolysis, we noted that this polypeptide was partially
degraded to smaller fragments during immunoprecipitation which,
however, were still able to bind DIG-PP-1
(Fig. 3C, lane2). It should
also be noted that the immunoprecipitated PP-1N holoenzymes were still
largely latent, as indicated by the severalfold increase of the
phosphorylase phosphatase activity by a preincubation with trypsin (not
illustrated).
Identification of R41 as NIPP-1
Since the recent
cDNA cloning of NIPP-1 revealed that the inhibitor(s) purified from
bovine thymus nuclei (16-18 kDa) were proteolytic fragments and
that recombinant NIPP-1 migrated during SDS-PAGE (10%) as a protein of
41 kDa,(
)we wondered whether R41 could be
identical to NIPP-1. We indeed noted that in the Mono Q fractions
containing PP-1N
, a polypeptide of 41 kDa was detected by
Western blotting with polyclonal antibodies against the C-terminal
domain of NIPP-1 (not shown). This 41-kDa polypeptide was no longer
detected when the primary antibodies were preincubated with an excess
of the synthetic peptide to which the antibodies were generated. As is
illustrated in Fig. 4, PP-1N
could be nearly
quantitatively immunoprecipated with the NIPP-1 antibodies and in the
immunoprecipitate R41 could be identified by labeling with
DIG-PP-1
.
with NIPP-1 antibodies. The peak fractions of Mono Q containing
PP-1N
were pooled and tested for immunoprecipation with
NIPP-1 antibodies. The immunoprecipitate was resuspended until the same
volume as the supernatant, and both fractions were assayed for
spontaneous and trypsin-revealed phosphorylase phosphatase activities (A). PanelB shows the labeling of the
supernatant and the immunoprecipitate with DIG-PP-1
as in Fig. 3C.
Subunit Structure of PP-1N
Gel filtration on
Superdex 75 showed that PP-1N migrated as a protein of
about 90 kDa (Fig. 5A). Combined with the above data,
these results suggest that PP-1N
is a heterodimer between
the catalytic subunit (37 kDa, see below) and NIPP-1. Gel filtration of
PP-1N
always yielded at least two peaks of activity with
apparent molecular masses of about 160 and 30-40 kDa (Fig. 5B). The first peak was largely latent and may
represent a heterodimer of the catalytic subunit and R111. The second
peak was fully active and is likely to be the free catalytic subunit,
generated by proteolysis or dissociation of the noncatalytic
subunit(s). The latter view is substantiated by findings that gel
filtration of stored samples of PP-1N
exclusively
yielded free catalytic subunit (not illustrated).
(upperpanel) or
PP-1N
(lowerpanel) were applied to a
Superdex-75 column, equilibrated in buffer C plus 0.1 M NaCl.
The fractions were assayed for spontaneous (
) and trypsin-revealed
(
) phosphorylase phosphatase activities. The arrows indicate the elution position of marker proteins, i.e. phosphorylase (185 kDa), aldolase (158 kDa), bovine serum albumin
(67 kDa), ovalbumin (43 kDa), and PP-1
(37
kDa).
There are five
known mammalian isoforms of
PP-1(1, 2, 3, 16) , and
it has been demonstrated that the
-,
-, and
-isoforms are present in rat liver(27) . Western analysis
with antibodies that rather specifically recognized the
-isoform
of PP-1
yielded a signal for both PP-1N
and
PP-1N
(Fig. 6). The presence of the
-isoform
is corroborated by findings that the two holoenzymes could be
immunoprecipitated with these antibodies (Fig. 3C).
Surprisingly, the catalytic subunit of PP-1N
migrated as
a slightly larger protein than recombinant PP-1C
and
than the catalytic subunit of PP-1N
. These differences
may reflect a distinct posttranslational modification or the presence
of two
-isoforms generated, for example, by alternative splicing.
Along the same line, it has been shown that there are two forms of
PP-1
that only differ by the presence or absence of an
amino-terminal 11-amino acid insert(16) .
-isoform of the
catalytic subunit in PP-1N. An aliquot of the Mono Q peak fractions of
PP-1N
and PP-1N
, as well as 50 ng of each
of the indicated recombinant isoforms of PP-1
were
subjected to SDS-PAGE (10%) and blotted onto Immobilon membranes.
Subsequently, the catalytic subunit was visualized with antibodies
against PP-1
, as explained under ``Experimental
Procedures.''
Substrate Specificity
The above data imply that
the noncatalytic subunit(s) of PP-1N and PP-1N
are inhibitory, using phosphorylase as substrate. Since the known
regulatory subunits of the cytoplasmic species of PP-1 can be either
inhibitory or stimulatory, depending on the substrate (1, 2, 3) , we have investigated whether this
also applies to PP-1N. For that purpose, we have compared the effect of
trypsinolysis of the nuclear holoenzymes on their activity toward
various substrates (Table 1). Depending on the substrate,
trypsinolysis of PP-1N
increased the phosphatase activity
2-13-fold. On the other hand, a preincubation of PP-1N
with trypsin only caused a 2-5-fold increase in the
phosphatase activities toward phosphorylase, myelin basic protein, and
histone H1 and had no effect on the casein and histone IIA phosphatase
activities. Table 1also shows that the activity of the catalytic
subunit toward all investigated substrates was either not affected or
only slightly increased by trypsinolysis, implying that the observed
effects of trypsin on the nuclear holoenzymes are largely or completely
due to the destruction of the noncatalytic subunit(s). In conclusion,
our data suggest that NIPP-1 and R111 are, if anything, inhibitory to
the dephosphorylation of the investigated substrates.
Regulation of PP-1N
We have previously shown that the active
fragments of NIPP-1 from bovine thymus show a decreased affinity for
PP-1by Reversible
Phosphorylation
after phosphorylation by protein kinase A or casein
kinase-2(11, 12) . In agreement with this result we
found that after incubation of hepatic PP-1N
with PKA
under phosphorylating conditions and subsequent denaturing
electrophoresis, NIPP-1 reproducibly bound less DIG-labeled catalytic
subunit (Fig. 7, upperleftpanel).
Interestingly, the remaining label was bound to a polypeptide, which
migrated somewhat slower during SDS-PAGE, indicating that the
electrophoretic mobility of NIPP-1 is decreased by phosphorylation with
PKA. A lesser DIG-PP-1
labeling was also obtained after
phosphorylation of blotted NIPP-1 with PKA. Subsequent incubation with
PP-2A
and PP-2B restored binding of DIG-PP-1
,
providing additional evidence that the observed effect was due to
phosphorylation of NIPP-1 and not, for example, due to a binding of PKA
or a (proteolytic) loss of NIPP-1. Incubation of PP-1N
or
blotted hepatic NIPP-1 with casein kinase-2 under phosphorylating
conditions did not affect the extent of binding of DIG-PP-1
to NIPP-1 (not shown).
by PKA decreased the affinity of NIPP-1 for
DIG-PP-1
, it did not affect the phosphorylase phosphatase
activity (not shown), suggesting that the decrease in the affinity was
not sufficient to cause a dissociation of the holoenzyme. However, a
2-4-fold increase in the phosphorylase phosphatase activity was
detected after phosphorylation of partially purified PP-1NR
with PKA or casein kinase-2 (not shown). This partially purified
enzyme was obtained by consecutive chromatographies of a nuclear
extract on Mono Q, heparin-Sepharose, and histone IIA-Sepharose (not
illustrated), and was almost certainly a proteolyzed species, as
indicated by observations that the associated NIPP-1 inhibitory
activity was heat stable.
-labeling method to investigate whether NIPP-1 is
also a substrate for PKA in vivo. For that purpose, rats were
injected for various times with glucagon after which liver nuclei were
prepared in the presence of phosphatase inhibitors to prevent
dephosphorylation of NIPP-1. In the lowerpanel of Fig. 7, it is shown that the administration of glucagon resulted
in a time-dependent decrease of the binding of DIG-PP-1
to
NIPP-1, with a maximal effect after 30-60 min. By 90 min the
binding of DIG-PP-1
had almost returned to the control
value. Again, all of the observed differences were abolished by
preincubation of the blots with a mixture of PP-2A
and
PP-2B. Also, Western analysis showed an equal concentration of NIPP-1
at all investigated time points (not shown), providing additional
evidence that the observed differences cannot be explained by a
different loading of NIPP-1.
or of the binding of
DIG-PP-1
to R111 through phosphorylation by PKA or casein
kinase-2.
PP-1N Is Controlled by Inhibitory
Polypeptides
In two previous studies it was concluded that PP-1
is present in hepatic nuclear extracts as free catalytic
subunit(7, 8) . This contrasts with the data of the
present work, showing an association of the catalytic subunit with
inhibitory polypeptides. One obvious reason for this discrepancy is our
use of lower NaCl concentrations (0.3 M) for the
solubilization of PP-1N versus 1-2 M in (7) and (8) , which presumably caused dissociation of
the catalytic subunit. Moreover, at NaCl concentrations above 0.3 M, histones are extracted(27) , which have been shown
to interfere with the phosphorylase phosphatase assay(1) . An
additional explanation for the generation of free PP-1 in
nuclear fractions is the extreme sensitivity of regulatory polypeptides
to endogenous proteases ( Fig. 1and Fig. 2). Perhaps such
proteolysis is mediated by the multicatalytic proteasome complex, which
was shown to be present in liver nuclei(28) . In agreement with
this proposal, we found that the degradation of the regulatory
polypeptides of PP-1 is blocked by leupeptin ( Fig. 1and Fig. 2), which has also been found to block the trypsin-like
activity associated with proteasomes. It cannot be excluded that
nuclear proteolysis provides a mechanism for regulating the activity of
PP-1N in vivo, as has been shown for other nuclear
proteins(29) .
-phosphorylated
histone H1 (30) or PKA-phosphorylated cAMP response element
binding protein, CREB(31) . Our investigations show that this
approach may yield a wrong estimate for the contribution of PP-1N.
Indeed, unless a host of protease inhibitors are added and fresh
material is used, one obtains free catalytic subunit whose substrate
specificity differs from that of holoenzymes (Table 1). It is
also important to note that PP-1N itself is controlled by reversible
phosphorylation (this study and Refs. 11 and 12). In the absence of
functional protein kinases (for lack of MgATP), PP-1N will be
(auto)dephosphorylated during tissue fractionation, and studies on such
fractions may therefore not be informative for the activity of native
PP-1N.
(Fig. 3). The
same approach has previously been used for the identification of the
PP-1
-binding subunit of purified PP-1M(26) . It
should be noted, however, that this approach does not necessarily allow
one to identify all PP-1
-binding polypeptides. Thus, we
have noted that inhibitor 2 and the active 16-18-kDa fragments of
NIPP-1 from bovine thymus do not bind DIG-PP-1
after
denaturing electrophoresis. Also, although it seems likely that the
heat-stable inhibitor(s) of PP-1
that are generated during
incubation of nuclei (Fig. 2) are proteolytic degradation
products of NIPP-1 and/or R111, we have been unable to detect the
accumulation of small, heat-stable inhibitor(s) with the
DIG-PP-1
-labeling technique (not shown).
The Subunit Structure of PP-1N
The copurification
and coimmunoprecipitation of NIPP-1 and PP-1N (Fig. 3-5), combined with the migration of the
holoenzyme during gel filtration as a protein of about 90 kDa (Fig. 5A), strongly indicate that native PP-1N
is a heterodimer of PP-1
and NIPP-1. We suggest that
PP-1N
represents the native hepatic homolog of
PP-1N
, which was previously identified in thymus nuclear
extracts(11, 12) . The latter migrated as a 50-kDa
polypeptide during gel filtration and was also proposed to be a
heterodimer of the catalytic subunit and NIPP-1. The smaller mass of
the thymus enzyme is likely to be explained by proteolysis of NIPP-1
during nuclear fractionation resulting in the generation of active
16-18-kDa fragments.
(Fig. 3)
suggest that R111 is a subunit of PP-1N
. From gel
filtration, we deduced an apparent molecular mass of about 160 kDa,
which agrees with a heterodimeric structure between PP-1
and R111. However, due to the extreme lability of the enzyme (Fig. 4B), it cannot be excluded that the native
phosphatase is larger and/or contains additional subunit(s).
and PP-1N
contain the
-isoform of the catalytic subunit (Fig. 6). Moreover, both species of PP-1N could be
immunoprecipitated with PP-1
-specific antibodies.
However, since only about 70% of the activities of PP-1N
and PP-1N
were precipitated with these antibodies,
it cannot be ruled out that a minor fraction of the holoenzymes
contains other isoform(s) of PP-1
.
Regulation of PP-1N
We found that
the phosphorylation of NIPP-1 by PKA was associated with a lesser
binding of PP-1 (Fig. 7). This agrees with similar
findings on PP-1N
from bovine thymus(11) . However, in
contrast with the thymus enzyme and with the partially purified liver
enzyme, which both contain a heat-stable proteolytic fragment of
NIPP-1, intact hepatic PP-1N
was not activated by
phosphorylation with PKA and/or casein kinase 2. This suggests that
native NIPP-1 is more tightly bound to the catalytic subunit than are
the heat-stable fragments of NIPP-1. Perhaps, phosphorylation by
additional protein kinases is required for dissociation of the native
holoenzyme. It can also be envisaged that dissociation and
reassociation of PP-1N
are independently controlled
processes and that PKA only controls the reassociation of the
holoenzyme.
,
catalytic subunit of PP-1; NIPP-1, nuclear inhibitor of PP-1; PP-1N,
nuclear PP-1; PP-1G, PP-1 associated with glycogen; PP-1M, PP-1
associated with myofibrils; PP-1E, PP-1 associated with endoplasmic
reticulum; PP-1S, soluble fraction of PP-1; PP-2A
, dimeric
form of protein phosphatase-2A, previously called PCS
or
PP2A
; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone;
TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; DIG,
digoxygenin; PAGE, polyacrylamide gel electrophoresis; PKA, protein
kinase A.
and Dr. E. Y. C
Lee for the recombinant isoforms of PP-1
. The
p34
used in this study was a gift of Drs. R.
Derua and J. Goris. We thank Dr. J. R. Vandenheede for advice on the
immunoprecipitation of PP-1N and Dr. S. Wera for comments on the
manuscript. Nicole Sente and Peter Vermaelen provided expert technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.