1 Institute of Medical Biochemistry, University of Oslo, PO Box 1112 Blindern,
0317 Oslo, Norway
2 Catholic University of Leuven, Division of Biochemistry, Campus Gasthuisberg,
Herestraat 49, 3000 Leuven, Belgium
* Author for correspondence (e-mail: philippe.collas{at}basalmed.uio.no)
Accepted 19 February 2003
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Summary |
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Key words: AKAP149, G1 phase, Nuclear envelope, Protein phosphatase, PP1
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Introduction |
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Growing evidence suggests that the NE serves not only as a barrier
separating the nucleus from the cytoplasm but also as a mediator of nuclear
functions. The NE consists of two concentric membranes, nuclear pores and the
nuclear lamina, a meshwork of intermediate filaments called A- and B-type
lamins (Gruenbaum et al.,
2000). The inner nuclear membrane (INM) harbors specific integral
proteins that provide attachment sites for the lamina, transcriptional
regulators, chromatin-associated proteins and DNA
(Vlcek et al., 2001
). The
discovery that mutations in genes coding for lamin A/C and emerin (an integral
protein of the INM) cause hereditary disorders affecting skeletal, cardiac and
adipose tissues (Vigouroux and Bonne,
2002
) suggests a role for the NE in the regulation of gene
expression. Lamins also play more than a structural role in the nucleus.
Nuclei reassembled in vitro without a lamina
(Jenkins et al., 1995
) or
disorganization of the lamina with dominant negative lamin mutants was shown
to alter DNA replication (Ellis et al.,
1997
; Spann et al.,
1997
; Moir et al.,
2000
). Intranuclear lamin foci also co-localize with RNA splicing
factors, suggesting that lamins may contribute to organizing the RNA
processing machinery (Jagatheesan et al.,
1999
).
Relatively few AKAPs have been identified at the NE. nAKAP150
(Zhang et al., 1996) and
AKAP95 (Coghlan et al., 1994
;
Collas et al., 1999
) have been
localized to the nuclear matrix and thus interact with the NE. A 255 kDa AKAP
(mAKAP) targets PKA and phosphodiesterase PDE4D3 near the ryanodine receptor
at the NE of myocytes and was proposed to modulate activity of the receptor
(Kapiloff et al., 2001
;
Dodge et al., 2001
). AKAP149
is a 149 kDa AKAP recently identified as a component of the endoplasmic
reticulum-NE system (Steen et al.,
2000
). AKAP149 also interacts with A- and B-type lamins
(Steen and Collas, 2001
),
suggesting that AKAP149 is associated with both the outer and inner nuclear
membranes.
PP1 belongs to the PPP family of protein Ser/Thr phosphatases. PP1
holoenzymes usually consist of a regulatory (R) and a catalytic subunit.
Similarly to AKAPs, the R subunits can serve as moieties targeting PP1 to or
near its substrate (Bollen,
2001). Most regulators of PP1 harbor a degenerate RVXF
motif (where X is any amino acid) that binds to a hydrophobic pocket
of PP1 (Bollen, 2001
;
Ceulemans et al., 2002a
). This
does not preclude, however, an association of PP1 with these R subunits via
additional binding motifs. PP1 regulates a variety of cellular processes
(Bollen, 2001
;
Cohen, 2002
) and is involved
in exit from mitosis (Tournebize et al.,
1997
; Sugiyama et al.,
2002
; Katayama et al.,
2001
). Nuclear PP1 has been implicated in the control of
transcription, pre-mRNA splicing and cell-cycle progression by
dephosphorylation of key proteins such as RNA polymerase II, SR-splicing
factors and the retinoblastoma protein (pRb)
(Boudrez et al., 2000
;
Riedl and Egly, 2000
;
Rubin et al., 2001
). AKAP149
interacts with a fraction of nuclear PP1 via a consensus `RVXF' motif
(155KGVLF159) and recruits the phosphatase to the NE
upon nuclear reconstitution in vitro
(Steen et al., 2000
) and at
the end of mitosis (Steen and Collas,
2001
). Among the late mitotic substrates of PP1 are B-type lamins
(Thompson et al., 1997
) and
recruitment of PP1 to nuclear membranes by AKAP149 is essential for B-type
lamin assembly and cell survival (Steen
and Collas, 2001
).
The role of PP1 at the NE upon nuclear reformation suggests a tight regulation of PP1 activity at the end of mitosis and possibly also in G1 phase. We show here that NE-associated AKAP149 anchors PP1 at the NE throughout G1 and dissociates at the G1/S phase transition upon serine phosphorylation of AKAP149. Premature disruption of the AKAP149-PP1 interaction in G1 results in intranuclear lamina solubilization and G1 arrest. We also demonstrate that AKAP149 is a novel B-type lamin specifying subunit of PP1.
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Materials and Methods |
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Cells and nuclei
HeLa cells were grown adherent in EMEM (Gibco-BRL) with 10% FCS
(Steen and Collas, 2001).
Cells were synchronized in M phase with 1 µM nocodazole for 18 hours. To
allow cell cycle re-entry, cells were washed and replated at
2.5x106 cells per 162 cm2 flask. `Time zero' after
mitotic release was time of replating. Nuclei were isolated by Dounce
homogenization (Steen et al.,
2000
) at indicated time points after replating. For in vitro
nuclear reconstitution assays, nuclei were isolated from confluent HeLa cell
cultures by Dounce homogenization (Collas
et al., 1999
). NEs were prepared from purified nuclei as described
previously (Steen et al.,
2000
).
Nuclear assembly assay
Condensed, membrane-free chromatin masses were prepared from HeLa nuclei
disassembled in mitotic extract as described previously
(Steen et al., 2000). After
sedimentation at 1000 g through 1 M sucrose, chromosomes (2
µl) were resuspended in 40 µl interphase cytosolic HeLa cell extract (at
5000 chromatin masses/µl), containing 4 µl mitotic membranes, an
ATP-regenerating system (1.2 µl) and 100 µM GTP (0.4 µl)
(Steen et al., 2000
).
RVXF or RAXF peptides (10 µM) were added to the extract
and the reaction was placed at 30°C for 2 hours. Nuclear assembly was
examined by phase contrast microscopy, membrane labeling with 10 µg/ml
DiOC6 and by immunofluorescence.
In vitro replication and quantification of DNA synthesis
DNA replication was assayed in vitro in isolated nuclei by incorporation of
[32P]dCTP in an S-phase extract using a method derived from
those reported previously (Krude et al.,
1997
; Stoeber et al.,
1998
). S-phase whole cell extracts were prepared from S-phase HeLa
cells collected 15 hours after release from mitotic arrest. Cells were lysed
by Dounce homogenization in cell lysis buffer
(Martins et al., 2000
), and
then briefly sonicated on ice to lyse nuclei and release soluble nuclear
components. The lysate was sedimented at 15,000 g for 15
minutes then at 200,000 g in a Beckman SW41 rotor for 2 hours
at 4°C. Protein concentration of the extract was 25-30 mg/ml. G0-phase
extracts were prepared as above from confluent HeLa cells serum-starved for
five days. Nuclei purified from G1-phase cells (and capable of import; data
not shown) were incubated at 30°C for 3 hours at 5000 nuclei/µl in 40
µl S-phase extract containing a mix of buffered dNTPs (40 mM Hepes, pH 7.8,
7 mM MgCl2, 0.1 mM each of dATP, dGTP, dTTP and dCTP; 2 µl)
(Krude et al., 1997
), 1 µl
[
32P]dCTP (3000 Ci/mmol; Nycomed-Amersham, Piscataway, NJ),
the ATP-regenerating system and 100 µM GTP. At the end of incubation,
samples were mixed with 1 volume of 20 mM Tris (pH 7.5) and 1 mg/ml proteinase
K and digested for 2 hours at 37°C
(Gant et al., 1999
). Samples
were mixed by pipetting and 5 µl aliquots were electrophoresed through 0.8%
agarose. Gel loading was assessed by ethidium bromide staining. Samples
contained equal numbers of nuclei and sedimentation steps were eliminated to
avoid loss of nuclei (Gant et al.,
1999
). Signals were detected by autoradiography of the gels.
Immunological procedures
Immunofluorescence analysis of methanol-fixed cells or nuclei was performed
as described previously (Steen and Collas,
2001). SDS-PAGE and immunoblotting analysis were carried out
(Steen et al., 2000
) using
indicated antibodies. AKAP149 was immunoprecipitated from HeLa NEs after
sonication and solubilization in immunoprecipitation buffer (10 mM HEPES, pH
7.5, 10 mM KCl, 2 mM MgCl2, 1% Triton X-100, 1 mM DTT and protease
inhibitors) (Steen and Collas,
2001
). Immune precipitates (IPs) were washed three times in
immunoprecipitation buffer and resuspended in 100 mM Tris-HCl (pH 7.5) for
phosphatase assays or in SDS-sample buffer for SDS-PAGE. AKAP149, NIPP1, PNUTS
and Sds22 were also immunoprecipitated from nuclear lysates as described
previously (Steen et al.,
2000
), then washed and dissolved in SDS-sample buffer. B-type
lamins were immunoprecipitated from solubilized NEs
(Steen and Collas, 2001
), IPs
were resuspended in 100 mM Tris-HCl (pH 7.5), phosphorylated in vitro (see
below) and used in phosphatase assays. For phosphorylation of
immunoprecipitated B-type lamins, phosphatase inhibitors (50 mM NaF, 5 mM
sodium pyrophosphate, 0.1 mM sodium orthovanadate) were included in the
immunoprecipitation buffer.
TUNEL analysis
Cells were fixed with 3% paraformaldehyde. Fragmented DNA was labeled with
fluorescein-conjugated dUTP using the Roche (Indianapolis, IN) In Situ Cell
Death Detection Kit. Cells were examined by fluorescence microscopy and images
analyzed with the AnalySIS software.
Nuclear microinjection
HeLa cells released from nocodazole-induced mitotic arrest were plated onto
coverslips. At indicated time points after replating, nuclei were injected
(Collas et al., 1999) with 25
pl PBS containing 10 µg/ml 150 kDa FITC-dextran or 0.1% phenol red, as
indicated, to visualize injections, and 100 nM of the indicated peptide. Cells
were cultured in EMEM/10% FCS for indicated time periods without or with BrdU
and processed for immunofluorescence or BrdU incorporation analysis (see
below). S-phase cells were injected 12 hours after release from mitotic
arrest. Between 45 and 55 cells were injected per treatment in two to three
replicates.
Bromodeoxyuridine labeling
Following release from nocodazole-induced mitotic arrest, cells (injected
or non-injected) were labeled with 100 µM BrdU (Sigma, St Louis, MO) for
indicated time periods in culture. BrdU incorporation was visualized with
FITC-conjugated anti-BrdU antibodies (Sigma) following methanol fixation.
Phosphatase assays
Dephosphorylation of 32P-labeled phosphorylase a was
done as described previously (Beullens et
al., 1998) using as a source of phosphatase an AKAP149-IP from
2x106 nuclei per treatment. To assess B-type lamin
dephosphorylation, B-type lamins were immunoprecipitated from 107
NEs. The IP was incubated for 30 minutes at 23°C in protein kinase C
phosphorylation buffer (200 mM NaCl, 10 mM MgSO4, 50 mM Tris-HCl,
pH 7.4, 100 µM CaCl2, 40 µg/ml phosphatidylserine, 20 µM
diacylglycerol, 1 mM DTT, 10 µM ATP) containing 1 µCi/ml
[
32P]ATP, 5 ng/µl rat
ß
PKC and
phosphatase inhibitors (50 mM NaF, 5 mM sodium pyrophosphate, 0.1 mM sodium
orthovanadate). Phosphorylated B-type lamin-IPs were sedimented, washed twice
in 50 mM Tris-HCl/50 mM NaCl/0.01% Tween 20 with phosphatase inhibitors and
once without inhibitors. IPs were then incubated with an AKAP149-IP from
107 cells in phosphatase assay buffer (25 mM Tris-HCl, pH 7.4, 3
mg/ml BSA, 1 mM DTT) for 15 minutes at 23°C with or without specified
inhibitors or peptides. B-type lamin dephosphorylation was measured by
scintillation counting of released 32P
(Beullens et al., 1998
).
Percentages of 32P release were compared by Chi-square analysis of
duplicate experiments.
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Results |
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|
AKAP149 is the only known binding protein of PP1 at the NE
Although most PP1 holoenzymes are heterodimers of a catalytic and an R
subunit, some contain two R subunits
(Bollen, 2001). Thus, we
explored whether established nuclear R subunits of PP1, i.e. PNUTS, Sds22 and
NIPP1 (Bollen and Beullens,
2002
), were also part of the AKAP149-PP1 complex. PNUTS, Sds22 and
NIPP1, present in HeLa nuclei, did not co-immunoprecipitate with AKAP149 from
G1-phase nuclear extracts (Fig.
2A). Also, overlay assays of nuclear AKAP149-IPs using recombinant
PP1 did not reveal any PP1-binding protein other than AKAP149 (data not
shown). This suggests that the AKAP149-PP1 complex of the NE does not contain
any other established nuclear PP1-R subunits.
|
Disruption of RVXF-motif-mediated association of PP1 with R
subunits often perturbs the activity of the holoenzyme but not always results
in the release of the catalytic subunit because most R subunits have multiple
phosphatase interaction sites (Egloff et
al., 1997). We determined the ability of the
RVXF-motif-containing AKAP149 peptide
150SSPKGVLFSS159 (`RVXF') to physically
dissociate PP1 from NIPP1, PNUTS or Sds22 in vitro. Ten µM RVXF
peptide dissociated PP1 from an AKAP149-IP, whereas a mutated version of this
peptide (RAXF) (Steen et al.,
2000
) was ineffective (Fig.
2B). However, the RVXF peptide did not dislocate the bulk
of PP1 from NIPP1- or PNUTS-IPs, despite the existence of RVXF motifs
in these subunits (Ceulemans et al.,
2002a
) (Fig. 2B,
bottom panel). RVXF peptides did not dissociate PP1 from Sds22,
consistent with the lack of an RVXF sequence in this subunit
(Ceulemans et al., 2002b
). We
concluded that the AKAP149-derived RVXF peptide was capable of
displacing PP1 from AKAP149-IPs, but not from other known nuclear PP1-R
subunits.
AKAP149 functions as a B-type lamin-specifying subunit of PP1
To determine whether AKAP149 regulated PP1 activity, we immunoprecipitated
the AKAP149-PP1 complex from NEs (using anti-AKAP149 antibodies) as a source
of phosphatase, and took advantage of the resistance of PP1 to trypsin
(Beullens et al., 1998) to
digest associated AKAP149 (Steen et al.,
2000
). Phosphatase activity of the complex before and after
trypsin digestion was measured using 32P-labeled glycogen
phosphorylase a as a substrate.
Fig. 3A shows that the complex
was not capable of dephosphorylating phosphorylase a. However,
trypsin digestion enhanced the phosphorylase phosphatase activity of the
complex by 12-fold (P<0.001), and this activity was blocked by 1
µM inhibitor-2 (I-2), a specific inhibitor of PP1. Co-incubation of the
AKAP149-PP1 complex with the peptide KNSRVTFSED, which comprises the
PP1-binding RVXF motif of NIPP1 and competes with other
RVXF-containing regulators for binding to PP1
(Beullens et al., 1999
), also
increased the phosphorylase phosphatase activity several-fold
(P<0.001). On the other hand, an RAXF or RAXA
peptide was without effect (Fig.
3A). This demonstrates that AKAP149 acts as an inhibitor of PP1
when glycogen phosphorylase a is used as a substrate.
|
To provide a physiological NE substrate for AKAP149-bound PP1, phosphatase
activity towards immunoprecipitated B-type lamins was measured.
Immunoprecipitated B-type lamins were phosphorylated and
32P-labeled with PKC (Goss et
al., 1994). The AKAP149-PP1 complex dephosphorylated B-type lamins
and this activity was blocked by 1 µM I-2
(Fig. 3B; P<0.001).
However, trypsin digestion abolished the B-type lamin phosphatase activity of
the complex (P<0.001), suggesting that AKAP149 stimulates
dephosphorylation of B-type lamins by PP1
(Fig. 3B). Disruption of the
AKAP149-PP1 interaction with RVXF peptides also reduced B-type lamin
dephosphorylation, whereas non-disrupting RAXF peptides had no or
little effect (Fig. 3B). These
data indicate that AKAP149 functions as a B-type lamin-specifying subunit of
PP1.
B-type lamin assembly requires AKAP149-mediated targeting of active
PP1
Targeting of PP1 to nuclear membranes by AKAP149 upon nuclear reassembly in
vitro and in vivo correlates with assembly of B-type lamins into the NE
(Steen et al., 2000;
Steen and Collas, 2001
). We
extended these studies to show that AKAP149-induced PP1 activity towards
B-type lamins indeed promotes lamin assembly. Nuclear membrane formation, PP1
targeting to the NE, and lamin assembly were elicited in a nuclear
reconstitution assay (Fig. 4A, top panels) (Steen et al.,
2000
). Consistent with our earlier findings, B-type lamin assembly
was abolished with the RVXF-motif-containing AKAP149 peptide
(P<0.01; Chi-square test), but not with the RAXF peptide
(Fig. 4). Moreover, when 1
µM I-2 was added to the assay one hour after initiation of nuclear
reformation, that is, after nuclear membranes were assembled but before lamin
polymerization occurred (data not shown), B-type lamin assembly was inhibited
despite the localization of PP1 at the NE
(Fig. 4, I-2;
P<0.01). In contrast, 100 nM okadaic acid, a PP2A inhibitor at
this concentration, did not affect lamin assembly. Thus, sole targeting of PP1
to the NE is not sufficient for B-type lamin assembly into the NE, and PP1
activity is also required. This is consistent with our earlier finding that
AKAP149 acts as a B-type lamin-specifier of PP1.
|
RVXF-motif-containing AKAP149 peptides abolish DNA
synthesis
To address the significance of the association of PP1 with AKAP149 in G1
phase, we examined the effect of disrupting the AKAP149-PP1 interaction on
progression into the cell cycle. AKAP149-derived RVXF peptides (100
nM), which dissociate PP1 from AKAP149 (see
Fig. 2B), or 100 nM control
RAXF peptides were injected into nuclei of HeLa cells in early G1,
that is, within two hours of release from a nocodazole-induced mitotic arrest.
Injections were confirmed by nuclear retention of a 150 kDa FITC-dextran
(Fig. 5A). After eight hours of
culture, cells injected with RVXF retained a round phenotype and
remained in doublets resembling the cells in G1 at the time of injection
(Fig. 5A). In contrast, mock-
or RAXF-injected cells were flattened and noticeably larger,
indicative of progression into the cell cycle. Moreover, culture of
RVXF-injected cells with 10 µM BrdU for eight hours indicated that
RVXF abolished DNA synthesis in >90% of the cells
(Fig. 5A). However, 80-90%
mock- or RAXF-injected cells underwent DNA synthesis, which was
inhibited by 50 µM aphidicolin. Notably, RVXF injection into
S-phase nuclei (12 hours after release from mitotic arrest) did not inhibit
DNA replication (Fig. 5A).
These data suggest that RVXF peptides injected into G1 nuclei inhibit
cell cycle progression.
|
The effect of RVXF peptides on initiation of replication in
G1-phase nuclei was examined in vitro. RVXF or control RAXF
peptides (100 µM) were incubated for one hour with nuclei purified from
G1-phase HeLa cells to allow their uptake into the nuclei. Nuclear uptake of
fluorescent RAXF peptides was verified by fluorescence microscopy
(data not shown). Mock (buffer)- or peptide-loaded G1 nuclei were incubated
for three hours at 37°C in S-phase HeLa cell extract containing
[32P]-dCTP, dNTPs, GTP and an ATP-regenerating system.
Synthesized DNA was examined by electrophoresis and autoradiography. G1-phase
nuclei underwent replication in the S-phase extract
(Fig. 5B, G1
S). Moreover,
as initiation of replication requires the cyclin A/Cdk2 complex
(Stoeber et al., 1998
), we
showed that DNA synthesis was inhibited with 10 µM of the Cdk2 inhibitor
olomoucine (Stoeber et al.,
1998
) (Fig. 5C).
Thus, DNA synthesis in G1-phase nuclei in our assay was due to initiation of
replication. Peptide buffer or RAXF peptides did not affect
replication (Fig. 5B). In
contrast, RVXF abolished replication nearly completely
(Fig. 5B). We ruled out the
hypothesis that replication observed in G1 nuclei represented an elongation
phase in already replicating nuclei, as G1 nuclei incubated in a extract from
G0-phase HeLa cells did not replicate (Fig.
5B, G1
G0). Moreover, when introduced into S-phase nuclei,
none of the peptides impaired DNA replication, indicating that the elongation
phase of DNA replication was not affected by the RVXF peptide
(Fig. 5D). These results
suggest that the dissociation of PP1 from AKAP149 mediated by RVXF
peptide in G1 phase affects initiation of replication in vitro.
AKAP149 peptides containing RVXF motif elicit intranuclear lamin
phosphorylation and solubilization in G1 phase
To identify possible reasons for RVXF peptide-mediated inhibition
of DNA replication, immunofluorescence analysis of the NE in G1-phase cells
within 30 minutes of intranuclear peptide injection was performed. In order
not to interfere with immunofluorescence, peptide injection was monitored with
phenol red (Fig. 6A). At the
time of injection, A- and B-type lamins, lamin B receptor (LBR) and
anti-mAb414-reactive nucleoporins (Nups) showed expected perinuclear
distributions (Fig. 6A, Input
G1 cells). Absence of soluble (phosphorylated) A- and B-type lamins at the
time of peptide injection was demonstrated by the lack of 32P
incorporation into the lamins when cells were metabolically labeled between
0.5 and 2 hours after release from mitotic arrest
(Fig. 6B, G1). This argued that
the lamins were already incorporated into the lamina at the time of peptide
injection. Note that cells labeled for 1.5 hours during mitotic block, as a
control, contained phosphorylated lamins
(Fig. 6B, M).
Immunofluorescence labeling patterns of LBR or anti-mAb414-reactive
nucleoporins were not altered by buffer (mock), RVXF or control
RAXF peptide injection (Fig.
6A), suggesting that the peptides did not induce gross changes in
NE structure. In contrast, lamin distribution was drastically affected by
RVXF peptides (Fig.
6A). In >90% of RVXF-peptide-injected cells, both A-
and B-type lamins were detected throughout the nucleoplasm
(Fig. 6A). This contrasted with
RAXF- or mock-injected cells, which displayed perinuclear lamin
staining (Fig. 6A). Lamina
disruption by RVXF peptides appeared to be specific for G1 because
the peptides did not alter lamina organization after injection into S-phase
nuclei (Fig. 6A).
|
Disruption of the nuclear lamina by RVXF peptides was also shown
by western blot analysis. However, in this case, early G1 cells were
transfected, rather than injected, with RVXF or RAXF
peptides using the lipophilic reagent, DOTAP
(Steen et al., 2000), starting
0.5 hour after release from mitotic arrest. After 1.5 hours of incubation with
the DOTAP-peptide mix, nuclei were purified by careful Dounce homogenization
(nuclei of RVXF-injected cells were fragile) and dissolved in SDS
sample buffer or processed for isolation of NEs. Immunoblotting analyses
indicate that nuclei contained AKAP149 and PP1 regardless of the peptide
transfected (Fig. 7A). However,
NEs were depleted of PP1 and both A- and B-type lamins
(Fig. 7A). Immunofluorescence
analysis of nuclei purified from cells transfected with RVXF
peptides, but not RAXF peptides, indeed revealed strong intranuclear
lamin labelling, whereas anti-LBR antibodies decorated the NE
(Fig. 7B).
|
To determine whether nuclei containing RVXF peptides harbored
soluble (phosphorylated) lamins, peptides were transfected as described above
into 2x107 early G1-phase cells that were simultaneously
metabolically labeled with 32P
(Courvalin et al., 1992).
Western blotting and autoradiography analysis of lamins immunoprecipitated
from nuclear lysates 1.5-2 hours after start of transfection showed that A-
and B-type lamins co-precipitated after RAXF peptide transfection
(Fig. 7C, Blot) and essentially
no lamin phosphorylation was detected, as expected from cells in G1
(Fig. 7C, 32P). In
contrast, RVXF peptides induced phosphorylation of A- and B-type
lamins which, in addition, did not co-immunoprecipitate
(Fig. 7C). Similar results were
obtained later in G1, when cells were 32P-labeled and transfected
with peptides between 5 and 6.5 hours after release from mitotic arrest (data
not shown). We concluded that RVXF peptides elicited lamin phosphorylation and
released polymerized lamins, rather than prevented assembly of soluble lamins,
when introduced into G1-phase nuclei. Nevertheless, the INM remains apparently
intact and nuclear pore complex distribution unaffected.
The duration of RVXF-induced G1 arrest and whether the cells eventually re-entered the cell cycle were determined by prolonged (17 hour) culture with BrdU following RVXF peptide injection. Rather than replicating, the DNA condensed into apoptotic-like structures (Fig. 8A; P<0.01 compared to the RAXF control; Chi-square test). Apoptosis of all arrested cells was evidenced by TUNEL analysis (Fig. 8). Chromatin condensation and apoptosis were not observed after peptide injection into S-phase nuclei (Fig. 8).
|
Collectively, our results indicate that RVXF peptides, first, trigger dissociation of PP1 from AKAP149 within G1 nuclei, second, induce phosphorylation of A- and B-type lamins, third, cause disassembly of the lamins into the nuclear interior, and fourth, inhibit DNA synthesis in vitro and in vivo. None of these phenotypes were detected upon RVXF peptide introduction into S-phase nuclei. This suggests that dissociation of PP1 from NE-bound AKAP149 in G1 phase drastically destabilizes the nuclear lamina and as a result can prevent DNA replication.
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Discussion |
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Other well-studied regulators of PP1 display similar properties. G-subunits anchor PP1 to glycogen particles and
increase phosphatase activity towards glycogen synthase, but also inhibit
dephosphorylation of glycogen phosphorylase
(Armstrong et al., 1998;
Liu and Brautigan, 2000
).
Similarly, MYPT-proteins target PP1 to myosin and enhance the
myosin-phosphatase activity of PP1 while decreasing PP1 activity towards other
substrates (Toth et al., 2000
;
Johnson et al., 1996
). The
distinct effects of these R subunits have been explained by their association
with different sets of binding sites on PP1
(Bollen, 2001
;
Cohen, 2002
). NE-bound AKAP149
might stimulate B-type lamin dephosphorylation by inducing conformational
changes in PP1 to allow a better binding of the lamins. Indeed, NE-associated
AKAP149 itself could also bind B-type lamins (it co-immunoprecipitates with
lamins in interphase) (Steen and Collas,
2001
) and target lamin phosphoserine(s)
(Ottaviano and Gerace, 1985
)
to the catalytic site of PP1. As specifically NE-associated AKAP149-IPs were
used in this study, it would be interesting to determine the substrates for
the AKAP149-PP1 holoenzyme co-fractionating with the endoplasmic reticulum
(R.L.S., M.B., H.B.L. et al., unpublished) and the nature of the regulation of
PP1 by AKAP149.
Regulation of the AKAP149-PP1 interaction at the NE
Regulation of PP1 holoenzymes involves modulation of subunit interactions,
which are often mediated by phosphorylation of the R subunits. In three
unrelated R subunits phosphorylation of serine residues near or within the
RVXF motif disrupts binding to PP1
(Beullens et al., 1999;
Liu and Brautigan, 2000
;
McAvoy et al., 1999
), altering
the activity of the holoenzyme or resulting in a release of PP1. Serine
phosphorylation of AKAP149 correlates with the release of PP1 from the
NE-bound AKAP. Our previous (Steen and
Collas, 2001
) and current data suggest that the AKAP149-PP1
interaction at the NE is controlled in a cell-cycle- and
phosphorylation-dependent manner. The role of AKAP149 phosphorylation on the
regulation of its association with PP1 is currently being examined.
At the G1/S phase transition, phosphorylation of NE-associated AKAP149
correlates with the release of PP1 from the AKAP149 complex and downregulates
PP1 activity towards assembled lamins. In spite of the down regulation of PP1,
B-type lamins remain hypophosphorylated, probably because B-type lamin kinases
are also downregulated. Furthermore, as S phase progresses, AKAP149 is
dephosphorylated by an unidentified protein Ser/Thr phosphatase and remains
dephosphorylated during the rest of interphase (R.L.S., M.B., H.B.L. et al.,
unpublished). Immunoprecipitation experiments indicate that dephosphorylation
of AKAP149 during early S phase is not associated with the reformation of an
AKAP149-PP1 complex at the NE, and the AKAP149-PP1 complex at the NE is not
reformed until the end of mitosis (R.L.S., M.B., H.B.L. et al., unpublished).
This suggests that the AKAP149-PP1 interaction at the NE is not solely
controlled by phosphorylation of AKAP149: binding of PP1 could also be
regulated by the occupation of PP1 binding sites by another protein, or by
allosteric regulators. In this respect, it should be noted that most R
subunits form multiple points of interaction with PP1; nevertheless,
disruption of a single interaction site can be sufficient to weaken or destroy
interaction with PP1 (Bollen,
2001).
AKAP149-mediated PP1 phosphatase activity towards B-type lamins in
G1
B-type lamins are likely to be substrates for dephosphorylation by the
AKAP149-PP1 holoenzyme, and this is expected to promote lamina polymerization
and completion of NE assembly (Thompson et
al., 1997; Steen and Collas,
2001
). Dephosphorylation of disassembled B-type lamins promotes
their polymerization into the reforming lamina during G1
(Gerace and Blobel, 1980
). In
contrast, B-type lamins that are synthesized de novo in S phase are not
phosphorylated and therefore do not require a dephosphorylation step for
polymerization. Consequently, the B-type lamin phosphatase activity of PP1
mediated by NE-associated AKAP149 appears to be dispensable beyond the G1/S
phase transition. This could account for the lack of effect of RVXF
peptides on NE organization in S-phase nuclei.
Our results argue that in G1, continuous dephosphorylation of B-type lamins
at the NE needs to take place to maintain lamins in a polymerized form.
Exogenous RVXF peptides have a dramatic effect on lamina structure:
they elicit phosphorylation of A- and B-type lamins and their disassembly into
the nuclear interior, as shown by 32P incorporation, immunolabeling
and lack of co-immunoprecipitation of A- and B-type lamins. Intranuclear, as
opposed to cytoplasmic, solubilization of the lamins may be explained by the
maintenance of intact nuclear membranes and of nuclear pore distribution,
based on immunostaining of LBR and nucleoporins. The detrimental effects of
RVXF peptides in G1 nuclei on lamina organization appear to be
irreversible. Apoptosis takes place several hours after the G1 arrest
phenotype is first observed. This contrasts with the rapid apoptosis occurring
after nuclear membrane formation at mitosis exit, when B-type lamin assembly
is prevented by the RVXF peptide
(Steen and Collas, 2001).
Interestingly, attempts to rescue the cells from G1 arrest simply by elevating
intranuclear PP1 concentration by injection of purified PP1 into nuclei of
arrested cells failed (R.L.S., M.B., H.B.L. et al., unpublished), suggesting
that the amount of intranuclear PP1 activity per se is not a factor hindering
cell cycle progression beyond G1.
Rather, we propose that the anchoring of PP1 in defined subnuclear loci is important for proper modulation of PP1 phosphatase activity towards specific substrates in G1-phase nuclei. Once the balance of PP1 activities is perturbed (by RVXF peptides) the cell does not enter S phase and commits to apoptosis. Thus, premature dissociation of PP1 from NE-associated AKAP149 in G1, and/or disruption of the intranuclear balance of PP1 activities with RVXF peptides are detrimental. It should be mentioned that although RVXF peptides do not disrupt the structure of known nuclear PP1 holoenzymes other than the AKAP149-PP1 complex (Fig. 2), mislocalization of AKAP149-bound PP1 and/or alteration of activity of other nuclear PP1-R holoenzymes by RVXF peptides may also account for the phenotypes reported.
We cannot at present exclude the possibility that AKAP149-PP1 is also
involved in dephosphorylation of key regulatory proteins in addition to
lamins, in particular in the late M and early G1 phases, when a burst of
protein dephosphorylation occurs (Bollen
and Beullens, 2002). These could in principle include
lamina-associated proteins of the INM and chromatin. There is considerable
evidence for essential functions of PP1 at these stages of the cell cycle in
many organisms, although the involved R subunits remain to be identified
(Tournebize et al., 1997
;
Hsu et al., 2000
;
Sugiyama et al., 2002
). For
example in G1, PP1 prevents premature entry into S phase by keeping pRb
hypophosphorylated (Rubin et al.,
2001
). AKAP149-PP1 might contribute to modulating pRb
phosphorylation and it remains possible that exogenous RVXF peptides
affect pRb phosphorylation in G1. AKAP149-PP1 might also be involved in the
attenuation of PKA-mediated CREB signaling, which results from CREB
dephosphorylation by Ser/Thr phosphatases including PP1
(Mayr and Montminy, 2001
).
These alternatives remain to be examined.
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Acknowledgments |
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