From the Duke University Medical Center, Department of Pharmacology and Cancer Biology, Durham, North Carolina 27710
Received for publication, September 6, 2000, and in revised form, October 26, 2000
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ABSTRACT |
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Entry into mitosis is regulated by the Cdc2
kinase complexed to B-type cyclins. We and others recently reported
that cyclin B1/Cdc2 complexes, which appear to be constitutively
cytoplasmic during interphase, actually shuttle continually into and
out of the nucleus, with the rate of nuclear export exceeding the
import rate (1-3). At the time of entry into mitosis, the import rate is increased, whereas the export rate is decreased, leading to rapid
nuclear accumulation of Cdc2/cyclin B1. Although it has recently been
reported that phosphorylation of 4 serines within cyclin B1 promotes
the rapid nuclear translocation of Cdc2/cyclin B1 at
G2/M, the role that individual phosphorylation sites
play in this process has not been examined (3, 4). We report here that
phosphorylation of a single serine residue (Ser113 of
Xenopus cyclin B1) abrogates nuclear export of cyclin B1. This serine lies directly within the cyclin B1 nuclear export sequence
and, when phosphorylated, prevents binding of the nuclear export
factor, CRM1. In contrast, analysis of phosphorylation site mutants
suggests that coordinate phosphorylation of all 4 serines (94, 96, 101, and 113) is required for the accelerated nuclear import of cyclin
B1/Cdc2 characteristic of G2/M. Additionally, binding of
cyclin B1 to importin- In all eukaryotes studied to date, the onset of both mitosis and
meiosis is regulated by the activation of MPF (maturation promoting factor), a heterodimeric complex that
contains the serine/threonine kinase Cdc2 and a B-type cyclin.
Coordination of the G2/M transition relies upon tight
regulation of the kinase activity of Cdc2/cyclin B. During interphase,
cyclin B is synthesized and forms complexes with the inactive Cdc2
monomer. Phosphorylation of complexed Cdc2 at two sites,
Thr14 and Tyr15, by the inhibitory kinases Wee1
and Myt1 keeps Cdc2 inactive until the time of entry into mitosis
(5-13). At the G2/M transition, dephosphorylation of Cdc2
by the dual specificity phosphatase, Cdc25, promotes Cdc2/cyclin B
activation, thus driving cells into mitosis (14-17).
The biological function of Cdc2/cyclin B is regulated not only at the
level of enzymatic activity but also by changes in subcellular localization. During interphase, Cdc2/cyclin B1 kinase complexes are
predominantly cytoplasmic; at the onset of mitosis, they transit precipitously to the nucleus, presumably to catalyze the nuclear events
of mitosis (18). Cyclin B2, in contrast, remains cytoplasmic throughout
the cell cycle (19). By deletion analysis, the region of cyclin B1
responsible for its interphase cytoplasmic localization maps to a
domain near the N terminus of the protein termed the cytoplasmic
retention sequence (CRS)1
(19). Recently, we and others reported that the CRS of cyclin B1
contains an autonomous nuclear export sequence that mediates the
nuclear export of cyclin B1 through binding of the nuclear transport
factor CRM1(1-3). Because impeding nuclear export through use of the
CRM1 inhibitor leptomycin B led to the marked nuclear accumulation of
cyclin B1, we concluded that the seemingly static cytoplasmic
localization of cyclin B1 during interphase actually reflects a balance
of ongoing nuclear import and more rapid nuclear export.
At the onset of mitosis, cyclin B1 is phosphorylated on several sites,
among which four conserved serines (Ser94,
Ser96, Ser101, and Ser113) within
the CRS have been proposed to play a role in mediating the biological
activity of cyclin B1 (3, 20). In Xenopus oocytes, mutation
of all four serines (to Ala) to abolish phosphorylation diminished the
ability of this mutant to induce oocyte maturation (the
G2/M transition) (20). In Cos cells,
phosphorylation-deficient cyclin B1 protein is cytoplasmic; in
contrast, mutation of all four serines to mimic constitutive
phosphorylation (Ser to Glu) drives cyclin B1 into the nucleus.
Moreover, appending a strong nuclear localization signal (NLS) to the
phosphorylation-deficient cyclin B1 restored its ability to promote
oocyte maturation (4). Consistent with these observations, we reported
that mutation of all four serines to Glu impaired the ability of the
CRS to bind to the nuclear export factor CRM1 and prevented cyclin B1 nuclear export, thus facilitating nuclear accumulation of cyclin B1
complexes at the G2/M transition (3).
Although cyclin B1 complexes appear to transit into the nucleus at a
continually slow rate during interphase, nuclear import of Cdc2/cyclin
B1 rapidly accelerates at the G2/M transition. As reported
recently by Pines and colleagues (21), changing all of the serine
residues within the CRS to Ala impedes nuclear import of cyclin B1,
whereas changing them all to Glu accelerates import, consistent with a
role for phosphorylation in both inhibiting nuclear export and
increasing nuclear import of cyclin B1. In this report, we further
investigate the role of cyclin B1 phosphorylation in nuclear transport.
Specifically, we were interested in determining whether all four
phosphorylation sites within the CRS must act in concert to regulate
cyclin trafficking and whether the nuclear import and export processes
were controlled by distinct phosphorylation sites. By analyzing CRM1
binding and nuclear transport properties of single and multiple site
mutants of cyclin B1 within the CRS, we have found that nuclear export
of cyclin B1 is controlled exclusively through phosphorylation of
Ser113 (Xenopus numbering), whereas
phosphorylation of all four serine residues within the CRS is required
to accelerate nuclear import. This offers the opportunity for
combinatorial control of Cdc2/cyclin B1 localization by distinct
kinases acting at distinct phosphorylation sites, allowing multiple
cellular inputs into the regulation of cyclin B1/Cdc2 localization.
In Vitro Translation--
Xenopus cyclin B1 wild
type, Ala, and Glu mutants were kindly provided by Dr. D. Donoghue
(University of California, San Diego) in the vector SP64T, containing
cyclin variants cloned downstream of an SP6 promoter.
35S-Labeled proteins were produced by use of the
SP6-coupled TNT reticulocyte system (Promega) according to
manufacturer's instructions.
Preparation of Xenopus Oocyte and Egg Extracts--
Oocyte
extracts were prepared as described previously (22). Interphase egg
extracts and mitotic extracts were prepared according to the protocols
of Smythe and Newport (23).
Oocyte Microinjection and Subfractionation--
Stage VI oocytes
of Xenopus laevis were prepared for
microinjection, as described (24). Oocytes were put in individual wells of the 96-well plate, oriented with animal pole upward. The plates were
placed on top of centrifuge bucket adaptor in Beckman table top and
spun for 10 min at 1000 × g at 18 °C. This step
enabled the nucleus to move closer to the animal pole, and sometimes a white spot (nucleus) can be visualized in the animal pole region. Eight
to ten nanoliters of protein sample was injected into oocyte nuclei.
Two injection controls for nuclear integrity were used: 14C-labeled bovine serum albumin (Amersham Pharmacia
Biotech) and in vitro translated 35S-labeled
GRP90 (a protein that does not have an NES or NLS). Red
reticulocyte lysate was coinjected to mark the injected nuclei. Successfully injected oocytes (those with red nuclei) were separated into nuclear and cytoplasmic fractions by manual dissection under mineral oil (32). Fractions from 5 to 10 oocytes were homogenized in
buffer (65 mM Tris at pH 7.6, 10 mM EGTA at pH
8.0, 1 mM phenylmethylsulfonyl fluoride, 5 ng/µl aprotinin/leupeptin) and spun for 5 min at 13,000 × g to remove insoluble material. The supernatants were precipitated with 3 volumes of acetone, pelleted, and dried. The pellets were resuspended in 10 µl of SDS-PAGE sample buffer per oocyte and analyzed by SDS-PAGE, followed by autoradiography or Western
blotting. In the injection experiments, the absolute time course of
cyclin B export varies between batches of oocytes (i.e. between frogs). In all experiments in which we compare import or export
rates of different cyclin mutants, samples to be compared were done
with the same batch of oocytes. Multiple repetitions of each experiment
confirmed that the relative rates of export or import of the mutants
were the same from experiment to experiment.
Construction of Various Cyclin B1 Phosphorylation
Mutants--
Xenopus cyclin B1 4-Glu mutant cDNA in the
pSP64T vector was used as the template (provided by Dr. D. Donoghue). A
PCR-based mutagenesis was used to mutate each Glu individually to Ala.
The 5' oligonucleotide encoding the N terminus of cyclin B1 was
5'-GGGGGAATTCAGAAAATGTCGCTACGAGTCACC-3', where an EcoRI site
was inserted before the start codon. The 3' oligonucleotide encoding
the C terminus of cyclin B1 was
5'-GGGGGATCCCATGAGTGGGCGGGCCATTTCCAC-3', where a BamHI site
was inserted after the stop codon. To mutate individual glutamic acids
to alanines, we utilized mutagenic primers and their reverse primers,
both of which contain the targeted mutagenesis sites. Using the 5'
primer along with one mutagenic primer, we produced a PCR fragment
extending from the 5' end (encoding the extreme N terminus) to the site
of mutation. We then generated a second PCR product using the
C-terminal primer and the corresponding reverse primer, producing a DNA
fragment extending from the mutation site to the C-terminal end. A
full-length mutant clone was generated with an additional round of PCR,
using a mixture of the N- and C-terminal encoding DNA fragments as
templates for PCR with the original 5' and 3' primers. The full-length
product was subcloned into the pSP64T vector. For individual mutants,
their mutagenic primers are listed: S94A/3E,
5'-GCTCAGGTTGAACCCAGCGCGCCAGAGCCAATGGAAAC-3'; S96A/3E,
5'-GGTTGAACCCAGCGAACCGGCGCCAATGGAAACAGAAGG-3'; S101A/3E, 5'-CAGAGCCAATGGAAACAGCCGGCTGCCTCCCTGATGAG-3'; and S113A/3E,
5'-GCTCTGCCAGGCTTTCGCGGACGTCCTCATTCACGTG-3'. For construction of
the S113E mutant of cyclin B1, the wild type cyclin B1 cDNA in
pSP64T was used as the template, and the PCR-based mutagenesis method
was used as described above. The mutagenic primer was
5'-GCTCTGCGAGGCTTTCGAGGATGTCCTCATTCACG-3'. All mutations were
confirmed by DNA sequencing.
Construction of the Various GST-CRS Fusion Proteins--
The
various CRS regions of cyclin B1 were generated by PCR.
Full-length cyclin B1 from the wild type and all the phosphorylation mutants described above were used as templates. The 5' oligonucleotide encoding the N-terminal region of the CRS was
5'-TTGGGATCCCTTAAAGTGATAGAA-3', where a BamHI site was
inserted to facilitate the subsequent cloning steps. The 3'
oligonucleotide encoding the C-terminal region of CRS was
5'-CATTGAATTCCCCTAATCATCAGCATC-3', where an EcoRI site was
inserted after the stop codon. The resulting CRS cDNAs were inserted into the pGexKG vector through BamHI and
EcoRI sites. The resulting constructs were confirmed by DNA sequencing.
Expression and Purification of Recombinant GST Fusion
Proteins--
All constructs were expressed in Topp3 E. coli (Stratagene). To increase solubility of recombinant proteins,
bacteria were grown to OD of 0.5 at 37 °C and then shifted to
18 °C. 0.4 mM isopropyl-1-thio- Pull-down Experiments--
GST fusion proteins were coupled to
glutathione-Sepharose beads and blocked with boiled fetal bovine serum
or 1% casamino acids plus 0.1% Tween 20 to block nonspecific
interactions of proteins with the Sepharose beads. The beads were then
incubated with either oocyte extract or in vitro translated
protein in boiled fetal bovine serum (or 1% casamino acids plus 0.1%
TWEEN 20) at 4 °C for an hour. The beads were then washed with EB
buffer, and the binding proteins were resolved by SDS-PAGE followed by
immunoblotting or autoradiography.
Nuclear Export of Cyclin B1 Is Controlled by Phosphorylation of
Ser113--
As described above, we have shown previously
that phosphorylation of four conserved serines within the CRS region of
cyclin B1 can regulate its nuclear export (3). Specifically, mutation of all four serines to Glu to mimic constitutive phosphorylation impairs the ability of cyclin B1 to bind to the nuclear export factor,
CRM1. Because all four of these serines (Ser94,
Ser96, Ser101, and Ser113) had been
mapped as phosphorylation sites in vivo (4), we wished to
determine the contributions of individual phosphorylation sites to the
regulation of cyclin B1 nuclear trafficking. In the most simple
scenario, if phosphorylation of a single site is required to prevent
the cyclin B1-CRM1 interaction, then rendering that site
nonphosphorylatable (through mutation to Ala) should permit interaction
with CRM1, even if all of the other CRS serine residues are
phosphorylated (or mutated to Glu to mimic constitutive
phosphorylation). Therefore, starting with a GST-CRS construct in which
all of the serine phosphorylation sites had been changed to Glu, we
mutated each site individually back to Ala and examined the resulting panel of mutant CRS regions for their ability to bind CRM1. As shown in
Fig. 1, changing Ser113, but
not any other site, to Ala (S113A/3E) conferred on cyclin B1 the
ability to bind to CRM1. These data suggest that phosphorylation of
Ser113 at the G2/M transition blocks binding of
cyclin B1 to CRM1. To assess whether Ser113 phosphorylation
alone is sufficient for this inhibition, we also changed only
Ser113 to Glu in the context of the wild type CRS (all
other sites Ser; S113E). This mutation was indistinguishable from the
4-Glu mutant in that it very efficiently prevented CRM1 binding (Fig.
1). These data indicate that phosphorylation of Ser113 is
primarily responsible for controlling the cyclin B1-CRM1
interaction.
To determine whether phosphorylation of Ser113 directly
modulates cyclin B1 nuclear export, we introduced the S113E and
S113A/3E mutations into the full-length wild type cyclin B1 to compare their nuclear export rates with 4-Glu and wild type cyclin B1 s. For
this purpose, the mutant and wild type cyclins were translated in
vitro in the presence of [35S]methionine and
injected into the nuclei of stage VI oocytes. 35S-labeled
GRP94, which contains neither an NES nor an NLS, was coinjected as a
control for maintenance of nuclear integrity during the microinjection
and microdissection procedures. At 0, 3, and 6 h after injection,
the oocytes were dissected manually into nuclear and cytoplasmic
fractions. After identification of successfully injected oocytes (by
the presence of the pigmented hemoglobin from the reticulocyte lysate),
nuclear and cytoplasmic fractions were analyzed by SDS-PAGE and
autoradiography for the presence of labeled protein. In agreement with
the CRM1 binding result, we found that introduction of the S113E
mutation into full-length cyclin B1 had an effect similar to that of
the 4-Glu mutation, significantly reducing the cyclin B1 nuclear export
rate (Fig. 2A). Moreover,
changing only Ser113 to Ala, in a context where all of the
other Ser were changed to Glu (S113A/3E) completely restored cyclin
B1nuclear export (Fig. 2B). It is interesting to note that
the S113A/3E mutant exports from nuclei even faster than the wild type
cyclin B1, whose export is slightly retarded by a low basal level of
nuclear Ser113-directed kinase activity (Fig. 2B
and data not shown). Collectively, these data strongly suggest that
nuclear export of cyclin B1 is controlled by phosphorylation of
Ser113.
Phosphorylation of All Four Serines within the CRS Is Required for
Acceleration of Cyclin B1 Nuclear Import--
As noted by Pines and
colleagues (1), the slow nuclear import rate observed when cyclin B1
nuclear export is inhibited during interphase cannot fully account for
the rapid nuclear accumulation of cyclin B1 observed at the
G2/M transition. Indeed, as they recently reported,
phosphorylation within the CRS of human cyclin B1 seems to accelerate
its nuclear import, as well as retarding its export (21). To confirm
that this was the case in the Xenopus system, we compared
the nuclear import rates of the wild type 4-Glu and 4-Ala mutants by
injecting 35S-labeled cyclin B1 variants into the cytoplasm
of oocytes pretreated with leptomycin B to block CRM1-mediated nuclear
export. At various time points after injection, the oocytes were
dissected into cytoplasmic and nuclear fractions, and the amount of
radiolabeled cyclin B1 in each fraction was quantitated.
14C-Labeled bovine serum albumin, which lacks both NES and
NLS sequences, was coinjected as a control for maintenance of nuclear
integrity during the injection and microdissection procedures. As shown in Fig. 3, changing Ser to Glu to mimic
constitutive phosphorylation dramatically accelerated the cyclin B1
nuclear import rate, confirming that phosphorylation within the CRS
both inhibits nuclear export and accelerates nuclear import.
To extend these studies and to understand how phosphorylation of
individual serines modulates cyclin B1 nuclear import, we again started
with the most uncomplicated scenario; if phosphorylation of a single
serine is absolutely required to accelerate the nuclear import of
cyclin B1 at the G2/M transition, then rendering that site
nonphosphorylatable (mutated to Ala) should slow down its import rate
to the level of wild type cyclin B1, even when all of the other sites
are mutated to Glu to mimic constitutive phosphorylation. Therefore, we
compared the nuclear import rates of various cyclin B1 mutants, in
which each serine was individually mutated to Ala, whereas all of the
other serines remained Glu. Surprisingly, we found that rendering any
of the four CRS serine residues unphosphorylatable decreased cyclin B1
nuclear import rates to wild type interphase levels (Fig.
4). Therefore, we conclude that
phosphorylation of all four serines (Ser94,
Ser96, Ser101, and Ser113) within
the CRS is required to increase the nuclear import rate of cyclin B1
prior to entry into mitosis.
Phosphorylation within the CRS Does Not Regulate Direct Binding to
Importin- The Ability of Cyclin B1 to Induce the G2/M Transition
Requires Phosphorylation of All Four Serines within the CRS--
As
reported by Li et al. (4), cyclin B1 bearing a 4-Glu mutant
CRS is a much more potent inducer of oocyte maturation
(G2/M transition as scored by GVBD) than are the wild type
or 4-Ala proteins. To analyze the effects of the individual
phosphorylation site mutations, the mutants in which single serines
were altered to Ala, whereas all of the other serines were altered to
Glu, were tested for their ability to induce GVBD following injection into stage VI G2-arrested oocytes. Consistent with our
observation that mutation of any single site to Ala compromised nuclear
import, none of the individual mutants were capable of driving the
G2/M transition as efficiently as the 4-Glu mutant (Table
I). These data strongly suggest that
phosphorylation of all four serines within the CRS is not only required
for optimal nuclear accumulation of cyclin B1 but is also required for
maximally efficient promotion of the G2/M transition.
Cyclin B1/Cdc2 biological activity is controlled at both the level
of enzymatic activity and subcellular distribution. Because premature
nuclear entry of this complex can have critical consequences for the
timing of entry into mitosis, the cell maintains a tight control on its
entrance into and exit from the nucleus. We show in this report that
phosphorylation of all four sites within the CRS is required for the
rapid nuclear entry of Cdc2/cyclin B1 characteristic of the
G2/M transition. In contrast, phosphorylation of a single
site (Ser113) suffices to inhibit cyclin B1 nuclear export.
Phosphorylation Site Effects on Interaction with Nuclear Transport
Factors--
The ability of a single site of serine phosphorylation to
inhibit cyclin B1 nuclear export is easily explainable given the fact
that Ser113 lies within the CRM1-binding nuclear export
sequence, immediately adjacent to a hydrophobic residue required for
NES activity (2, 3, 21). Phosphorylation of this site introduces a
charge that most likely interferes with recognition of the sequence by CRM1. It is not as clear, however, how phosphorylation within the CRS
accelerates nuclear import of Cdc2/cyclin B1. Although direct
interaction of cyclin B1 with importin- Phosphorylation of Cyclin B and Entry into Mitosis--
The
presence of four phosphorylation sites that must be coordinately
controlled to promote nuclear translocation of cyclin B1 offers the
possibility for multiple regulatory inputs into cyclin localization. We
and others have data to suggest that Ser96 is a bona
fide Cdc2 phosphorylation
site2 and Ser94
is also a potential Cdc2 site based on sequence consensus (30). Although the kinase(s) responsible for phosphorylation of serines 101 and 113 has not yet been identified, these sites do not fit the
consensus for Cdc2-catalyzed phosphorylation. Moreover, the sequences
flanking Ser101 and Ser113 are not similar to
each other, suggesting that more than one kinase may phosphorylate
these sites. Thus, it is seems likely that two or more kinases
cooperate to trigger nuclear translocation of the Cdc2/cyclin B1
complex at G2/M. Because all four sites must be
phosphorylated for rapid cyclin B1 nuclear import, phosphorylation of
cyclin B1 is likely to occur first in the cytoplasm. This is consistent
with our previously published observation that the vast majority of
CRS-directed kinase activity is cytoplasmic rather than nuclear.
Preliminary data from our laboratory suggest that phosphorylation of
Ser113 may rely upon prior phosphorylation of one or all of
the other serines,2 providing an additional layer of
assurance that nuclear export of Cdc2/cyclin B1 will not be prematurely
turned off by Ser113 phosphorylation of cyclin B1 shuttling
through the nucleus during interphase.
This stringent regulation of cyclin B1 trafficking prevents premature
mitotic entry and may be a component of the mechanism that cells use to
prevent the onset of mitosis in the presence of damaged or incompletely
replicated DNA. It has been reported that Cdc2/cyclin B1 is cytoplasmic
in G2-arrested cells bearing damaged DNA (2). Indeed,
forcibly localizing Cdc2/cyclin B1 to the nucleus by appending a
classical NLS can trigger premature mitotic events in DNA
damage-arrested cells harboring a Cdc2 allele insensitive to inhibitory
phosphorylation (31). Similarly, Toyoshima et al. (2)
reported that a nuclear export-defective cyclin B1 mutant was more
effective than wild type cyclin B1 in cooperating with caffeine to
override a DNA damage-induced G2 arrest. Given these
effects of cyclin B1 subcellular localization on checkpoint function,
it will be interesting, in the future, to determine whether
G2/M checkpoint pathways directly modulate the
phosphorylation state of the cyclin B1 CRS.
, the factor known to be responsible for the
slow interphase nuclear entry of cyclin B1, appears to be unaffected by
the phosphorylation state of cyclin B. These data suggest that a
distinct import factor must be recruited to enhance nuclear entry of
Cdc2/cyclin B1 at the G2/M transition.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside was added to
induce protein expression at 18 °C overnight. Bacteria were pelleted
and resuspended in lysis buffer (50 mM Tris, pH 7.5, 10 mM KCl, 1 mM EDTA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride). 1% Triton-X and 300 mM NaCl were added to increase protein solubility. Cells
were lysed twice using a French Press and spun at 17,000 × g for 30 min. The supernatants were diluted 1:1 with buffer
(10 mM Hepes, pH 8.0, and 1 mM DTT) to reduce
the Triton concentration and incubated with glutathione-Sepharose beads
at 4 °C for 1-2 h. Beads were pelleted, washed with buffer (10 mM Hepes, pH 8.0, 300 mM NaCl, and 1 mM DTT), and kept in storage buffer (10 mM
Hepes, pH 8.0, 50% glycerol, and 1 mM DTT) at
20 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Phosphorylation of Ser113 within
the CRS of cyclin B1 impairs its ability to bind to the nuclear export
factor, CRM1. Mutant variants of the isolated CRS were fused to
the C terminus of GST. These mutants include variants in which: 1) all
four Ser were changed to Glu (4-Glu), 2) each individual Ser was
changed to Ala within the context of the all Glu background (S94A/3E,
S96A/3E, S101A/3E, and S113A/3E), and 3) only Ser113 was
changed to Glu (S113E). Equal amounts of the various mutant GST-CRS
fusion proteins or the wild type (WT) GST-CRS protein were
coupled to glutathione-Sepharose beads. 20 µl of each resin was
incubated in the presence or absence of oocyte extract (+ or ) for 60 min, and the beads were then pelleted and washed. The bead-bound
material was analyzed by SDS-PAGE followed by immunoblotting with
anti-CRM1 antibody.
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Fig. 2.
Phosphorylation of cyclin B1 on
Ser113 inhibits cyclin B1 nuclear export.
A, 35S-labeled S113E or 4-GLU mutant cyclin B1
proteins were coinjected into Xenopus oocyte nuclei with
35S-labeled GRP94 as a control protein. At 0, 3, or 6 h after injection, the oocytes were dissected manually into cytoplasmic
and nuclear fractions, extracted, and analyzed by SDS-PAGE followed by
autoradiography. The graph represents a quantitation of the data above
showing the percentage of cyclin B1 remaining in nuclei after 0, 3, or
6 h. B, the nuclear export rates of
35S-labeled wild type (WT) cyclin B1 and
S113A/3E mutants were compared as described in A. The
graph represents a quantitation of the data, showing the percentage of
cyclin B1 remaining in nuclei after 0, 3, or 6 h. T,
total; C, cytoplasmic; N, nuclear.
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Fig. 3.
The Glu mutant of cyclin B1 is imported into
nuclei more efficiently than the wild type or Ala proteins.
A, oocytes were incubated with 200 nM leptomycin
B in MB buffer for 2 h before injection. 40 nl of
35S-labeled in vitro translated cyclin B1 4-Glu,
wild type (WT), or 4-Ala proteins were injected into the
cytoplasm of oocytes along with 14C-labeled bovine serum
albumin as control. Injected oocytes were dissected into cytoplasmic
and nuclear fractions at the indicated times after injection, and
proteins were analyzed by SDS-PAGE and autoradiography.
B, the graph represents a quantitation of the data in
A, showing the percentage of cyclin B1 in the nuclear
fraction at the indicated times. C, cytoplasmic;
N, nuclear.
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Fig. 4.
Phosphorylation of Ser94,
Ser96, Ser101, and Ser113 are all
required to accelerate cyclin B1 nuclear import. A,
oocytes were incubated with 200 nM leptomycin B in MB
buffer for 2 h before injection. 40 nl of 35S-labeled
in vitro translated cyclin B1 4-Glu, wild type
(WT), S94A/3E, S96A/3E, S101A/3E, or S113A/3E protein was
injected into the oocyte cytoplasm. Injected oocytes were
dissected into cytoplasmic and nuclear fractions at the indicated times
after injection and proteins were analyzed by SDS-PAGE and
autoradiography. B, the graph represents a quatitation of
the data in A, showing the percentage of cyclin B1 in the
nuclear fraction at the indicated times. T, total;
C, cytoplasmic; N, nuclear.
--
We recently reported that the interphase nuclear
import of cyclin B1 is mediated by direct binding to the nuclear
transport factor, importin-
(26). It has also been suggested that
cyclin B1 can "piggyback" into the nucleus at G2/M by
binding to another cyclin protein, cyclin F (25). However, because
cyclin F binding to cyclin B1 is reportedly unaffected by
phosphorylation of cyclin B1, it seems unlikely that the
phosphorylation-induced increase in cyclin B1 nuclear entry is due to
enhanced cyclin F interactions (25). Therefore, we sought to determine
whether the enhanced import of the 4-Glu mutant and the diminished
import of the Ala mutants might reflect changes in the efficiency of
the importin-
-cyclin B1 interaction. Accordingly, we incubated
radiolabeled, in vitro translated cyclin B1 variants with
GST-importin-
immobilized on glutathione-Sepharose and examined the
degree to which the CRS mutations affected importin-
binding. In
multiple experiments, the characteristic, differential import rates of
the various mutants were not reflected in differences in importin
binding (Fig. 5). Therefore,
phosphorylation does not appear to enhance nuclear import of cyclin B1
by increasing the affinity of cyclin B1 for importin-
.
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Fig. 5.
Direct binding of cyclin B1 to
importin- is not affected by the cyclin B1
phosphorylation state. A, the cyclin B1 mutants
analyzed in Fig. 4 were translated in vitro in the presence
of [35S]methionine and incubated in phosphate-buffered
saline containing 0.1% Tween 20 and 1% casamino acids (as a blocking
agent) for 1 h at 4 °C along with GST-importin-
protein
linked to glutathione-Sepharose. These beads were pelleted, washed
extensively, resolved by SDS-PAGE, and processed for autoradiography.
B, quantitation of the data in A. Shown are
percentages of input cyclin B1 protein retrieved on the importin-
beads.
The ability of various Cyclin B1 phosphorylation mutants to induce
GVBD
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
appears to account for the
interphase nuclear import of cyclin B1, it is not clear whether
importin-
is involved in the more rapid nuclear entry of cyclin B1
at G2/M (26). That this interaction may indeed be important
for G2/M nuclear translocation of cyclin is suggested by
the fact that immunodepletion of importin-
from mitotic cytosolic extracts compromises the ability of these extracts to promote cyclin B1
nuclear translocation in a semi-permeabilized cell nuclear transport
assay (27). In our assays, there was no discernible difference in the
ability of the various phosphorylation site mutants to interact with
importin-
. The assays shown in Fig. 5 were, however, performed in
buffer with isolated, recombinant importin-
in the absence of
ancillary factors. Hence, if importin-
recruits a novel factor
responsible for modulating interaction with cyclin B1, it would not be
picked up in this particular assay. That being said, we have observed
no consistent changes in importin-
binding of these mutants, even
when the incubations were performed in the presence of either
interphase or mitotic cytosol (data not shown). Similarly, although it
has been suggested that cyclin B1 can piggyback into the nucleus
through interaction with nuclear-bound cyclin F, this interaction is
not modulated by phosphorylation within the CRS (25). Therefore, we
suspect either that phosphorylation of cyclin B1 induces interaction
with a distinct transport factor other than importin-
or that the
altered efficiency of nuclear entry at the G2/M is not
controlled at the level of cyclin B1-import factor interactions. Before
it was discovered that the CRS region of cyclin B1 provides a binding
site for CRM1, it was proposed that the CRS provided an anchoring site
for cyclin B1 in the cytoplasm and, in this way, prevented its untimely
nuclear entry. One intriguing possibility is that cyclin B1 does,
indeed, have a cytoplasmic retention mechanism (in addition to a
nuclear export mechanism) and that this mechanism is regulated by
phosphorylation within the CRS. Under these circumstances,
unphosphorylated cyclin B1 would still have the capacity to interact
with transport factors but the "anchor" would in some way
physically prevent the rapid nuclear translocation characteristic of
mitotic entry. Because leptomycin B treatment of cells does eventually
allow the quantitative accumulation of cyclin B1 in the nucleus, such a
hypothetical retention mechanism would have to be "leaky" enough to
allow slow interphase import or be selective enough to interfere with
the import mechanism used at mitosis, without inhibiting the
importin-
-mediated interphase import. An alternative, although
perhaps unlikely, explanation for the nuclear accumulation of cyclin B1
in the presence of leptomycin B is that a factor required for
restricting the nuclear import of unphosphorylated cyclin B1 must
itself be continually exported from the nucleus. Such a possibility has
also been suggested for the Cdc2 activator Cdc25, whose interphase
nuclear import is restricted by binding to 14-3-3 proteins yet rapidly
accumulates in nuclei upon leptomycin B treatment (28, 29).
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ACKNOWLEDGEMENT |
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We thank Danny Lew for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1GM60500 and the American Cancer Society Grant RPG-95-078-04-CCG (to S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Whitehead Inst., Nine Cambridge Center,
Cambridge, MA 02142.
§ Present address: IRC, University of Ulsan, San 29, Moo Keo Dong, Nam Ku, Ulsan, S. Korea.
¶ Scholar of the Leukemia and Lymphoma Society. To whom correspondence should be addressed: Duke University Medical Center, Dept. of Pharmacology and Cancer Biology, Box 3813, C370 LSRC, Research Dr., Durham, NC 27710. Tel.: 919-613-8624; Fax: 919-681-1005; E-mail: kornb001@mc.duke.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M008151200
2 H. Song and S. Kornbluth, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CRS, cytoplasmic retention sequence; NLS, nuclear localization signal; NES, nuclear export sequence; GVBD, germinal vesicle breakdown; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; GST, glutathione S-transferase; DTT, dithiothreitol.
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