(Received for publication, August 19, 1994; and in revised form, October 17, 1994)
From the
During each cell cycle, the nucleus of higher eukaryotes undergoes a dramatic assembly and disassembly. These events can be faithfully reproduced in vitro using cell-free extracts derived from Xenopus eggs. Such extracts contain three major N-acetylglucosaminylated proteins, p200, p97, and p60. All three become assembled into reconstituted nuclear pores. Here we show that p200, p97, and p60 exist in eggs in soluble high molecular mass complexes of 1000, 450, and 600 kDA, respectively. The bulk of p60 is stably associated with proteins of 58 and 54 kDa, while p200 is associated with a fraction of p60 in a separate complex lacking p58 and p54. Upon examining the behavior of these proteins in the cell cycle, we find that p200 and p97 are highly phosphorylated at mitosis, both in vivo and in vitro. Moreover, in extracts that cycle between interphase and mitosis, p200 and p97 are specifically phosphorylated at mitosis. Corresponding with their mitotic phosphorylation, both p200 and p97 are specific substrates for purified mitotic Cdc2 kinase, whereas nucleoporin p60 is not. Analysis indicates that the size of the complexes containing the pore N-acetylglucosamine glycoproteins does not change during mitosis, suggesting that such complexes represent stable multicomponent modules into which the nucleus disassembles at mitosis.
The mitotic reorganization of the eukaryotic cell architecture ensures the correct partitioning of the cellular contents to each of the daughter cells. These mitotic changes are seen most vividly in the dramatic alterations of the cell nucleus where the chromosomes condense, the mitotic spindle assembles, and the nuclear envelope breaks down(1, 2, 3) . There is currently a great deal of evidence to indicate that movement of the cell cycle into mitosis is regulated by the Cdc2 protein kinase(4) . The phosphorylation of key cellular proteins by Cdc2 kinase, or activated downstream kinases, is known to correlate with many of the major mitotic rearrangements. Determining the targets of the Cdc2 kinase is, therefore, a means to identify proteins that initiate cell cycle rearrangements(5) . For example, one of the non-histone chromosomal scaffold proteins, topoisomerase II, is required for chromosome condensation and shows increased activity during mitosis(6, 7, 8, 9) . The increase in topoisomerase II activity is associated with an increase in its phosphorylation state as a result of either protein kinase C (10) or casein kinase II(11, 12) , the latter of which is a substrate for the Cdc2 kinase. Similarly, a number of proteins that bind to and modulate microtubule dynamics have been isolated, and some of these proteins (MAP4, p220, and p47) are phosphorylated in a cell cycle-dependent fashion(13, 15, 16, 17, 18) .
Another key mitotic reorganization event seen in higher eukaryotes is the disassembly of the nuclear envelope. Specifically, the nuclear lamina network depolymerizes, the double nuclear membranes vesiculate, and the pore complexes disassemble(19, 20, 21, 22) . Of all of the mitotic rearrangements that occur, the disassembly of the nuclear lamina is the best understood. The lamina is composed of polymerized intermediate filament-like proteins that are arranged between the inner nuclear membrane and chromatin. The lamin proteins form homo- and heteropolymers through strong coiled/coil interactions both in vivo and in vitro(23, 24) . Despite these strong interactions the lamina is a very dynamic network that both completely disassembles during mitosis and incorporates new lamin proteins during nuclear growth in interphase(20, 25) . Lamins have been shown to be phosphorylated during interphase and hyperphosphorylated during mitosis (21) . Similarly, lamin-associated proteins of the inner nuclear membrane are also phosphorylated during interphase and mitosis (26, 27, 28, 29) . Phosphorylation may regulate the dynamics of the lamin network in three ways: 1) by affecting the association between individual lamin monomers(30) , 2) by regulating the nuclear import of lamin monomers(31) , and 3) by affecting the association between lamins and their putative inner nuclear membrane receptors(26, 29, 32) . Purified Cdc2 kinase can phosphorylate and dissociate lamin polymers formed in vitro(33) . In addition, either purified Cdc2 kinase or protein kinase C will phosphorylate and disassemble the lamin network of detergent-treated nuclei(34, 35) , lending support to the hypothesis that mitotic phosphorylation induces disassembly of the nuclear lamina.
The molecular mechanism of disassembly of the other nuclear envelope structures, such as the nuclear membrane and nuclear pore complexes, is much less well understood. A variety of in vitro assays have demonstrated that mitotic phosphorylation negatively regulates the binding of membrane vesicles to chromatin(29, 36, 37) . It appears that once the nuclear membrane has vesiculated, phosphorylation of membrane components by kinases downstream from Cdc2 prevents the reassociation of vesicles with chromatin until interphase resumes. However, almost nothing is known about the actual mechanism of membrane vesicularization or, indeed, nuclear pore disassembly.
In previous studies we used an in vitro nuclear reconstitution system to identify and characterize a family of nuclear pore glycoproteins required for nuclear transport(38, 39) . In this reconstitution system, nuclei assemble when sperm chromatin is mixed with the cytosolic and membrane fractions of a Xenopus egg lysate(40, 41) . Reconstituted nuclei are fully functional in that they accumulate nuclear proteins(42, 43) , replicate their DNA(44, 45) , and respond to mitotic signals by condensation of the chromatin and disassembly of the nuclear envelope(46, 47, 48, 49) . This system has proved to be uniquely useful for biochemically dissecting the various steps in both the assembly and mitotic disassembly of the nucleus(36, 40, 41, 45, 50, 51) .
A major open question is the manner in which the nuclear pore
complex assembles and disassembles with each cell cycle. In this study
we have used interphase and mitotic egg extracts in a first step to
investigate whether known nuclear pore proteins are phosphorylated in a
cell cycle-dependent manner. The specific proteins examined are the
nuclear pore glycoproteins, which are modified with O-linked
GlcNAc residues. Such proteins can be identified or isolated by their
binding to the lectin, wheat germ agglutinin (WGA). ()Moreover, WGA-coated ferritin binds to both the
cytoplasmic and nuclear faces of the nuclear pore, as does a series of
anti-pore monoclonal antibodies that react with one or more members of
this glycoprotein
family(38, 52, 53, 54, 55, 56, 57) . (
)Previously we had shown that this family of glycoproteins
could be depleted from nuclear reconstitution extracts derived from Xenopus egg lysates using WGA-Sepharose(39) . Nuclei
assembled in such depleted extracts are defective in nuclear
transport(39, 58, 59) . In this study we find
that two prominent members of the glycoprotein family, p200 and p97,
are specifically phosphorylated during mitosis. They are phosphorylated
both in vivo when eggs are injected with
[
P]ATP, and in vitro when mitotic
egg lysates are labeled with [
P]ATP.
Moreover, we find that p200 and p97 are specific targets for
immunopurified mitotic Cdc2 kinase, while the pore protein p60 is not.
The distinct complexes that contain each of these glycoproteins will be
discussed.
Alternatively, specific members
of the labeled WGA-binding proteins were immunoprecipitated by stopping
the reaction with the addition of 5 µl of 20% SDS, 1 ml of HENP (5
mM Hepes, pH 7.4, 150 mM NaCl, 50 mM NaF, 40
mM -glycerophosphate, 10 mM pyrophosphate, 5
mM EDTA, 0.5% Triton X-100), 20 µl of protein A-Sepharose
(Sigma P9424), and 20 µl of either anti-p200, anti-p97, or anti-rat
p62 crude antiserum (64) for 1 h at 4 °C. The Sepharose was
washed in HENP (10
1 ml), TN (4
1 ml), and the bound
proteins eluted with SDS-gel loading buffer.
The relative
phosphorylation state of the WGA-binding proteins in a cycling extract
was monitored every 10 min by pulse labeling an aliquot of the extract
with [P]ATP for a 10-min period.
Specifically, a cycling extract was prepared and divided into 42-µl
aliquots that were all placed at 22 °C at time 0 to initiate
cycling, which occurs due to the translation of cyclin from the
endogenous mRNA pool(62, 65) . Every 10 min, 2 µl
of an aliquot was removed and frozen in liquid nitrogen for subsequent
H1 kinase assays. The remaining 40 µl was immediately labeled with
[
P]ATP to a final concentration of 4
mCi/ml. After 10 min, the labeling of the aliquot was stopped, and the
WGA-binding proteins were isolated as described above. To monitor the
levels of Cdc2 kinase in the cycling extract, histone H1 kinase assays
were performed using the frozen aliquots as described by Smythe and
Newport (61) .
Subsequent gel filtration was performed by fast protein liquid
chromatography with a Superose 6 column (Pharmacia Biotech Inc.).
WGA-binding proteins were purified from 2-4 ml of either
interphase cytosol or mitotic cytosol made by the conversion of
interphase cytosol with GST-cyclin. These proteins were applied to the
column equilibrated with phosphate-buffered saline, 0.05% Triton X-100.
Fractions (0.6 ml) were collected at a flow rate of 0.4 ml/min,
trichloroacetic acid-precipitated, and resolved on gels as above. The
proteins were transferred to PVDF and probed with I-WGA.
Because iodinated WGA often did not label the minor WGA-binding
proteins very well, we routinely stained our protein blots with India
ink first, as it labels these proteins more sensitively than does WGA.
To compare the total amount of the WGA-binding proteins present in mitotic (unactivated) and interphase (activated) eggs, a group (100) of each type of eggs was collected and homogenized as outlined above. The crude lysate was mixed with 30 µl of WGA-Sepharose in 1 ml of PIB containing 0.1% SDS, 0.1% Triton X-100, and 0.1% sodium deoxycholate for 6 h at 4 °C. The Sepharose was washed, and the bound proteins were eluted with SDS gel loading buffer. Equal amounts of eluate from the mitotic and activated eggs were electrophoresed on an 8% polyacrylamide gel, blotted to PVDF, probed with horseradish peroxidase-conjugated WGA, and visualized using chemiluminescence.
Purified Cdc2 kinase was prepared by immunoprecipitation
with the highly specific anti-Cdc2 C-terminal peptide antibody
D65(66) . This antibody specifically binds to p34 Cdc2 kinase
and not the related p32 Cdk2 kinase(66) . Protein-A Sepharose
(20 µl) was mixed with either D65 (10 µg) or nonspecific rabbit
IgG (10 µg) in 1 ml of kinase wash buffer (KWB: 10 mM Hepes, pH 7.4, 80 mM -glycerophosphate, 250 mM NaCl, 5 mM NaF, 5 mM EGTA, 0.1% Triton X-100).
Interphase cytosol (25 µl) or cytosol that had been preincubated
(50 min) with GST-cyclin to induce high levels of Cdc2 kinase activity
was added, and the samples were mixed at 4 °C for 1.5 h. The
Sepharose beads were extensively washed with KWB (5
1 ml) and
then with the kinase reaction buffer (10 mM Hepes, pH 7.4, 5
mM EGTA, 10 mM MgCl
, 40 mM
-glycerophosphate; 4
0.5 ml).
The ability of the
immunocomplexes to phosphorylate the WGA-binding proteins was tested by
mixing the Cdc2-antibody-Sepharose beads with 20 µl of the eluted
WGA-binding proteins and [P]ATP (0.3
mCi/µmol ATP and ATP at 0.4 mM). To determine which
phosphorylated proteins originated from the immunoprecipitated Cdc2
complexes themselves, a parallel set of Cdc2-antibody-Sepharose beads
were mixed with 20 µl of HSB
buffer alone containing
[
P]ATP (0.3 mCi/µmol ATP and ATP at 0.4
mM). All reactions were incubated for 10 min at 22 °C and
stopped by the addition of SDS-gel loading buffer. The samples were
electrophoresed on an 8% polyacrylamide gel and processed for
autoradiography.
In order to examine the relative Cdc2/H1 kinase
levels present on the Cdc2-antibody-Sepharose beads, histone H1 (0.4
mg/ml) was added in place of the WGA-binding proteins in the same
reaction buffer (HSB, 0.4 mM ATP,
[
P]ATP at 0.3 mCi/µmol ATP).
Interphase extracts of Xenopus eggs can be
made mitotic by the addition of the purified recombinant fusion
protein, GST-cyclin-13(63) . Conversion to mitosis is
indicated by the induction of H1 kinase activity or can be visualized
microscopically by the nuclear envelope breakdown and chomatin
condensation of added test nuclei. To examine the nuclear pore
glycoproteins described above for evidence of mitotic phosphorylation,
the relative phosphorylation states of the WGA-binding proteins from
interphase and mitotic lysates was analyzed. Cytosol with and without
added GST-cyclin was labeled with [
P]ATP
for 10 min. The WGA-binding proteins were affinity purified using
WGA-Sepharose, eluted with SDS, and electrophoresed on a polyacrylamide
gel. An autoradiogram of this gel (Fig. 1) demonstrates that the
three major WGA-binding proteins (p200, p97, and p60) are highly
phosphorylated in a mitotic extract (lane2), and
only lightly so in an interphase extract (lane1). We
repeatedly found that p97 and p200 were phosphorylated to similar
extents and that p60 was much less phosphorylated. In order to be
certain of the identity of these phosphoproteins, they were
immunoprecipitated from radiolabeled mitotic extracts using antisera
specific for Xenopus p200 (lane3), p97 (lane4), or rat p62 (lane5). The
latter antisera cross-reacts with the Xenopus p60
homolog(64) . All three major GlcNAc-containing phosphoproteins
were immunoprecipitated by their specific antisera. The total amount of
the WGA-binding proteins present in both the interphase and mitotic
extracts was found to be identical as determined by Western blotting
(not shown), indicating that the increase in phosphorylated protein
observed at mitosis was due to increased phosphorylation, not increased
protein synthesis.
Figure 1:
The WGA-binding
proteins are phosphorylated in mitotic egg extracts. Interphase cytosol
was preincubated with (+) or without(-) GST-cyclin for 50
min at 22 °C. The individual reactions were then pulse-labeled with
[P]ATP for 10 min. The labeled cytosol was
mixed with WGA-Sepharose (lanes1 and 2) or
with protein-A Sepharose containing antisera against p200 (lane3), p97 (lane4), or p60 (lane5). The Sepharose beads were thoroughly washed, and the
bound proteins were eluted with SDS, electrophoresed on an 8%
polyacrylamide gel, and processed for
autoradiography.
Whereas the above results indicate that the turnover of phosphate in the mitotic cytosol is much higher than in interphase cytosol, we wished to know if the overall phosphorylation state of the proteins was increased. Posttranslational modifications such as phosphorylation often lead to changes in the mobility of a protein on an SDS-polyacrylamide gel. To examine this possibility, we compared p200 and p97 isolated from a) interphase extracts, b) mitotic extracts made by adding GST-cyclin to an interphase extract, and c) mitotic extracts made using an egg lysis buffer that preserves the naturally high levels of Cdc2 kinase present in mitotically arrested eggs(6) . The mobilities were compared both before and after treatment with potato acid phosphatase (Fig. 2). Both p200 and p97 proved to migrate more slowly when isolated from mitotic extracts (lanes2 and 4) than from interphase extracts (lane1). The mobility of p200 and p97 isolated from mitotic extracts could be increased by treatment with potato acid phosphatase (lanes3 and 5), indicating that the change in mobility at mitosis is due to phosphorylation. Note that the mobilities of both p200 and p97 are quantitatively shifted at mitosis, demonstrating that all of the p200 and p97 in the extract becomes mitotically phosphorylated. The mobility of the interphase form of p200 (lane1) appears intermediate between that of the mitotic and phosphatase-treated samples. It is possible that the interphase form is quantitatively phosphorylated on a small number of sites, which is nevertheless below the number of sites phosphorylated at mitosis. In this immunoblot assay, the mobility of p60 did not change at mitosis.
Figure 2: The 200- and 97-kDa proteins are quantitatively phosphorylated during mitosis. The WGA-binding proteins from aliquots (100 µl) of interphase cytosol (lane1), interphase cytosol supplemented with GST-cyclin (lanes2 and 3), or mitotic cytosol (lanes4 and 5) were isolated by binding to WGA-Sepharose (20 µl). The WGA-Sepharose and bound proteins were thoroughly washed and treated with potato acid phosphatase (PAP, lanes3 and 5) or PAP buffer (lanes1, 2, and 4) at 30 °C for 60 min. After washing, the WGA-binding proteins were solubilized in SDS, electrophoresed on a 25-cm long, 7.5% polyacrylamide gel, and transferred to PVDF membrane. The proteins were detected using horseradish peroxidase-conjugated WGA and chemiluminescence.
Having
observed an alteration in the phosphorylation state of the nucleoporins
p200 and p97, we wished to know whether any other biochemical
properties of these proteins were altered at mitosis. The rat
WGA-binding nucleoporin, p62, can be extracted from pores as a
multimeric complex with at least two other nucleoporins, p54 and p58 (64, 68, 69) . To determine if the soluble
WGA-binding proteins from Xenopus egg lysates also exist in
large multimeric complexes and, if so, whether these associations
change during mitosis, we measured their apparent size by gel
filtration. The WGA-binding proteins were affinity purified from egg
lysates using WGA-Sepharose and eluted with the competing sugar. This
mixture was fractionated on a Sephacryl S300 column; the eluted
proteins were precipitated with trichloroacetic acid, electrophoresed
on a polyacrylamide gel, transferred to PVDF membrane, and probed with I-WGA. By comparing the elution position of the
WGA-binding proteins to those of a set of known protein standards, we
could estimate the size of these soluble glycoproteins. All three major
WGA-binding proteins were seen to exist in distinct high molecular
weight complexes (Fig. 3). The peak of p60 elution was in
fraction 16, which corresponds to an apparent size of approximately 600
kDa. Like the rat p62 homolog, the Xenopus p60
co-chomatographed with WGA-binding proteins of 54 and 58 kDa, although
p54 is poorly recognized by
I-WGA. Note that there is a
protein of 52 kDa whose peak of elution is in fractions 19 and 20. On
this exposure it is difficult to resolve the 52 and 54 kDa bands, but
they are clearly distinguished on lighter exposures.
Figure 3:
The soluble WGA-binding proteins exist as
large molecular weight complexes. The WGA-binding proteins from 4 ml of
egg cytosol were applied to a calibrated HR S-300 Sephacryl column.
Fractions (1 ml), were collected, trichloroacetic acid-precipitated,
electrophoresed on a polyacrylamide gel, transferred to PVDF membrane,
and probed with I-WGA. Only the column fractions that
contain the WGA-binding proteins (8-25) are shown. The
position of p200, p97, and the p60 complex (p60, p58, and p54), are
indicated to the left of the gel, as are the positions of the
minor WGA-binding proteins K (140 kDa), L (125 kDa), M (110 kDa), and D (82 kDa), and a dot indicates the position of a 52-kDa protein. The p200 protein
elution peak occurs in fractions 11 and 12, the p97 protein elution
peak occurs in fractions 19 and 20, while the p60 complex elution peak
occurs in fraction 16.
The elution of p200 peaked in fractions 11 and 12 giving it an estimated molecular mass of 1000 kDa, while the elution of p97 peaked in fractions 19 and 20 giving it an estimated molecular mass of 450 kDa. We noted that a certain amount of the p60 nucleoporin co-chomatographed with the p200 complex. We have consistently seen an association between a small amount of p60 and p200 by fractionation on gel filtration columns, ion exchange columns, and by co-immunoprecipitation with specific antibodies that do not cross-react between the two proteins (not shown). Interestingly, the p54 and p58 proteins that co-elute with the bulk of p60 are not present in the complex containing p60 and p200. Likewise, the p200 protein is seen to co-chomatograph with a number of minor WGA-binding proteins labeled K (140 kDa), L (125 kDa), and M (110 kDa). These proteins do not cofractionate with p200 when examined by either ion exchange chomatography or by immunoprecipitation, so they are not considered to be part of the p200 high molecular weight complex. These same results were obtained when gel filtration was performed using a Superose 6 column.
Given that both p200 and p97 are highly phosphorylated at mitosis, we wished to know if their protein/protein interactions would change during mitosis as indicated by a change in the size of the complexes eluting from the gel filtration column. When the WGA-binding proteins from interphase and mitotic lysates were fractionated on a gel filtration column, there was no apparent difference in the positions at which the proteins eluted, indicating that the size of these complexes did not change significantly during mitosis (Fig. 4). We conclude that the nucleoporins p200, p97, and p60 are contained in complexes with apparent molecular masses of 1000, 450, and 600 kDa, respectively. Most importantly, the soluble interphase form of the complexes does not change upon conversion of cytosol to a mitotic state, indicating that these complexes likely represent the subunits into which the nuclear pore disassembles at mitosis.
Figure 4:
The p60, p97, and p200 complexes are
maintained in a mitotic extract. The WGA-binding proteins either from
interphase cytosol (A) or from cytosol that had been converted
to a mitotic state (B) were applied to a Superose 6 column.
Fractions were collected, subjected to SDS-polyacrylamide gel
electrophoresis, transferred to PVDF, and probed with I-WGA as in Fig. 3. The lanes marked S contain 1% of the material loaded onto the column. Only
fractions containing the WGA-binding proteins are
shown(12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) .
The bars denote the positions of p200, p97, and p60. The p97
peaks in fraction 22, and p60 peaks in fractions 20 and 21. Smearing in
the high molecular weight region of the WGA blots made it difficult to
discern where the peak of elution of the p200 complex was. An India ink
stain of the blots (not shown) clearly showed that the peak of elution
of p200 (as well as of K, L, and M) was in
fraction 16. Both the mitotic and interphase WGA-binding proteins elute
from the gel filtration column at the same
position.
When parallel cycling extracts
were pulse labeled for successive 10-min intervals with
[P]ATP and examined, the phosphorylation
state of the WGA-binding proteins clearly varied in a cell
cycle-dependent manner (Fig. 5). Phosphorylation of p200 and p97
in the cycling extract peaked at 60 min, corresponding in time to the
peak in H1 kinase activity (lane6). This was also
visualized to be the time of mitosis by examining parallel samples
containing test nuclei which at 60 min underwent chomosome condensation
and nuclear envelope breakdown (not shown). After mitosis, the level of
H1 kinase activity and the WGA-binding protein kinase activity abruptly
dropped (lane7) and then slowly rose during the
second interphase period (lane9). The p200 protein
is seen to be phosphorylated during the interphase state and is highly
phosphorylated during mitosis, whereas p97 phosphorylation is more
restricted to the peak of mitosis. In cycling extracts, much less
phosphorylated p60 was observed compared with that of p97 and p200.
Taken together, the results indicate that in vitro both the
p200 and p97 WGA-binding proteins become hyperphosphorylated at
mitosis, while relatively low levels of p60 phosphorylation are
observed.
Figure 5:
The p200 and p97 glycoproteins are
phosphorylated during mitosis in cycling egg extracts. Parallel
aliquots (42 µl) of a ``cycling'' extract were placed at
22 °C at time 0` to initiate the cycling of the extracts. Every 10
min a different aliquot was treated such that a 2-µl sample was
removed for H1 kinase assays, and the remaining sample (40 µl) was
pulse-labeled with [P]ATP for a 10-min
period. The WGA-binding proteins from each such sequentially labeled
aliquot (0`-10`, 10`-20`, 20`-30`, etc.) were
affinity purified with WGA-Sepharose, solubilized with SDS,
electrophoresed on an 8% polyacrylamide gel, and processed for
autoradiography. Time 0` gives the H1 kinase levels in the extract when
it was on ice. Mitosis is seen to occur at 60 min in a parallel aliquot
of extract containing test nuclei. This corresponds to the peak in H1
kinase activity and the peak in the phosphorylation of the WGA-binding
proteins.
Xenopus egg extracts seem to accurately represent
the in vivo conditions found in the intact egg. Nevertheless,
we wished to be sure that both p200 and p97 are in fact mitotically
phosphorylated in vivo. This is typically determined by
isolating the proteins from radiolabeled tissue culture cells that have
been synchronized and arrested in either mitosis or
interphase(72) . Although antibodies specific for p200 and p97 (Fig. 1) have been raised and used to characterize the location
of these proteins in reconstituted nuclei and tissue culture
cells, our attempts to synchronize the slow growing Xenopus A6 tissue culture cells failed. In addition, the
antibodies do not cross-react significantly with other species, so more
easily synchronizable cell lines could not be used. Therefore, intact
eggs and oocytes were used to look for cell cycle-dependent changes in
the phosphorylation state of the WGA-binding proteins in vivo. Xenopus oocytes are arrested in an interphase state, while
eggs are arrested at the second meiotic metaphase of the cell cycle.
Oocytes, preloaded with [
P]phosphate, can be
matured into eggs in vitro by incubation in
progesterone(73, 74) . When we compared the relative
phosphorylation state of the WGA-binding proteins present in comparable
numbers of preloaded oocytes and eggs, we found that p200 and p97 are
much more highly phosphorylated in the egg than in the oocyte (not
shown). Immunoblots indicate that there is also more p200 and p97
protein in the eggs than in the oocytes (not shown). This most likely
reflects the translation of maternal mRNA during maturation.
To
compare equivalent amounts of protein from mitotic and
interphase-arrested cells, we microinjected
[P]ATP into either activated (interphase)
or nonactivated (mitotic) eggs. Equal numbers of activated or
nonactivated eggs were injected with [
P]ATP
in an EGTA-containing buffer to prevent the unwanted activation of the
nonactivated egg group by the injection needle(75) . After a
10-min labeling period, the WGA-binding proteins were extracted from
each group of eggs and electrophoresed on a polyacrylamide gel (Fig. 6a). It is clear that WGA-binding proteins from
mitotic eggs (lane2) are more highly phosphorylated
than those from interphase eggs (lane1). Again as
with the cycling extracts, p200 was found to be slightly phosphorylated
in the interphase egg and highly phosphorylated in mitosis, while p97
only appears to be phosphorylated at mitosis. A protein migrating at 60
kDa was heavily phosphorylated in the experiment shown; however, the
level of phosphorylation for this protein varied substantially between
experiments. When the WGA-binding proteins extracted from nonactivated
and activated eggs were electrophoresed on a polyacrylamide gel,
blotted to PVDF, and probed with horseradish peroxidase-conjugated WGA,
equivalent amounts of protein were found in both types of eggs (Fig. 6b). Thus p200 and p97 are consistently highly
phosphorylated during mitosis both in vitro and in
vivo.
Figure 6:
The 200- and 97-kDa glycoproteins are
mitotically phosphorylated in vivo. PanelA,
a group (28) of activated (interphase, lane1) and nonactivated (mitotic, lane2)
eggs were microinjected with 50 nl of
[P]ATP (133 µCi/µl). After 10 min
at 22 °C, the eggs were homogenized and the glycoproteins were
affinity purified using WGA-Sepharose. The proteins were eluted using
SDS, electrophoresed on an 8% polyacrylamide gel, and processed for
autoradiography. The position of molecular mass markers are indicated
to the left of the panel, while the position of the
three major WGA-binding proteins are indicated between panelsA and B. PanelB, a group
(100) of activated (interphase, lane1) and
nonactivated (mitotic, lane2) eggs were homogenized,
and the glycoproteins were affinity purified using WGA-Sepharose. The
proteins were eluted with SDS, and equal amounts of eluate from the
activated and nonactivated eggs were electrophoresed on an 8%
polyacrylamide gel, blotted to PVDF, probed with horseradish
peroxidase-conjugated WGA, and visualized by chemiluminescence. The
proteins in A and B were electrophoresed at different
times.
Figure 7:
Cdc2
kinase specifically phosphorylates the p200 and p97 nuclear pore
proteins in vitro. PanelA, protein
A-Sepharose bound with nonspecific rabbit IgG (lanes1, 2, 5, and 6), or anti-Cdc2
specific antibodies (lanes3, 4, 7,
and 8) was used to immunoadsorb proteins from interphase egg
cytosol (lanes1-4), or egg cytosol that was
supplemented with GST-cyclin for 50 min to induce mitotic conversion
and high levels of Cdc2 kinase activity (lanes5-8). After extensive washing of the protein
A-Sepharose, WGA-binding proteins specifically eluted from
WGA-Sepharose with high sugar buffer (HSB) were added to
the reactions in lanes2, 4, 6, and 8. No WGA-binding proteins, only the HSB
buffer
alone, was added to the reactions in lanes1, 3, 5, and 7. All reactions were then labeled
with [
P]ATP for 10 min. The reactions were
stopped using SDS-gel loading buffer, electrophoresed on an 8%
polyacrylamide gel, and processed for autoradiography. Radiolabeled
p200 and p97 were observed only when mixed with anti-Cdc2 beads
isolated from a mitotically converted extract (lane8). Phosphorylated WGA-binding proteins isolated from
mitotic cytosol labeled with [
P]ATP in the
presence of 2 µM okadaic acid served as markers (laneM). PanelB, the H1 kinase activity was
assayed for each condition in panelA by adding
histone H1 in HSB
(lanes2, 4, 6, and 8) or HSB
buffer alone (lanes1, 3, 5, and 7) along with
[
P]ATP to a parallel set of protein-A beads
as used in panelA. After 10 min, the H1 kinase
reactions were solubilized in SDS, electrophoresed on a 10%
polyacrylamide gel, and processed for autoradiography. A small amount
of H1 kinase activity was seen when Cdc2 was immunoprecipitated from
interphase extracts, due to the residual cyclin present in interphase
cytosol (Fig. 8B, lane4). On much longer
exposures, some phosphorylation of p97 can be seen using this kinase
(not shown).
It was previously found that there are three major soluble N-acetylglucosamine-modified proteins, p200, p97, and p60,
present in Xenopus eggs and that each of these proteins
becomes associated with nuclei reconstituted in
vitro(38, 39) . Both p200 and p60 have been
localized to the nuclear pore complex by immunoelectron
microscopy(52, 79) , whereas p97 is a
component of the nuclear pore, as well as internal structures.
In the present study we have found that the major WGA-binding
proteins are phosphorylated in a cell cycle-specific manner. The p97
and p200 nucleoporins are highly phosphorylated under all mitotic
conditions examined both in vivo and in vitro. The
phosphorylation of the p60 nucleoporin was always less than that of
p200 and p97 in mitotic egg lysates, was only occasionally seen in
cycling egg extracts, and was inconsistently observed in microinjected
eggs. We do not know why p60 phosphorylation was so inconsistent, but
because of this we do not consider p60 to be stably mitotically
phosphorylated like p200 and p97. Upon further analysis, we find that
p200 and p97 are substrates for the mitotic Cdc2 kinase and are the
first nucleoporins demonstrated to be mitotically phosphorylated.
In higher eukaryotes the proteins of the nuclear pores are seen by immunofluorescence to be dispersed throughout the cytoplasm during mitosis(52, 80) . It is reasonable to expect that, like the other nuclear Cdc2 kinase substrates such as the lamins, mitotic phosphorylation plays a role in the mitotic disassembly of the pore complex(5) . We have attempted a number of assays to measure the effects of mitotic phosphorylation, most relevantly the thiophosphorylation of the WGA-binding proteins in vitro to determine whether such mitotic phosporylation results in a failure to incorporate into reconstituted nuclei. However, these assays have not yet yielded definitive results.
In analyzing the higher order
structures into which the glycoproteins assemble, we found that each of
the three major WGA-binding proteins exist in the egg cytosol as part
of distinct high molecular weight complexes. Like the rat p62
nucleoporin complex(64, 68, 69) , the bulk of Xenopus p60 is complexed with proteins of 58 and 54 kDa. This
is interesting considering that the Xenopus complex exists in
the egg cytosol as part of the soluble disassembled pore components
derived from the mitotically disassembled oocyte nucleus, while the rat
complex was biochemically extracted from intact interphase nuclei using
mild detergents. The p200 nucleoporin exists as a higher molecular mass
complex of 1000 kDa, as judged by gel filtration, which contains
p200, p60, and possibly other unknown components. The p97 protein is
contained in a 450-kDa complex; other protein components of this
complex, if they exist, are unknown. The size of the complexes are the
same in interphase and mitotic extracts, suggesting that they are in
their most disassembled state and represent stable subunits into which
the nuclear pores disassemble. With respect to other such complexes,
Dabauvalle et al.(59) reported a 254-kDa complex in Xenopus containing p62, which we did not observe. It has also
been reported that the NUP153 pore protein is part of a separate large
(16 S) complex in Xenopus egg cytosol (81) . Together,
these results suggest that the pore mitotically disassembles into
discrete multicomponent subunits. Further analysis of such subunits
should prove greatly informative both in identifying new pore proteins,
as well as in elucidating the nearest neighbor interactions among the
100 different proteins present in the nuclear pore.
When the
WGA-binding proteins from a Xenopus egg lysate are
electrophoresed on a polyacrylamide gel and stained with Coomassie
Blue, the p60 complex, p97, and p200 appear in roughly equimolar
amounts and are among the most prominent proteins present. ()However, in addition to these major soluble glycoproteins,
there are a number of less abundant WGA-binding proteins present in the
egg lysates (Fig. 3). Proteins other than nucleoporins are also
modified by GlcNAc additions. In fact, the interior of the nucleus is a
major location for GlcNAc-modified proteins(82) . For example,
RNA polymerase II and many RNA polymerase II transcription factors
contain GlcNAc modifications(83, 84) , as do
chomosomal proteins (85) and a nuclear tyrosine
phosphatase(86, 87) . Indeed, certain monoclonal
antibodies with a broad specificity for GlcNAc-containing proteins
exhibit strong intranuclear staining(53) . When long
autoradiographic exposures of labeled WGA-binding proteins isolated
from mitotic extracts were examined, the less abundant WGA-binding
proteins were also seen to be phosphorylated. The localization and
extent of phosphorylation of these proteins will be a future area to be
explored.
It is also interesting to note that a number of the minor WGA-binding proteins are revealed to be phosphorylated when egg lysates are treated with the phosphatase inhibitor, okadaic acid (Fig. 7, laneM). Under these conditions, highly phosphorylated forms of the three major WGA-binding proteins, as well as a number of the minor WGA-binding proteins, accumulate. The kinase or kinases responsible for this phosphorylation are not known, but it is not likely to be the Cdc2 kinase since okadaic acid alone does not increase H1 kinase activity when added to the lysates (not shown). Moreover, p60, which is heavily phosphorylated in okadaic acid-treated lysates, is not a substrate for Cdc2 kinase in vitro (Fig. 7). The role of the phosphorylation events revealed by okadaic acid is not known, but this finding demonstrates that the phosphorylation of pore proteins and other WGA-binding proteins may also occur during interphase. It is possible that okadaic acid acts to stabilize normally transient interphase phosphorylation that is important for the function of these proteins. There exists precedent for such phosphorylation events playing a crucial role in regulating the nuclear import of both lamins and the SV40 T antigen(31, 88, 89) . Other transient interphase phosphorylation events are also known to transmit growth regulatory signals from the cell surface to the nucleus of a cell(90) . Indeed, it has been found that the maximum size of nuclear transport probes that can be transported though the pore increases as cells are stimulated to divide(91) , indicating that the nuclear pore complex is regulated by growth controls. The mechanism of this regulation is not yet known, but may well involve phosphorylation. In assessing other putative roles for pore protein phosphorylation, it is known that nuclear pores are not only assembled into the nuclear envelope at telophase during postmitotic reassembly of the nucleus, but also can insert into existing nuclear envelopes during S phase, when both the size of the nucleus and the number of pores doubles. It is possible therefore that transient interphase phosphorylation of pore proteins not only regulates the function of these proteins in nuclear transport but also their assembly. Clearly, this is a preliminary discussion of such hypotheses. It will be interesting to examine the specific roles of interphase and mitotic phosphorylation in future work.