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The Rockefeller University Press, 0021-9525/98/06/1207/9 $2.00
The Journal of Cell Biology, Volume 141, Number 5, June
1, 1998 1207–1215
http://www.jcb.org 1207
Differential Subcellular Localization of Protein
Phosphatase-1
a
,
g
1, and
d
Isoforms during Both Interphase
and Mitosis in Mammalian Cells
Paul R. Andreassen,* Françoise B. Lacroix,* Emma Villa-Moruzzi,
‡
and Robert L. Margolis*
*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,
a
,
g
1,
and
d
, have nearly identical catalytic domains, but they
vary in sequence at their extreme NH
2
and COOH ter-
mini. 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 mam-
malian 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
a
associates with the nuclear matrix, PP-1
g
1 con-
centrates in nucleoli in association with RNA, and PP-1
d
localizes to nonnucleolar whole chromatin. During
mitosis, PP-1
a
is localized to the centrosome, PP-1
g
1
is associated with microtubules of the mitotic spindle,
and PP-1
d
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.
R
eversible
phosphorylation of protein substrates
plays an essential role in general metabolic regula-
tion. The overall state of phosphorylation of sub-
strates regulates such fundamental processes as gene ex-
pression, cell cycle progression, and maintenance of the
differentiated state. The state of phosphorylation of spe-
cific substrates is, in turn, maintained by a highly regulated
balance between specific protein kinases and protein phos-
phatases. 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 con-
trolled 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 control-
ling a variety of metabolic events through substrate speci-
ficity, which is sometimes exquisitely selective. In contrast,
there are relatively few families of serine/threonine phos-
phatases, 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 as-
sembly (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 sub-
units 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 pos-
sible that the different isoforms may be targeted individu-
ally by association with unique regulatory subunits.
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.
1.
Abbreviations used in this paper
: CENP-A, centromere protein-A;
CREB, cAMP response element binding; PP-1, protein phosphatase-1.
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Published June 1, 1998
The Journal of Cell Biology, Volume 141, 1998 1208
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 anti-
bodies (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 mam-
malian cells in culture, to determine if individual isoforms
show evidence of independent targeting.
We find that PP-1
a
,
g
1, and
d
localize to distinct subcel-
lular compartments during both interphase and mitosis.
All PP-1 isoforms are present in nuclei, as well as in the cy-
toplasm, during interphase. Within the nucleus, PP-1
a
as-
sociates with the nuclear matrix, whereas PP-1
g
1 localizes
to the nucleolus, and PP-1
d
is associated with whole chro-
matin. During mitosis, the PP-1 isoforms also localize dif-
ferentially. PP-1
a
localizes to centrosomes, while PP-1
g
1
is associated with microtubules of the mitotic spindle. In
contrast, we find that PP-1
d
is strongly localized to chro-
mosomes. 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 se-
rum (Hyclone Labs, Logan, UT). All cells were maintained in a humid in-
cubator at 5% CO
2
and 37
8
C.
Antibodies
Peptide affinity-purified rabbit isoform-specific antibody to PP-1
a
(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
g
1 and
PP-1
d
have been previously described (Villa-Moruzzi et al., 1996). Anti–
b
-tubulin ascites antibody (TUB 2.1) was from Sigma Chemical Co. (St.
Louis, MO). Human autoimmune serum B.S., which recognizes cen-
tromere 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
2
20
8
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 Ap-
paratus (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 extrac-
tion, HeLa cells were grown on poly-lysine–coated coverslips for a mini-
mum 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 MgCl
2
, and 1 mM EGTA
containing 1 mM PMSF for 2 min (Zeng et al., 1994). After permeabiliza-
tion, RNA and DNA were digested with 100
m
g/ml RNase A and 100
m
g/ml
DNase I (Sigma Chemical Co.), respectively, for 20 min in the buffer de-
scribed above. Digested chromatin was extracted 5 min with 250 mM am-
monium sulfate in the same buffer after digestion of DNA. Permeabiliza-
tion, 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 im-
munoblotted. Exponentially growing Manca cells were collected by cen-
trifugation and washed with 3.75 mM Tris-HCl, pH 7.4, 15 mM KCl, 3.75 mM
NaCl, 125
m
M spermidine, 37.5
m
M spermine, 250
m
M EDTA, and 50
m
M
EGTA with 30% (vol/vol) glycerol, 15 mM
b
-mercaptoethanol, 10
m
M
aprotinin, 10
m
M leupeptin, and 100
m
M PMSF. Cells were then resus-
pended in 5 ml of the same buffer containing 0.2% Triton X-100 and incu-
bated at 4
8
C for 30 min. Cells were homogenized by 20 strokes of a
Dounce-A pestle, after which nuclei were determined to be free of cyto-
plasm by phase-contrast microscopy. Nuclei were collected by centrifuga-
tion (1,000
g
, 10 min), and the nuclear and cytoplasmic fractions were ad-
justed to equivalent volumes with sample buffer for SDS-PAGE.
Mitotic Spindle.
The association of PP-1 isoforms with the mitotic spin-
dle was determined by comparing the residual fractions in which microtu-
bules were either stabilized with taxol (Schiff and Horwitz, 1980) or depo-
lymerized 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
m
g/ml) or
nocodazole (1
m
g/ml) for 16 h. Mitotic indices of detached cells were
greater than 90%. After collection, cells were permeabilized 2 min at 37
8
C
in 60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl
2
, pH 6.9
(PHEM; Gorbsky and Ricketts, 1993) containing 0.2% Triton X-100, 10
m
g/ml aprotinin, 10
m
g/ml leupeptin, and 0.1 mM PMSF. Residual pellets
were then collected at 37
8
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 di-
gestion of a chromosome fraction. Residual cell pellets containing chro-
mosomes were prepared by lysis of selectively detached mitotic cells after
treatment with 1.0
m
g/ml nocodazole. Cells were permeabilized in PHEM
containing 0.1% NP-40, 10
m
g/ml aprotinin, 10
m
g/ml leupeptin, and 0.1 mM
PMSF. After 2 min, cells were collected by centrifugation and digested 30
min at 37
8
C with 40
m
g/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
m
g/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
m
g/ml aprotonin, 10
m
g/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., Arling-
ton 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 Col-
lege, Valhalla, NY).
Results
Specificity of PP-1 Isoform Antibodies
The human
a
,
g
1, and
d
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
a
)
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Andreassen et al.
Isoform-specific Subcellular Localization of PP-1
1209
(Peruski et al., 1993) have greater than 97% homology be-
tween 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 iso-
forms may be of significance, as the COOH-terminal iso-
form-specific sequences are very highly conserved amongst
mammalian species (Sasaki et al., 1990; Barker et al., 1994).
The presence of COOH-terminal isoform-specific se-
quence has allowed the production of antibodies that spe-
cifically 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 pro-
teins (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 pre-
vious results (Puntoni and Villa-Moruzzi, 1997), the
a
,
g
1,
and
d
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 NH
2
-terminal or COOH-terminal se-
quence divergence might target the different isoforms to
specific sites where they might have unique functions. We
therefore tested for differences in localization of the anti-
gens by immunofluorescence microscopy. In whole cell
images generated from serial sections collected by confo-
cal microscopy, we find that each isoform is evident both
in the cytoplasm and nuclei of HeLa cells during inter-
phase (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 de-
tectable signal either in nuclei or the cytoplasm (data not
shown).
We used optical sections obtained by confocal micros-
copy 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
a
,
g
1, and
d
all are present in HeLa cell nuclei but that they
localize to distinct nuclear compartments. Image overlay
of PP-1
a
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 dis-
cernible by strong propidium iodide staining. PP-1
a
also
concentrates on the centrosome, which is adjacent to the
nucleus in interphase cells (Fig. 3
A
). PP-1
d
is also ex-
cluded from nucleoli but displays a more homogeneous
distribution elsewhere in the nucleus than PP-1
a
. In con-
trast, PP-1
g
1 localizes preferentially to the nucleolus,
yielding a yellow coloration where propidium iodide and
PP-1
g
1 overlap (Fig. 3
A
). A previous immunolocaliza-
tion 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 distri-
butions of the PP-1 isoforms observed in HeLa cells are
also present in other mammalian cell lines. In addition to
Figure 1. Isoforms of PP-1
are most divergent at their
COOH termini. (A) The se-
quences of human PP-1 a,
g1, and d have been aligned
and compared in this sche-
matic representation. Each
amino acid difference be-
tween any pair of PP-1 iso-
forms (Barker et al., 1994) is
indicated by a solid vertical
line. There is a modest
amount of divergence at the
NH2 terminus, but most di-
vergence occurs proximal to
the COOH terminus. There
are few differences within
the conserved catalytic do-
main (amino acids 42–98 in
human PP-1 a; Peruski et al.,
1993), indicated by a horizontal bar above the diagram. Single letter code sequences of PP-1 a, g1, and d at their divergent COOH ter-
mini are shown below the diagram. The peptide sequences used for the generation of isoform-specific antibodies are underlined. (B)
Cross-blots of the PP-1 a, g1, and d antibodies against all three isoforms demonstrate that each antibody is isoform specific. (C) Anti-
bodies generated against COOH-terminal peptides from PP-1 a, g1, and d each specifically recognize a 37-kD protein corresponding to
PP-1 in blots of lysates from either interphase or mitotic HeLa cells. Bars at the left margin indicate size markers: 105, 82, 45, 33, 29, and
19 kD, respectively.
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The Journal of Cell Biology, Volume 141, 1998 1210
our study of PP-1 isoform distribution in HeLa, which are
epitheloid carcinoma cells, we have also examined the nu-
clear localization of PP-1 isoforms in transformed human
lymphocyte (Manca) cells (Nishikori et al., 1984) and in
nontransformed CHO fibroblasts and rat embryonal fibro-
blasts (REF-52) (data not shown). Immunoblots demon-
strate the same antibody specificity for PP-1 a, g1, and d 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 a, g1,
and d in HeLa cells during mitotic metaphase using optical
sections (shown as two-color overlays in Fig. 3 A). PP-1 a
shows a strong association with the centrosome but is ex-
cluded from chromosomes whose position is indicated by
propidium iodide stain. PP-1 g1 is present throughout the
mitotic spindle but shows a higher concentration near the
centrosomes (Fig. 3 A). Like PP-1 a, PP-1 g1 is excluded
from chromosomes (especially evident in separated chan-
nels; see Fig. 3 B). PP-1 d, 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 iso-
forms in greater detail in Fig. 3 B. Here, double-label im-
munofluorescence microscopy for PP-1 a and antitubulin
shows that PP-1 a is apparently centrosome associated
(Fig. 3 B, top). Localization of PP-1 a to the spindle poles
has been confirmed by digital overlay of the images for
PP-1 a and tubulin (data not shown). By contrast, compar-
ison of PP-1 g1 and tubulin distribution demonstrates that
PP-1 g1 associates with microtubules of the mitotic spin-
dle, but with an apparently higher concentration near the
centrosomes (Fig. 3 B, middle). PP-1 g1 remains associ-
ated with the spindle throughout mitosis, becoming con-
centrated near the spindle poles during telophase (data
not shown). It is clear from the separated images that nei-
ther PP-1 a nor PP-1 g1 associates with chromosomes. By
contrast, PP-1 d 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 a, g1,
and d with the centrosome, mitotic spindle, and chromo-
somes, respectively, are not sensitive to detergent extrac-
tion (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 summa-
rized in Table I.
The localization of each PP-1 isoform to nuclei was con-
firmed 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 cyto-
plasm 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 cytoplas-
mic fraction, thus demonstrating that the cytoplasmic frac-
tion 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 equiv-
alently between the nucleus and cytoplasm.
PP-1 has been implicated in the regulation of spliceosome
activity, cAMP response element binding (CREB)-depen-
dent 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 distribu-
tion of the PP-1 isoforms in nuclei suggests that the differ-
ent isoforms might independently control these distinct
processes. To further analyze the distribution of the differ-
ent PP-1’s within distinct nuclear compartments, we per-
formed indirect immunofluorescence microscopy after nu-
clear permeabilization and extraction of either RNA or
chromatin (Fig. 5).
After permeabilization (Fig. 5 A), PP-1 a and d still
show exclusion from nucleoli, while PP-1 g1 is found en-
riched in the nucleolar compartment. Upon digestion with
RNase A (Fig. 5 B), PP-1 g1 is extracted from nucleoli,
Figure 2. PP-1 a, g1, and d are each localized to both cytoplasm
and nuclei in interphase HeLa cells. Images, representing whole
cells, were generated from optical sections throughout the cell
depth that were collected by confocal microscopy. A detailed
comparison of isoform distributions in nuclei by optical sections
of interphase cells is shown in Fig. 3 A. Bar, 10 mm.
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Andreassen et al. Isoform-specific Subcellular Localization of PP-1 1211
and the signal is also diminished elsewhere in nuclei. We
note, however, that perinuclear cytoplasmic PP-1 g1 signal
is consistently augmented by RNase treatment. By con-
trast, PP-1 a and d are not sensitive to RNase A digestion.
Bulk chromatin can be removed from nuclei by the com-
bination of digestion with DNase I and salt extraction (Fig.
5 C) (Berezney and Coffey, 1977). Indirect immunofluo-
rescence microscopy after extraction of chromatin reveals
that the nuclear signal of PP-1 d is completely extracted by
this treatment. We conclude that PP-1 d is chromatin asso-
ciated. In addition to being sensitive to RNase treatment,
PP-1 g1 signal, including that in the nucleolus, is elimi-
nated by treatment with DNase I and ammonium sul-
phate. By contrast, the nuclear signal of nonnucleolar PP-1
a remains after extraction of either RNA or bulk chroma-
tin. We conclude, therefore, that a subset of PP-1 a is asso-
ciated with the nuclear matrix (Berezney and Coffey,
1977). These results demonstrate that the PP-1 a, g1, and d
isoforms localize not only to different sites but also to dif-
ferent molecular compartments within HeLa nuclei.
Fractions from HeLa cells were immunoblotted to con-
firm the differential localization of the PP-1 isoforms dur-
ing mitosis (Fig. 6). To determine association with the mi-
Figure 3. PP-1 isoforms are differentially localized both during interphase and mitosis in HeLa cells. (A) Images for PP-1 a, g1, and d
(green) merged with respective images of propidium iodide counterstain (red) are shown for cells at interphase and at mitotic
metaphase. All primary antibodies were detected with FITC-conjugated secondary antibodies. Each isoform is present in the nucleus at
interphase. PP-1 g1 localizes to the nucleolus, whereas PP-1 a and d distribute elsewhere in the nucleus. At mitotic metaphase, PP-1 a,
g1, and d antigens are distributed to the centrosome, mitotic microtubules, and chromosomes, respectively. Nucleoli and chromosomes
are strongly stained by propidium iodide at interphase and mitosis, respectively. Images are yellow where the green and red signals over-
lap. A centrosome adjacent to the nucleus of an interphase cell is labeled with PP-1 a–specific antibody. (B) Separated images show PP-1
isoforms are specifically associated either with the mitotic spindle, centrosomes, or chromosomes of HeLa cells. (Top) Double-label im-
munofluorescence images of PP-1 a (left) and antitubulin (right) in a metaphase cell demonstrate that PP-1 a is concentrated at the cen-
trosome. (Middle) Double-label immunofluorescence images of PP-1 g1 (left) and antitubulin (right) show that PP-1 g1 is localized to
spindle microtubules at metaphase. (Bottom) PP-1 d (left) remains associated with chromosomes throughout mitosis, as shown here for
a telophase cell. The propidium iodide counterstain (right) confirms that the cell is in telophase. Bars, 10 mm.
Table I. PP-1 Isoform Localization
PP-1 aPP-1 g 1 PP-1 d
Interphase nuclear matrix, centrosomes nucleoli chromatin
Mitosis centrosomes mitotic spindle chromosomes
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The Journal of Cell Biology, Volume 141, 1998 1212
totic spindle, cells were treated with either taxol, which
stabilizes microtubule assembly (Schiff and Horwitz, 1980),
or nocodazole, which induces depolymerization of micro-
tubules (Jordan et al., 1992). After drug treatment, mitotic
cells were selectively detached and were permeabilized
with a microtubule-stabilizing buffer (Gorbsky and Rick-
etts, 1993). Western blots show PP-1 g1 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 d is equally abundant in the cell pellets of
both taxol- and nocodazole-treated cells. PP-1 a is not de-
tectable in cell pellets from cells treated with either taxol
or nocodazole (Fig. 6 A). We conclude that each PP-1 iso-
form is distributed distinctly in mitotic cells and that the
distribution of PP-1 g1, but not of PP-1 a or d, is uniquely
microtubule dependent.
After lysis of cells arrested in mitosis (Fig. 6, A and B),
both PP-1 g1 and d are present in the cell pellet, which
contains both cytoskeleton and chromosomes, as deter-
mined by immunofluorescence microscopy and immuno-
blotting (data not shown). The association of PP-1 d with
chromosomes has been confirmed by its solubilization af-
ter digestion of lysed cells with DNase I (Fig. 6 B). The
release of PP-1 d into the supernatant is specific to DNase
treatment and does not occur in a mock-digestion without
DNase I. PP-1 a is absent from the cell pellet after perme-
abilization, and PP-1 g1, 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 d, in part, with
chromosomes.
Discussion
PP-1 has many important regulatory roles in cell metabo-
lism (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 previ-
ous study of the subcellular localization of PP-1 used an
Figure 4. Each PP-1 isoform is present
in isolated nuclei. Immunoblots of
Manca whole cell lysates, of isolated
nuclei, and of the released cytoplasmic
fraction are shown. All lanes were
loaded on the basis of cell equivalence.
The presence of each PP-1 isoform
both in nuclei and cytoplasm is demon-
strated by immunoblots probed for PP-1
a, g1, and d, respectively. Samples
were also exposed to antitubulin anti-
bodies to show the purity of isolated
nuclei, and to CENP-A antiserum to
show the purity of the cytoplasmic
fractions.
Figure 5. Nuclear extraction demonstrates that the different PP-1
isoforms localize to distinct nuclear compartments. HeLa cells
were fixed and prepared for immunofluorescence microscopy ei-
ther (A) after permeabilization with Triton X-100, (B) permeabi-
lization followed by RNA digestion, or (C) permeabilization fol-
lowed by chromatin extraction through treatment with DNase I
and then 0.25 M ammonium sulfate. PP-1 a, g1, and d are all
present in nuclei after permeabilization. PP-1 g1 is sensitive to
RNA extraction, PP-1 d is extracted with chromatin, and PP-1 a
resists extraction through association with the nuclear matrix.
Propidium iodide counterstain reveals both the extraction of nu-
cleolar RNA by RNase treatment and the extraction of chroma-
tin by the combination of DNase I and salt. Bar, 10 mm.
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Andreassen et al. Isoform-specific Subcellular Localization of PP-1 1213
antibody raised against the common catalytic domain se-
quence and thus did not distinguish between PP-1 iso-
forms (Fernandez et al., 1992). In contrast, using isoform-
specific antibodies raised against distinct COOH-terminal
sequences, we have found an intricate differential distribu-
tion pattern of PP-1 isoforms among different cellular or-
ganelles.
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 identi-
fied. 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), indepen-
dent targeting and regulation of PP-1 isoforms could per-
mit PP-1 to specifically and independently control multi-
ple 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 a, g1, and d show different sub-
cellular localization both during interphase and mitosis
(Table I). During interphase, each of the isoforms is con-
centrated in distinct nuclear compartments. For example,
PP-1 g1 is concentrated at nucleoli, while PP-1 a and d ap-
pear to be excluded from nucleoli. Within this compart-
ment, PP-1 a is partly associated with the nuclear matrix,
while PP-1 d is associated with the DNase-extractable
chromatin fraction. During mitosis, PP-1 d is associated
with chromosomes. By contrast, PP-1 g1 is associated with
microtubules of the mitotic spindle, while PP-1 a is associ-
ated 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 pro-
cesses 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 identi-
fied (Tang et al., 1991; Shimizu et al., 1994; Faux and Scott,
1996). It is clear that different isoforms can share associa-
tion 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 tar-
gets by association with unique targeting subunits. The
subunits that might target PP-1 catalytic isoforms to dis-
tinct sites within nuclei and that might direct PP-1 d to
chromosomes, PP-1 a to centrosomes, and PP-1 g1 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 mul-
tiple 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 regula-
tion of several site-specific processes previously ascribed
generically to PP-1. For example, CREB-dependent phos-
phorylation, which regulates CREB-dependent transcrip-
tion, is in turn regulated by PP-1 (Hagiwara et al., 1992).
Given the association of PP-1 d 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 re-
quired for spliceosome assembly (Mermoud et al., 1994;
Misteli and Spector, 1996). Splicing factors display a punc-
tate distribution and are associated with the nuclear ma-
trix (Bisotto et al., 1995; Misteli and Spector, 1996). These
are also characteristics of PP-1 a distribution, and they
suggest that PP-1 a might uniquely play a role in pre-
mRNA splicing. Similarly, the predominant localization of
Figure 6. PP-1 isoforms distribute to different compartments in
mitotic HeLa cells as assayed in immunoblots. PP-1 g1, but not
PP-1 a or d, is enriched in microtubule fractions after cell lysis
(A). Residual pellets from cells treated with taxol contain assem-
bled microtubules, but pellets from cells treated with 1.0 mg/ml
nocodazole (Noc.) are devoid of microtubules. PP-1 g1 is prefer-
entially associated with the residual cell pellet that contains mi-
crotubules after treatment with taxol. Whole cell lysates and re-
sidual cell pellets from cells treated with either taxol or
nocodazole and loaded on the basis of equal cell number are
shown. PP-1 d is associated with chromosomes, as determined by
release from the residual cell pellet after digestion with DNase I
(B). Mitotic cells were collected by selective detachment, perme-
abilized, and incubated in digestion buffer either with (1) or
without (2) DNase I, and supernatants (Sup.) were loaded onto
gels. The residual cell pellet after lysis, loaded on the basis of
equal cell number, is shown for reference. The majority of PP-1 d
is present in the cell supernatant from nocodazole-treated cells
(see Fig. 6 A). Therefore, the blot of PP-1 d shown here was ex-
posed extensively to bring out the pellet fraction to examine its
extractability by digestion with DNase I.
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Published June 1, 1998
The Journal of Cell Biology, Volume 141, 1998 1214
PP-1 g1 to nucleoli suggests it might have a specific func-
tion 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 d, and not PP-1 a or PP-1 g1, is associated with
chromosomes, we suggest that this isoform may be the
protein phosphatase responsible for the dephosphoryla-
tion of histone H1 and the regulation of decondensation of
chromosomes at the end of mitosis. If true, it would be ex-
pected that PP-1 d would remain associated with chromo-
somes through the end of mitosis when chromosome de-
condensation occurs, as we have observed (Fig. 3 B).
A role for PP-1 in the regulation of chromosome segre-
gation 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 g1 might control chromo-
some 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 dynam-
ics during exit from mitosis in Xenopus extracts (Tournebize
et al., 1997).
Alternatively, either PP-1 a or PP-1 g1 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 spin-
dle is the site of degradation of cyclin B and of p34cdc2 ki-
nase 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 phos-
phorylate (Villa-Moruzzi, 1992) and inactivate PP-1 (Dohad-
wala et al., 1994; Puntoni and Villa-Moruzzi, 1997), degra-
dation 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 activa-
tion, either PP-1 g1, which is associated with microtubules,
or PP-1 a, which is associated with the centrosome, might
regulate anaphase by local action at these sites.
The PP-1 a, g1, and d isoforms are products of distinct
genes (Barker et al., 1993, 1994). Both PP-1 a and PP-1 g1,
but apparently not PP-1 d, are expressed at elevated levels
in certain human tumors (Sogawa et al., 1994a,b). It is pos-
sible that these two isoforms have distinct roles in cell cy-
cle regulation not shared by PP-1 d. 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.
We are grateful to E.F. da Cruz e Silva and P. Greengard for providing af-
finity-purified anti–PP-1 a 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).
Received for publication 15 August 1997 and in revised form 24 March
1998.
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