* Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), 38027 Grenoble cedex 1, France; and Department of
Biomedicine, University of Pisa, 56126 Pisa, Italy
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein phosphatase-1 (PP-1) is involved in
the regulation of numerous metabolic processes in
mammalian cells. The major isoforms of PP-1, ,
1,
and
, have nearly identical catalytic domains, but they
vary in sequence at their extreme NH2 and COOH termini. With specific antibodies raised against the unique
COOH-terminal sequence of each isoform, we find that
the three PP-1 isoforms are each expressed in all mammalian cells tested, but that they localize within these
cells in a strikingly distinct and characteristic manner.
Each isoform is present both within the cytoplasm and in the nucleus during interphase. Within the nucleus,
PP-1
associates with the nuclear matrix, PP-1
1 concentrates in nucleoli in association with RNA, and PP-1
localizes to nonnucleolar whole chromatin. During
mitosis, PP-1
is localized to the centrosome, PP-1
1 is associated with microtubules of the mitotic spindle,
and PP-1
strongly associates with chromosomes. We
conclude that PP-1 isoforms are targeted to strikingly
distinct and independent sites in the cell, permitting
unique and independent roles for each of the isoforms
in regulating discrete cellular processes.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
REVERSIBLE phosphorylation of protein substrates
plays an essential role in general metabolic regulation. The overall state of phosphorylation of substrates regulates such fundamental processes as gene expression, cell cycle progression, and maintenance of the
differentiated state. The state of phosphorylation of specific substrates is, in turn, maintained by a highly regulated
balance between specific protein kinases and protein phosphatases. In addition to the complex controls that regulate
the state of activation of different kinases and phosphatases,
the phosphorylation status of substrates can also be controlled by selective targeting of kinases and phosphatases
to subcellular loci (Hubbard and Cohen, 1993; Faux and
Scott, 1996
).
The problem of targeting is particularly important for
phosphatases. Human cells are estimated to have as many
as 2,000 protein kinases (Hunter, 1995). Thus, a large
number of serine/threonine kinases are capable of controlling a variety of metabolic events through substrate specificity, which is sometimes exquisitely selective. In contrast,
there are relatively few families of serine/threonine phosphatases, and therefore they each must have a much
broader range of targets. The fact that relatively few
serine/threonine protein phosphatases are known raises
the intriguing possibility that a small number of protein
phosphatases might specifically regulate a large number of
phosphorylation events and cellular processes by being
targeted to various subcellular loci (Faux and Scott, 1996
).
Protein phosphatase-1 (PP-1),1 an important family of
serine/threonine phosphatases, is conserved in sequence
among eukaryotes and regulates numerous independent
processes in mammalian cells (Cohen, 1989; Shenolikar,
1994
). In fibroblasts, PP-1 is required for spliceosome assembly (Mermoud et al., 1994
; Misteli and Spector, 1996
),
for dephosphorylation of histone H1 (Paulson et al., 1996
), for maintenance of the tumor suppressor pRb in an active
state (Alberts et al., 1993
; Ludlow et al., 1993
), and for
anaphase progression and exit from mitosis (Fernandez
et al., 1992
).
The PP-1 family has three major 37-kD catalytic subunit
isoforms in mammalian cells (Sasaki et al., 1990; Barker et al.,
1994
). These isoforms exhibit 90% or greater identity in
overall amino acid composition. It has recently been found
that the different isoforms can all be expressed in the same
cell (Puntoni and Villa-Moruzzi, 1997
). Regulatory subunits that target PP-1 to myosin or glycogen in muscle and
to nuclei in fibroblasts have been identified (Tang et al.,
1991
; Shimizu et al., 1994
; Faux and Scott, 1996
). It is possible that the different isoforms may be targeted individually by association with unique regulatory subunits.
The different PP-1 isoforms contain a strong sequence
divergence in their COOH-terminal 30 amino acids, and
this has allowed the production of isoform-specific antibodies (da Cruz e Silva et al., 1995; Villa-Moruzzi et al.,
1996
). Here we have used isoform-specific antibodies to
perform immunofluorescent localization studies in mammalian cells in culture, to determine if individual isoforms show evidence of independent targeting.
We find that PP-1 ,
1, and
localize to distinct subcellular compartments during both interphase and mitosis.
All PP-1 isoforms are present in nuclei, as well as in the cytoplasm, during interphase. Within the nucleus, PP-1
associates with the nuclear matrix, whereas PP-1
1 localizes
to the nucleolus, and PP-1
is associated with whole chromatin. During mitosis, the PP-1 isoforms also localize differentially. PP-1
localizes to centrosomes, while PP-1
1
is associated with microtubules of the mitotic spindle. In
contrast, we find that PP-1
is strongly localized to chromosomes. Our results thus present the possibility that each
of the PP-1 isoforms is independently regulated and has
distinct cell targets and roles in cellular regulation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture
HeLa cells were grown as monolayers in DME (GIBCO BRL, Paisley,
UK). Manca (human non-Hodgkin's lymphoma) cells (Nishikori et al.,
1984) were grown in suspension in RPMI 1640 medium (GIBCO BRL).
HeLa and Manca cell cultures were supplemented with 5% bovine calf serum (Hyclone Labs, Logan, UT). All cells were maintained in a humid incubator at 5% CO2 and 37°C.
Antibodies
Peptide affinity-purified rabbit isoform-specific antibody to PP-1 (RU34) (da Cruz e Silva et al., 1995
) was a generous gift from Drs. Edgar
da Cruz e Silva and Paul Greengard (Rockefeller University, New York).
Peptide affinity-purified rabbit isoform-specific antibodies to PP-1
1 and
PP-1
have been previously described (Villa-Moruzzi et al., 1996
). Anti-
-tubulin ascites antibody (TUB 2.1) was from Sigma Chemical Co. (St.
Louis, MO). Human autoimmune serum B.S., which recognizes centromere protein-A (CENP-A), has been described previously (Palmer et al.,
1987
). Secondary antibodies included FITC-conjugated affinity-purified
goat anti-rabbit IgG antibodies from Cappel Laboratories (West Chester,
PA) and cyanine-3-conjugated goat anti-mouse IgG antibodies from
Jackson ImmunoResearch Laboratories (West Grove, PA).
Immunofluorescence Microscopy
HeLa cells were grown on poly-lysine-coated coverslips for a minimum of
24 h before fixation. Cells were fixed with 1% paraformaldehyde-PBS for
2 min, followed by 20°C methanol for 10 min and treatment with 0.5%
NP-40 in PBS for 2 min. Fixation with 2% paraformaldehyde alone gave
similar results. Washes with PBS, incubation with primary and secondary
antibodies, and counterstaining with propidium iodide were as described
previously (Andreassen and Margolis, 1994
).
Images were collected with a MRC-600 Laser Scanning Confocal Apparatus (BioRad Microscience Division, Herts, UK) coupled to a Nikon Optiphot microscope (Melville, NY). Composite whole cell images were generated from serial optical sections representing the entire depth of field using Comos software (BioRad Microscience Division).
Nuclear Extraction for Microscopy
For immunofluorescent localization of PP-1 isoforms after nuclear extraction, HeLa cells were grown on poly-lysine-coated coverslips for a minimum of 48 h and then subjected to permeabilization and cell extraction.
For extraction, cells were lysed with 0.5% Triton X-100 in 10 mM Pipes,
pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 1 mM EGTA
containing 1 mM PMSF for 2 min (Zeng et al., 1994). After permeabilization, RNA and DNA were digested with 100 µg/ml RNase A and 100 µg/ml
DNase I (Sigma Chemical Co.), respectively, for 20 min in the buffer described above. Digested chromatin was extracted 5 min with 250 mM ammonium sulfate in the same buffer after digestion of DNA. Permeabilization, digestion, and extraction were all performed at ambient temperature.
After each step, cells were fixed for immunofluorescence microscopy as
described above.
Preparation of Cellular Fractions for Immunoblotting
Nuclear Isolation.
To determine the presence of each PP-1 isoform in both
nuclear and cytoplasmic fractions, Manca cells were fractionated by a
modification of the procedure described by Palmer et al. (1987) and immunoblotted. Exponentially growing Manca cells were collected by centrifugation and washed with 3.75 mM Tris-HCl, pH 7.4, 15 mM KCl, 3.75 mM
NaCl, 125 µM spermidine, 37.5 µM spermine, 250 µM EDTA, and 50 µM
EGTA with 30% (vol/vol) glycerol, 15 mM
-mercaptoethanol, 10 µM
aprotinin, 10 µM leupeptin, and 100 µM PMSF. Cells were then resuspended in 5 ml of the same buffer containing 0.2% Triton X-100 and incubated at 4°C for 30 min. Cells were homogenized by 20 strokes of a
Dounce-A pestle, after which nuclei were determined to be free of cytoplasm by phase-contrast microscopy. Nuclei were collected by centrifugation (1,000 g, 10 min), and the nuclear and cytoplasmic fractions were adjusted to equivalent volumes with sample buffer for SDS-PAGE.
Mitotic Spindle.
The association of PP-1 isoforms with the mitotic spindle was determined by comparing the residual fractions in which microtubules were either stabilized with taxol (Schiff and Horwitz, 1980) or depolymerized with nocodazole (Jordan et al., 1992
) after permeabilization in a
microtubule-stabilizing buffer (Gorbsky and Ricketts, 1993
). Mitotic cells
were selectively detached after treatment with either taxol (5 µg/ml) or
nocodazole (1 µg/ml) for 16 h. Mitotic indices of detached cells were
greater than 90%. After collection, cells were permeabilized 2 min at 37°C
in 60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl2, pH 6.9 (PHEM; Gorbsky and Ricketts, 1993
) containing 0.2% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.1 mM PMSF. Residual pellets
were then collected at 37°C by centrifugation (300 g, 2 min), and fractions
were prepared in sample buffer for SDS-PAGE.
Chromosomes. The association of each PP-1 isoform with chromosomes was determined by its release into the soluble fraction after nuclease digestion of a chromosome fraction. Residual cell pellets containing chromosomes were prepared by lysis of selectively detached mitotic cells after treatment with 1.0 µg/ml nocodazole. Cells were permeabilized in PHEM containing 0.1% NP-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.1 mM PMSF. After 2 min, cells were collected by centrifugation and digested 30 min at 37°C with 40 µg/ml DNAse I. Soluble and residual fractions were then separated by centrifugation (300 g, 2 min) and analyzed as above.
Immunoblotting
Interphase HeLa cells were collected by trypsinization and mitotic cells
by selective detachment after arrest with 0.04 µg/ml nocodazole. Cells
were then lysed in 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 5 mM EGTA,
0.1% NP-40, 10 µg/ml aprotonin, 10 µg/ml leupeptin, and 1.0 mM PMSF
for 30 min on ice. Lysates were resolved on 12% polyacrylamide gels, and
gel-separated proteins were then transferred to nitrocellulose sheets using
a semidry blotting apparatus, blocked with 5% nonfat milk, incubated
overnight with primary antibodies, washed, and then incubated with
HRP-conjugated goat anti-rabbit IgG secondary antibodies, as previously
described (Andreassen and Margolis, 1994). Protein-antibody complex
was detected by enhanced chemiluminescence (Amersham Corp., Arlington Heights, IL).
Isoform specificity of each antibody was tested by immunoblotting
against equivalent amounts of each recombinant PP-1 isoform (Zhang et al.,
1993; protein kindly provided by Dr. E.Y.C. Lee, New York Medical College, Valhalla, NY).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Specificity of PP-1 Isoform Antibodies
The human ,
1, and
isoforms of PP-1 have 90% or
greater identity in amino acid sequence (Barker et al.,
1994
). The catalytic domains (amino acids 42-298 of PP-1
)
(Peruski et al., 1993
) have greater than 97% homology between isoforms and have similar activities in vitro (Zhang
et al., 1993
). A small number of amino acid substitutions
are conserved among mammalian species in the first 35 amino acids of sequence, upstream of the catalytic domain (Fig. 1 A). However, by far the greatest sequence variance
occurs in the COOH-terminal 25-33 amino acids. The
COOH-terminal divergence in sequence between isoforms may be of significance, as the COOH-terminal isoform-specific sequences are very highly conserved amongst
mammalian species (Sasaki et al., 1990
; Barker et al., 1994
).
|
The presence of COOH-terminal isoform-specific sequence has allowed the production of antibodies that specifically recognize each of the isoforms in mammalian cells
(da Cruz e Silva et al., 1995; Villa-Moruzzi et al., 1996
).
Each of the antibodies used in our study is specific for a
single isoform. The specificity has been tested by cross-blotting procedures in which each antibody was used to
probe each of the isoforms, expressed as recombinant proteins (Fig. 1 B; see also da Cruz e Silva et al., 1995
; Villa-Moruzzi et al., 1996
). Furthermore, each of the isoform-specific antibodies recognizes a single protein of 37 kD in
both interphase and mitotic HeLa whole cell extracts (Fig.
1 C) and is therefore specific for PP-1. In accord with previous results (Puntoni and Villa-Moruzzi, 1997
), the
,
1,
and
isoforms of PP-1 are all expressed in HeLa cells.
Differential Localization of PP-1 Isoforms in HeLa Cells at Interphase and Mitosis
Although the PP-1 isoforms are highly homologous and
have nearly identical catalytic domains, it is possible that
either the conserved NH2-terminal or COOH-terminal sequence divergence might target the different isoforms to
specific sites where they might have unique functions. We
therefore tested for differences in localization of the antigens by immunofluorescence microscopy. In whole cell
images generated from serial sections collected by confocal microscopy, we find that each isoform is evident both in the cytoplasm and nuclei of HeLa cells during interphase (Fig. 2). This is consistent with studies showing both
nuclear and cytoplasmic PP-1 activity in interphase HeLa
cells (Puntoni and Villa-Moruzzi, 1997). Controls treated
without anti-PP-1 primary antibody do not display a detectable signal either in nuclei or the cytoplasm (data not
shown).
|
|
We used optical sections obtained by confocal microscopy to examine in greater detail the specific localization
of each PP-1 isoform both during interphase and mitosis in
HeLa (Fig. 3 A). During interphase, we find that PP-1 ,
1, and
all are present in HeLa cell nuclei but that they
localize to distinct nuclear compartments. Image overlay
of PP-1
against a propidium iodide counterstain shows
that this isoform has a nonuniform distribution in nuclei
(Fig. 3 A) and is excluded from nucleoli, which are discernible by strong propidium iodide staining. PP-1
also
concentrates on the centrosome, which is adjacent to the
nucleus in interphase cells (Fig. 3 A). PP-1
is also excluded from nucleoli but displays a more homogeneous
distribution elsewhere in the nucleus than PP-1
. In contrast, PP-1
1 localizes preferentially to the nucleolus, yielding a yellow coloration where propidium iodide and
PP-1
1 overlap (Fig. 3 A). A previous immunolocalization study had demonstrated that PP-1 localizes to nuclei
in rat embryo fibroblasts, but it used antibodies that did
not distinguish between PP-1 isoforms (Fernandez et al.,
1992
). Cytoplasmic signal is less concentrated than nuclear
signal and is not uniformly detected in the optical sections
shown in Fig. 3 A.
We have determined that the distinctive nuclear distributions of the PP-1 isoforms observed in HeLa cells are
also present in other mammalian cell lines. In addition to
our study of PP-1 isoform distribution in HeLa, which are
epitheloid carcinoma cells, we have also examined the nuclear localization of PP-1 isoforms in transformed human
lymphocyte (Manca) cells (Nishikori et al., 1984) and in
nontransformed CHO fibroblasts and rat embryonal fibroblasts (REF-52) (data not shown). Immunoblots demonstrate the same antibody specificity for PP-1
,
1, and
in
Manca cell extracts as in HeLa extracts (data not shown).
Our results with these other cell lines show that interphase
distributions of the isoforms are identical to those in HeLa
cells.
We have also examined the localization of PP-1 ,
1,
and
in HeLa cells during mitotic metaphase using optical
sections (shown as two-color overlays in Fig. 3 A). PP-1
shows a strong association with the centrosome but is excluded from chromosomes whose position is indicated by
propidium iodide stain. PP-1
1 is present throughout the
mitotic spindle but shows a higher concentration near the
centrosomes (Fig. 3 A). Like PP-1
, PP-1
1 is excluded from chromosomes (especially evident in separated channels; see Fig. 3 B). PP-1
, by contrast with the other two
isoforms, is present predominantly on chromosomes and
does not show any localization to the mitotic spindle.
We have examined the mitotic distributions of PP-1 isoforms in greater detail in Fig. 3 B. Here, double-label immunofluorescence microscopy for PP-1 and antitubulin
shows that PP-1
is apparently centrosome associated
(Fig. 3 B, top). Localization of PP-1
to the spindle poles
has been confirmed by digital overlay of the images for
PP-1
and tubulin (data not shown). By contrast, comparison of PP-1
1 and tubulin distribution demonstrates that PP-1
1 associates with microtubules of the mitotic spindle, but with an apparently higher concentration near the
centrosomes (Fig. 3 B, middle). PP-1
1 remains associated with the spindle throughout mitosis, becoming concentrated near the spindle poles during telophase (data
not shown). It is clear from the separated images that neither PP-1
nor PP-1
1 associates with chromosomes. By
contrast, PP-1
is present on metaphase chromosomes
(Fig. 3 A), and it remains associated with chromosomes
throughout mitosis, as is evident in a telophase image (Fig.
3 B, bottom). The differential associations of PP-1
,
1,
and
with the centrosome, mitotic spindle, and chromosomes, respectively, are not sensitive to detergent extraction (data not shown). The distribution of each isoform
observed during mitosis is also conserved in Manca, CHO,
and REF-52 cells (data not shown). We conclude that the
immunofluorescence data demonstrate that the different
PP-1 isoforms are targeted to markedly different sites in
both interphase and mitotic cells. These data are summarized in Table I.
|
The localization of each PP-1 isoform to nuclei was confirmed by immunoblots of nuclei isolated from Manca
cells (Fig. 4). Manca cells, which are transformed human
lymphocytes (Nishikori et al., 1984), were used for this
procedure since their nuclei can be separated cleanly from
cytoplasm. Each PP-1 isoform localizes to nuclei in Manca
cells, with sublocalization similar to that of HeLa cells
(data not shown). Effective separation of nuclei from cytoplasm has been confirmed by immunoblots for tubulin, which show that isolated nuclei are devoid of cytoskeletal
and cytoplasmic contamination (Fig. 4). By contrast, the
centromeric protein CENP-A (Palmer et al., 1987
) is
present in isolated nuclei but is absent from the cytoplasmic fraction, thus demonstrating that the cytoplasmic fraction is devoid of nuclear contamination. Each of the three
PP-1 isoforms is present both in isolated nuclei and in the
cytoplasmic fraction, and all are distributed roughly equivalently between the nucleus and cytoplasm.
|
PP-1 has been implicated in the regulation of spliceosome
activity, cAMP response element binding (CREB)-dependent transcription, and control of S phase progression
(Hagiwara et al., 1992; Walker et al., 1992
; Mermoud et al.,
1994
; Misteli and Spector, 1996
). The differential distribution of the PP-1 isoforms in nuclei suggests that the different isoforms might independently control these distinct
processes. To further analyze the distribution of the different PP-1's within distinct nuclear compartments, we performed indirect immunofluorescence microscopy after nuclear permeabilization and extraction of either RNA or
chromatin (Fig. 5).
|
After permeabilization (Fig. 5 A), PP-1 and
still
show exclusion from nucleoli, while PP-1
1 is found enriched in the nucleolar compartment. Upon digestion with
RNase A (Fig. 5 B), PP-1
1 is extracted from nucleoli,
and the signal is also diminished elsewhere in nuclei. We
note, however, that perinuclear cytoplasmic PP-1
1 signal
is consistently augmented by RNase treatment. By contrast, PP-1
and
are not sensitive to RNase A digestion.
Bulk chromatin can be removed from nuclei by the combination of digestion with DNase I and salt extraction (Fig.
5 C) (Berezney and Coffey, 1977). Indirect immunofluorescence microscopy after extraction of chromatin reveals
that the nuclear signal of PP-1
is completely extracted by
this treatment. We conclude that PP-1
is chromatin associated. In addition to being sensitive to RNase treatment,
PP-1
1 signal, including that in the nucleolus, is eliminated by treatment with DNase I and ammonium sulphate. By contrast, the nuclear signal of nonnucleolar PP-1
remains after extraction of either RNA or bulk chromatin. We conclude, therefore, that a subset of PP-1
is associated with the nuclear matrix (Berezney and Coffey,
1977
). These results demonstrate that the PP-1
,
1, and
isoforms localize not only to different sites but also to different molecular compartments within HeLa nuclei.
Fractions from HeLa cells were immunoblotted to confirm the differential localization of the PP-1 isoforms during mitosis (Fig. 6). To determine association with the mitotic spindle, cells were treated with either taxol, which
stabilizes microtubule assembly (Schiff and Horwitz, 1980),
or nocodazole, which induces depolymerization of microtubules (Jordan et al., 1992
). After drug treatment, mitotic cells were selectively detached and were permeabilized
with a microtubule-stabilizing buffer (Gorbsky and Ricketts, 1993
). Western blots show PP-1
1 is associated with
the microtubule-containing cell pellet from taxol-treated
cells, but it is much diminished in pellets from nocodazole-treated cells that are devoid of microtubules (Fig. 6 A). By
contrast, PP-1
is equally abundant in the cell pellets of
both taxol- and nocodazole-treated cells. PP-1
is not detectable in cell pellets from cells treated with either taxol
or nocodazole (Fig. 6 A). We conclude that each PP-1 isoform is distributed distinctly in mitotic cells and that the
distribution of PP-1
1, but not of PP-1
or
, is uniquely
microtubule dependent.
|
After lysis of cells arrested in mitosis (Fig. 6, A and B),
both PP-1 1 and
are present in the cell pellet, which
contains both cytoskeleton and chromosomes, as determined by immunofluorescence microscopy and immunoblotting (data not shown). The association of PP-1
with
chromosomes has been confirmed by its solubilization after digestion of lysed cells with DNase I (Fig. 6 B). The
release of PP-1
into the supernatant is specific to DNase
treatment and does not occur in a mock-digestion without
DNase I. PP-1
is absent from the cell pellet after permeabilization, and PP-1
1, which is weakly present in the
pellet fraction (Fig. 6 A), is not preferentially solubilized
by digestion with DNase I (Fig. 6 B). The evidence thus
supports the unique association of PP-1
, in part, with
chromosomes.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PP-1 has many important regulatory roles in cell metabolism (Cohen, 1989; Shenolikar, 1994
). Although the PP-1
family is composed of several highly conserved catalytic
isoforms, it has not been evident that these isoforms might
have distinct functions within a single cell. Here we report
that the different isoforms of PP-1 are all present within
each of several different cell types from different species.
Furthermore, the PP-1 isoforms each localize to distinct
sites both in mitotic cells and in interphase nuclei. A previous study of the subcellular localization of PP-1 used an
antibody raised against the common catalytic domain sequence and thus did not distinguish between PP-1 isoforms (Fernandez et al., 1992
). In contrast, using isoform-specific antibodies raised against distinct COOH-terminal
sequences, we have found an intricate differential distribution pattern of PP-1 isoforms among different cellular organelles.
PP-1 can be targeted and regulated by association with
targeting subunits (Hubbard and Cohen, 1993; Stuart et al.,
1994
; Faux and Scott, 1996
). Our finding of independent
localization of PP-1 isoforms suggests that each catalytic
isoform may be differentially localized by association with
one or more unique targeting subunits. Assuming this is
true, many PP-1-targeting subunits remain to be identified. At each specific site, PP-1 is likely to have unique substrates. Because the targeting subunits are themselves
subject to regulation, for example by phosphorylation
(MacKintosh et al., 1988
; Beullens et al., 1993
), independent targeting and regulation of PP-1 isoforms could permit PP-1 to specifically and independently control multiple cellular processes. Finally, given the demonstrated role
of PP-1 in the regulation of mitosis (Doonan and Morris, 1989
; Ohkura et al., 1989
; Axton et al., 1990
; Fernandez
et al., 1992
) and the distinct localizations we have found,
our results suggest a specific and independent role for
each PP-1 isoform in mitosis.
Isoforms of the PP-1 Catalytic Subunit Are Differentially Targeted
We have found that PP-1 ,
1, and
show different subcellular localization both during interphase and mitosis
(Table I). During interphase, each of the isoforms is concentrated in distinct nuclear compartments. For example,
PP-1
1 is concentrated at nucleoli, while PP-1
and
appear to be excluded from nucleoli. Within this compartment, PP-1
is partly associated with the nuclear matrix,
while PP-1
is associated with the DNase-extractable chromatin fraction. During mitosis, PP-1
is associated
with chromosomes. By contrast, PP-1
1 is associated with
microtubules of the mitotic spindle, while PP-1
is associated with the centrosome. These results suggest that PP-1
isoforms achieve specificity by targeting to different sites
within the cell. Furthermore, these results strongly suggest
that there is an isoform-specific regulation of various processes that have thus far been attributed to PP-1 activity
without distinction of the isoform involved.
The catalytic subunits of PP-1 are localized by targeting
subunits (Hubbard and Cohen, 1993; Stuart et al., 1994
;
Faux and Scott, 1996
). In mammalia, subunits that target
PP-1 to glycogen, myofibrils, and nuclei have been identified (Tang et al., 1991
; Shimizu et al., 1994
; Faux and Scott,
1996
). It is clear that different isoforms can share association with certain targeting subunits (Alessi et al., 1993
).
However, our results suggest it is equally probable that
specific catalytic subunits can be directed to different targets by association with unique targeting subunits. The
subunits that might target PP-1 catalytic isoforms to distinct sites within nuclei and that might direct PP-1
to
chromosomes, PP-1
to centrosomes, and PP-1
1 to the
mitotic spindle are presently unknown.
Because PP-1-targeting subunits can regulate substrate
specificity and sensitivity to inhibitory proteins (Hubbard
and Cohen, 1993), the function of PP-1 must be considered
in the context of the specific catalytic-regulatory subunit
complex. The targeting subunit itself can be regulated by
phosphorylation (MacKintosh et al., 1988
; Beullens et al.,
1993
), suggesting that PP-1 isoforms localized at unique
sites can be independently regulated. Each isoform of the
catalytic subunit of PP-1 could possibly associate with multiple targeting subunits, yielding further specificity of PP-1
targeting and regulation.
The specific localization of PP-1 isoforms that we have
observed implicates a specific PP-1 isoform in the regulation of several site-specific processes previously ascribed
generically to PP-1. For example, CREB-dependent phosphorylation, which regulates CREB-dependent transcription, is in turn regulated by PP-1 (Hagiwara et al., 1992).
Given the association of PP-1
with chromatin, we suggest that this isoform may specifically regulate transcription.
Other nuclear processes might also be regulated by site-specific activity of PP-1 isoforms. For example, PP-1 is required for spliceosome assembly (Mermoud et al., 1994
;
Misteli and Spector, 1996
). Splicing factors display a punctate distribution and are associated with the nuclear matrix (Bisotto et al., 1995
; Misteli and Spector, 1996
). These
are also characteristics of PP-1
distribution, and they
suggest that PP-1
might uniquely play a role in pre-mRNA splicing. Similarly, the predominant localization of
PP-1
1 to nucleoli suggests it might have a specific function in ribosome processing (Beullens et al., 1996
).
During mitosis, a PP-1 activity has recently been found
to be associated with chromosomes and to be involved in
the dephosphorylation of histone H1 (Paulson et al., 1996).
Since PP-1
, and not PP-1
or PP-1
1, is associated with
chromosomes, we suggest that this isoform may be the
protein phosphatase responsible for the dephosphorylation of histone H1 and the regulation of decondensation of
chromosomes at the end of mitosis. If true, it would be expected that PP-1
would remain associated with chromosomes through the end of mitosis when chromosome decondensation occurs, as we have observed (Fig. 3 B).
A role for PP-1 in the regulation of chromosome segregation and mitotic exit has been demonstrated previously
(Doonan and Morris, 1989; Ohkura et al., 1989
; Axton et al.,
1990
; Fernandez et al., 1992
). In Drosophila, mutation of a
single isoform of PP-1 causes mitotic arrest. By localizing
to spindle microtubules, PP-1
1 might control chromosome segregation by regulating microtubule dynamics.
Such localization is interesting in light of a recent report of
a specific role for PP-1 in the control of microtubule dynamics during exit from mitosis in Xenopus extracts (Tournebize et al., 1997
).
Alternatively, either PP-1 or PP-1
1 might play a role
at the metaphase-anaphase transition. Either of these PP-1
isoforms could be involved in checkpoint mechanisms that
monitor mitotic spindle function and delay the onset of
anaphase. Microinjection of mammalian fibroblasts with
non-isoform-specific PP-1 antibodies induces arrest at
metaphase (Fernandez et al., 1992
). Also, the mitotic spindle is the site of degradation of cyclin B and of p34cdc2 kinase inactivation (Kubiak et al., 1993
; Andreassen and
Margolis, 1994
; Tugendreich et al., 1995
), both of which
normally occur at the onset of anaphase (Pines and
Hunter, 1991
; Hunt et al., 1992
). Since p34cdc2 can phosphorylate (Villa-Moruzzi, 1992
) and inactivate PP-1 (Dohadwala et al., 1994
; Puntoni and Villa-Moruzzi, 1997
), degradation of cyclin B at the onset of anaphase might lead to
the activation of PP-1 as a requirement for the onset or
completion of anaphase (Kwon et al., 1997
). Upon activation, either PP-1
1, which is associated with microtubules,
or PP-1
, which is associated with the centrosome, might
regulate anaphase by local action at these sites.
The PP-1 ,
1, and
isoforms are products of distinct
genes (Barker et al., 1993
, 1994
). Both PP-1
and PP-1
1,
but apparently not PP-1
, are expressed at elevated levels
in certain human tumors (Sogawa et al., 1994a
,b). It is possible that these two isoforms have distinct roles in cell cycle regulation not shared by PP-1
. Our work now makes
it important to pursue an understanding of the distinct site-specific roles that each of the PP-1 isoforms must play in
regulation of the cell cycle, and possibly in tumorigenesis.
![]() |
Footnotes |
---|
Received for publication 15 August 1997 and in revised form 24 March 1998.
Address all correspondence to Robert L. Margolis, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), 41 avenue des Martyrs, 38027 Grenoble cedex 1, France. Tel.: 33-4-76-88-96-16. Fax: 33-4-76-88-54-94.We are grateful to E.F. da Cruz e Silva and P. Greengard for providing affinity-purified anti-PP-1 antibody, and Dr. E.Y.C. Lee for providing PP-1
recombinant proteins.
This work was supported in part by grants from the Association pour la Recherche sur le Cancer and from the International Human Frontiers of Sciences Program (R.L. Margolis) and the Associazione Italiana Ricerca sul Cancro (E. Villa-Moruzzi).
![]() |
Abbreviations used in this paper |
---|
CENP-A, centromere protein-A; CREB, cAMP response element binding; PP-1, protein phosphatase-1.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Alberts, A.S., A.M. Thorburn, S. Shenolikar, M.C. Mumby, and J.R. Feramisco. 1993. Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. Proc. Natl. Acad. Sci. USA. 90: 388-392 [Abstract]. |
2. | Alessi, D.R., A.J. Street, P. Cohen, and P.T.W. Cohen. 1993. Inhibitor-2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 into a conformation with the specificity and regulatory properties of the native enzyme. Eur. J. Biochem. 213: 1055-1066 [Abstract]. |
3. | Andreassen, P.R., and R.L. Margolis. 1994. Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells. J. Cell Biol. 127: 789-802 [Abstract]. |
4. | Axton, J.M., V. Dombradi, P.T.W. Cohen, and D.M. Glover. 1990. One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell. 63: 33-46 |
5. |
Barker, H.M.,
S.P. Craig,
N.K. Spurr, and
P.T.W. Cohen.
1993.
Sequence of human protein serine/threonine phosphatase 1 ![]() |
6. |
Barker, H.M.,
N.D. Brewis,
A.J. Street,
N.K. Spurr, and
P.T.W. Cohen.
1994.
Three genes for protein phosphatase 1 map to different human chromosomes: sequence expression and gene localisation of protein serine/threonine phosphatase 1 ![]() ![]() |
7. |
Berezney, R., and
D.S. Coffey.
1977.
Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei.
J. Cell Biol.
73:
616-637
|
8. |
Beullens, M.,
A. Van Eynde,
M. Bollen, and
W. Stalmans.
1993.
Inactivation of
nuclear inhibitory polypeptides of protein phosphatase-1 (NIPP-1) by protein kinase A.
J. Biol. Chem
268:
13172-13177
|
9. | Beullens, M., W. Stalmans, and M. Bollen. 1996. Characterization of a ribosomal inhibitory polypeptide of protein phosphatase-1 from rat liver. Eur. J. Biochem. 239: 183-189 [Abstract]. |
10. |
Bisotto, S.,
P. Lauriault,
M. Duval, and
M. Vincent.
1995.
Colocalization of a
high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes.
J. Cell Sci.
108:
1873-1882
|
11. | Cohen, P.. 1989. The structure and regulation of protein phosphatases. Annu. Rev. Biochem 58: 453-508 |
12. | da Cruz e Silva, E.F., C.A. Fox, C.C. Ouimet, E. Gustafson, S.J. Watson, and P. Greengard. 1995. Differential expression of protein phosphatase 1 isoforms in mammalian brain. J. Neurosci. 15: 3375-3389 [Abstract]. |
13. | Dohadwala, M., E.F. da Cruz e Silva, F.L. Hall, R.T. Williams, D.A. Carbonaro-Hall, A.C. Nairn, P. Greengard, and N. Berndt. 1994. Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases. Proc. Natl. Acad. Sci. USA. 91: 6408-6412 [Abstract]. |
14. | Doonan, J.H., and N.R. Morris. 1989. The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1. Cell. 57: 987-996 |
15. | Faux, M.C., and J.D. Scott. 1996. More on target with protein phosphorylation: conferring specificity by location. Trends Biochem. Sci. 21: 312-315 |
16. | Fernandez, A., D.L. Brautigan, and N.J.C. Lamb. 1992. Protein phosphatase type 1 in mammalian cell mitosis: chromosomal localization and involvement in mitotic exit. J. Cell Biol. 116: 1421-1430 [Abstract]. |
17. | Gorbsky, G.J., and W.A. Ricketts. 1993. Differential expression of a phosphoepitope at the kinetochores of moving chromosomes. J. Cell Biol. 122: 1311-1321 [Abstract]. |
18. | Hagiwara, M., A. Alberts, P. Brindle, J. Meinkoth, J. Feramisco, T. Deng, M. Karin, S. Shenolikar, and M. Montminy. 1992. Transcriptional attenuation following cAMP induction requires PP-1 mediated dephosphorylation of CREB. Cell 70: 105-113 |
19. | Hubbard, M.J., and P. Cohen. 1993. On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem. Sci. 18: 172-177 |
20. | Hunt, T., F.C. Luca, and J.V. Ruderman. 1992. The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo. J. Cell Biol. 116: 707-724 [Abstract]. |
21. | Hunter, T.. 1995. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell. 80: 225-236 |
22. | Jordan, M.A., D. Thrower, and L. Wilson. 1992. Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles: implications for the role of microtubule dynamics in mitosis. J. Cell Sci. 102: 401-416 [Abstract]. |
23. | Kubiak, J.Z., M. Weber, H. de Pennart, N.J. Winston, and B. Maro. 1993. The metaphase II arrest in mouse oocytes is controlled through microtubule- dependent destruction of cyclin B in the presence of CSF. EMBO (Eur. Mol. Biol. Organ.) J. 12: 3773-3778 [Abstract]. |
24. |
Kwon, Y.-G.,
S.Y. Lee,
Y. Choi,
P. Greengard, and
A.C. Nairn.
1997.
Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by
cdc2 kinase.
Proc. Natl. Acad. Sci. USA.
94:
2168-2173
|
25. | Ludlow, J.W., C.L. Glendening, D.M. Livingston, and J.A. DeCaprio. 1993. Specific enzymatic dephosphorylation of the retinoblastoma protein. Mol. Cell. Biol. 13: 367-372 [Abstract]. |
26. | MacKintosh, C., D.G. Campbell, A. Hiraga, and P. Cohen. 1988. Phosphorylation of the glycogen-binding subunit of protein phosphatase-1G in response to adrenalin. FEBS Lett 234: 189-194 |
27. | Mermoud, J.E., P.T.W. Cohen, and A.I. Lamond. 1994. Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO (Eur. Mol. Biol. Organ.) J 13: 5679-5688 [Abstract]. |
28. | Misteli, T., and D.L. Spector. 1996. Serine/threonine phosphatase 1 modulates the subnuclear distribution of pre-mRNA splicing factors. Mol. Biol. Cell. 7: 1559-1572 [Abstract]. |
29. | Nishikori, M., H. Hansen, S. Jhanwar, J. Fried, P. Sordillo, B. Koziner, K. Lloyd, and B. Clarkson. 1984. Establishment of a near tetraploid B-cell lymphoma line with duplication of the 8;14 translocation. Cancer Genet. Cytogenet 12: 39-50 |
30. | Ohkura, H., N. Kinoshita, S. Miyatani, T. Toda, and M. Yanagida. 1989. The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell. 57: 997-1007 |
31. | Palmer, D.K., K. O'Day, M.H. Wener, B.S. Andrews, and R.L. Margolis. 1987. A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones. J. Cell Biol. 104: 805-815 [Abstract]. |
32. |
Paulson, J.R.,
J.S. Patzlaff, and
A.J. Vallis.
1996.
Evidence that the endogenous
histone H1 phosphatase in HeLa mitotic chromosomes is protein phosphatase 1, not protein phosphatase 2A.
J. Cell Sci.
109:
1437-1447
|
33. | Peruski, L.F. Jr., B.E. Wadzinski, and G.L. Johnson. 1993. Analysis of the multiplicity, structure, and function of protein serine/threonine phosphatases. Adv. Prot. Phos. 7: 9-30 . |
34. | Pines, J., and T. Hunter. 1991. Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J. Cell Biol. 115: 1-17 [Abstract]. |
35. |
Puntoni, F., and
E. Villa-Moruzzi.
1997.
Protein phosphatase-1![]() ![]() ![]() |
36. |
Sasaki, K.,
H. Shima,
Y. Kitagawa,
S. Irino,
T. Sugimura, and
M. Nagao.
1990.
Identification of members of the protein phosphatase 1 gene family in the rat
and enhanced expression of protein phosphatase 1![]() |
37. | Schiff, P.B., and S.B. Horwitz. 1980. Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Natl. Acad. Sci. USA. 77: 1561-1565 [Abstract]. |
38. |
Shenolikar, S..
1994.
Protein serine/threonine phosphatases![]() |
39. |
Shimizu, H.,
M. Ito,
M. Miyahara,
K. Ichikawa,
S. Okubo,
T. Konishi,
M. Naka,
T. Tanaka,
K. Hirano,
D.J. Hartshorne, and
T. Nakano.
1994.
Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase.
J.
Biol. Chem
269:
30407-30411
|
40. |
Sogawa, K.,
T. Yamada,
Y. Funamoto,
K. Kohno,
H. Nishikawa,
F. Kishida,
F. Hamazaki,
N. Yamashita, and
K. Matsumoto.
1994a.
Selective increase in
expression of isoform PP1 ![]() |
41. | Sogawa, K., T. Yamada, T. Masaki, H. Nishikawa, Y. Cai, S. Oka, H. Norimatsu, and K. Matsumoto. 1994b. Enhanced expression of catalytic subunits of protein phosphatase type 1 and high S-phase fraction in liposarcoma. Res. Commun. Mol. Path. Pharm 85: 359-362 |
42. | Stuart, J.S., D.L. Frederick, C.M. Varner, and K. Tatchell. 1994. The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit. Mol. Cell. Biol. 14: 896-905 [Abstract]. |
43. |
Tang, P.M.,
J.A. Bondor,
K.M. Swiderek, and
A.A. DePaoli-Roach.
1991.
Molecular cloning and expression of the regulatory (RG1) subunit of the glycogen-associated protein phosphatase.
J. Biol. Chem.
266:
15782-15789
|
44. |
Tournebize, R.,
S.S.L. Andersen,
F. Verde,
M. Dorée,
E. Karsenti, and
A.A. Hyman.
1997.
Distinct roles of PP1 and PP-2A-like phosphatases in control
of microtubule dynamics during mitosis.
EMBO (Eur. Mol. Biol. Org.) J.
16:
5537-5549
|
45. | Tugendreich, S., J. Tomkiel, W. Earnshaw, and P. Hieter. 1995. CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition. Cell. 81: 261-268 |
46. | Villa-Moruzzi, E.. 1992. Activation of type-1 protein phosphatase by cdc2 kinase. FEBS Lett. 304: 211-215 |
47. |
Villa-Moruzzi, E.,
F. Puntoni, and
O. Marin.
1996.
Activation of protein phosphatase-1 isoforms and glycogen synthase kinase-3![]() |
48. | Walker, D.H., A.A. DePaoli-Roach, and J.L. Maller. 1992. Multiple roles for protein phosphatase 1 in regulating the Xenopus early embryonic cell cycle. Mol. Biol. Cell. 3: 687-698 [Abstract]. |
49. | Zeng, C., D. He, and B.R. Brinkley. 1994. Localization of NuMA protein isoforms in the nuclear matrix of mammalian cells. Cell Motil. Cytoskel. 29: 167-176 |
50. |
Zhang, Z.,
G. Bai,
M. Shima,
S. Zhao,
M. Nagao, and
E.Y.C. Lee.
1993.
Expression and characterization of rat protein phosphatases -1![]() ![]() ![]() ![]() |