From the University of Texas Health Science Center at San Antonio, Department of Molecular Medicine, Institute of Biotechnology, San Antonio, Texas 78245-3207
Received for publication, September 29, 2000, and in revised form, October 19, 2000
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ABSTRACT |
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The regulation of the vertebrate cell cycle is
controlled by the function of cyclin-dependent kinases
(CDKs), cyclins, and CDK inhibitors. The Xenopus
laevis kinase inhibitor, p27Xic1 (Xic1) is a member
of the p21Cip1/p27Kip1/p57Kip2 CDK
inhibitor family and inhibits CDK2-cyclin E in vitro as
well as DNA replication in Xenopus egg extracts. Xic1 is
targeted for degradation in interphase extracts in a manner dependent
on both the ubiquitin conjugating enzyme, Cdc34, and nuclei. Here we
show that ubiquitination of Xic1 occurs exclusively in the nucleus and
that nuclear localization of Xic1 is necessary for its degradation. We
find that Xic1 nuclear localization is independently mediated by
binding to CDK2-cyclin E and by nuclear localization sequences within
the C terminus of Xic1. Our results also indicate that binding of Xic1
to CDK2-cyclin E is dispensable for Xic1 ubiquitination and
degradation. Moreover, we show that amino acids 180-183 of Xic1 are
critical determinants of Xic1 degradation. This region of Xic1 may
define a motif of Xic1 essential for recognition by the ubiquitin
conjugation machinery or for binding an alternate protein required for degradation.
The progression of the vertebrate cell cycle is positively
regulated by cyclin-dependent kinases
(CDKs)1 associated with their
cyclin partners and is negatively regulated by
cyclin-dependent kinase inhibitors (CKIs) (reviewed in
Refs. 1-4). The mammalian cell cycle transition between G1
and S phases requires the activity of CDK2-cyclin E (5-7). The
function of this kinase is negatively modulated by the CDK2 inhibitor,
p27Kip1 in quiescent cells and in G1 phase
cells (2, 8-12). Entry into S phase is accompanied by the
ubiquitin-dependent proteolysis of p27Kip1 by a
protein complex consisting of Cdc34 and SCFp45Skp2
(13-21). The ubiquitin conjugating enzyme Cdc34 (UBC3) was first identified in budding yeast as a protein essential for the
G1 to S phase transition (22, 23). The SCF
(Skp1, cullin, F-box) complex is
comprised of the F-box binding protein, p19-Skp1; the cullin family
member protein, Cul1; the p27Kip1 F-box protein,
p45Skp2; and the ring finger protein, Rbx1/Roc1/Hrt1
(reviewed in Refs. 24-27). p27Kip1 must be phosphorylated
on threonine 187 by CDK2-cyclin E before binding to the F-box protein
p45Skp2 (15, 20). This binding targets p27Kip1
to the Cdc34-SCF complex for polyubiquitination followed by subsequent degradation by the 26S proteasome (15, 20). p27Kip1 can
also be degraded upon the ectopic overexpression of the c-Jun and Jun D
coactivator protein, Jab1 (28, 29). This mode of p27Kip1
degradation is mediated by the proteasome-dependent export of p27Kip1 from the nucleus and subsequent ubiquitination and
degradation in the cytoplasm (28).
In the frog, Xenopus laevis, two CDK2 inhibitors of the
Cip/Kip family have been identified that exhibit homology to mammalian p27Kip1, p57Kip2, and p21Cip1.
Xenopus inhibitor of
CDK (p27Xic1 or Xic1) and
kinase inhibitor from
Xenopus (p28Kix1 or Kix1)
share ~90% amino acid sequence identity with each other and
preferentially inhibit the activity of CDK2-cyclin E (30, 31). Xic1 and
Kix1 also inhibit chromosomal nuclear DNA synthesis in interphase egg
extracts and bind both CDK-cyclin complexes and proliferating cell
nuclear antigen (PCNA) (30, 31).
Xic1 is degraded in Xenopus interphase egg extracts in a
manner dependent on Cdc34, polyubiquitination, and the proteasome (32,
33). Furthermore, the Cdc34-dependent degradation of Xic1
is only observed in the presence of nuclei and increasing amounts of
nuclei result in progressively shorter half-lives of Xic1 (32). The
requirement for nuclei in ubiquitin-dependent degradation
of Xic1 is intriguing, because mammalian p27Kip1 is readily
ubiquitinated and degraded in whole cell extracts without the need for
intact nuclei, whereas p21Cip1 is degraded in nuclei by the
proteasome but without a need for ubiquitination (15, 19, 20, 34).
To better define the requirement for nuclei in Xic1 degradation, we use
Xenopus interphase extracts and transport-competent nuclei
to demonstrate that Xic1 ubiquitination and degradation require
transport of Xic1 into the nucleus. We also identify the sequences of
Xic1 that are required for efficient nuclear transport and show that
nuclear localization is mediated by CDK2-cyclin E binding to Xic1 and
Xic1 nuclear localization sequences (NLSs). We also show that binding
to CDK2-cyclin E is dispensable for Xic1 ubiquitination and degradation
in the nucleus. Finally, we demonstrate that the basic residues
spanning amino acids 180-183 are critical for Xic1 degradation and
ubiquitination. We propose that this region of Xic1 is essential for
either binding the ubiquitin conjugation machinery or interacting with
an alternate as yet unidentified regulatory protein.
Preparation of Xenopus Interphase Egg Extract, Xenopus
Demembranated Sperm Chromatin, His6-Ubiquitin, and
Methyl-ubiquitin--
X. laevis egg interphase extract (low
speed supernatant (LSS)) and demembranated sperm chromatin were
prepared as described previously (32, 35, 36). Mouse ubiquitin was
cloned into pQE30 (Qiagen) from a 9.5/10.5 days postcoital mouse
embryo library with an N-terminal tag of 6 histidine residues
(His6-ubiquitin). Mouse ubiquitin protein was expressed in
M15 bacterial cells and purified on nickel-nitrilotriacetic
acid-agarose (Qiagen) under native conditions. Ubiquitin (Sigma) was
methylated as described previously and used at a final concentration of
2 mg/ml (37).
DNA Constructs, Site-directed Mutagenesis, and DNA
Sequencing--
Mutations of the Xic1 gene were generated by using the
pCS2+/Xic1 plasmid and the QuikChange site-directed mutagenesis kit (Stratagene) (32). Xic1 point mutations were generated by making substitutions of the amino acids indicated: NLS1 = R166A, K167A, and R168A; NLS2 = K180A, K182A, and K183A; NLS3 = R206A,
K207A, and K208A; c = R33A and L35A; k = F65A and F67A; ck- = R33A, L35A, F65A, and F67A. Following are the oligonucleotide primers
(Integrated DNA Technologies) utilized to generate the
specified mutations: NLS1,
5'-CTAACACATCTACACAGCGCGCGGCCGCGGAGATCACCACTCCCATC and
5'-GATGGGAGTGGTGATCTCCGCGGCCGCGCGCTGTGTAGATGTGTTAG; NLS2 (ARAA),
5'-CATCACCGATTATTTCCCTGCCCGGGCAGCGATACTGAGTGCCAAGCC and
5'-GGCTTGGCACTCAGTATCGCTGCCCGGGCAGGGAAATAATCGGTGATG; NLS2 (RRRR),
5'-CACCGATTATTTCCCTAGACGTCGACGGATACTGAGTGCCAAGC and
5'-GCTTGGCACTCAGTATCCGTCGACGTCTAGGGAAATAATCGGTG; NLS3,
5'-CTGGAACAGACCCCCGCGGCAGCGATTCGATGAAACC and
5'-GGTTTCATCGAATCGCTGCCGCGGGGGTCTGTTCCAG; c,
5'-GGAGGGGAGCCTGTGCGAATGCCTTCGGTCCTATCG and
5'-CGATAGGACCGAAGGCATTCGCACAGGCTCCCCTCC; and k,
5'-GTCAGAGGTGGAACGCTGACGCTGAAAGTGGCACCC and
5'-GGGTGCCACTTTCAGCGTCAGCGTTCCACCTCTGAC. All point mutations were
confirmed by DNA sequencing using the Amplicycle Sequencing Kit
(PerkinElmer Life Sciences). The entire coding sequence of the
Xic1-NLS2 (ARAA) subclone was sequenced to confirm that it did not
contain any other mutations.
In Vitro Transcription and Translation--
Wild type and mutant
Xic1 were in vitro transcribed and translated from the SP6
promoter in pCS2+ using [35S]methionine (New Life Science
Products) and the TNT-coupled reticulocyte lysate system (Promega). The
amounts of protein for all Xic1 mutants were compared with the
Xic1-wild type (WT) by SDS-polyacrylamide gel electrophoresis
(PAGE) and PhosphorImager analysis. The amounts of protein for all Xic1
mutants were normalized and added at levels relatively equal to the
Xic1-WT level.
Coimmunoprecipitation Assay--
LSS (20 µl) was mixed with 1 µl of [35S]methionine-labeled Xic1-WT and precleared
with 7.5 µl of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech)
for 30 min at 23 °C. After preclearing, the supernatant was
incubated with anti-cyclin E, anti-CDK2, or normal rabbit serum for 30 min at 23 °C, and then 7.5 µl of protein A-Sepharose beads were
added, and the incubation was continued for 1 h at 23 °C. The
protein A-Sepharose beads were pelleted by centrifugation and washed
four times with EB (80 mM Nuclei Spin Down Assay--
Nuclei were allowed to form by
incubating demembranated sperm chromatin (10 ng/µl) in 25 µl of LSS
containing 0.1 µg/µl cycloheximide and an energy regenerating
system (60 mM phosphocreatine, 1 mM ATP, 150 µg/ml creatine phosphokinase) at 23 °C for 1 h. Following nuclei formation, 0.5 µl/25 µl
[35S]methionine-labeled WT-Xic1 was added for nuclear
transport assays, whereas 2 µl/25 µl
[35S]methionine-labeled GST-Xic1-WT was added for
ubiquitination assays, followed by incubation at 23 °C for 45 min.
For nuclear transport and certain ubiquitination experiments,
methyl-ubiquitin was included at a final concentration of 2 mg/ml. The
samples were then diluted 12-fold with NIB (50 mM KCl, 50 mM HEPES-KOH, 5 mM MgCl2, 0.5 mM spermine 4HCl, 0.15 mM spermidine 3HCl) and overlaid onto a 300-µl cushion of NIB containing 15% sucrose. The
samples were then centrifuged in a Sorvall RC-5B at 5000 × g in a HB-6 rotor at 4 °C for 10 min. A 20-µl aliquot
of supernatant was collected as the cytoplasmic fraction. The pelleted
nuclei were washed and recentrifuged twice with 1 ml of NIB containing 250 mM sucrose at 3300 × g for 8 min at
4 °C. Samples were subjected to SDS-PAGE and quantitated by
PhosphorImager analysis. The amounts of
[35S]methionine-labeled mutant Xic1 proteins were
quantitated by PhosphorImager analysis and added at a volume equal to
the relative amount of radioactively labeled Xic1-WT or GST-Xic1-WT
protein. In general, all [35S]methionine-labeled samples
were used between final dilutions of 1:15 to 1:72. Chromatin spin down
assays were conducted by including 0.1% Nonidet P-40 in NIB (38). The
percentage of nuclear transport was calculated as the amount of Xic1 in
the nucleus divided by the sum of the amount of Xic1 in the nucleus and
the cytosol multiplied by 100%. All the samples were normalized to 100% of the WT Xic1 transported to the nucleus. For quantitation analyses, each sample was measured at least two or three times, and the
standard error of the mean for each sample was calculated and displayed
as error bars.
Degradation Assay and
Dephosphorylation--
[35S]Methionine-labeled Xic1 (0.5 µl) was incubated in LSS (8 µl) containing cycloheximide, an energy
regenerating system, and ubiquitin (1.25 mg/ml) with or without
demembranated sperm chromatin (10 ng/µl) at 23 °C for 0, 1, and
3 h. Aliquots of 1.4 µl were removed from the samples for
analysis by SDS-PAGE followed by quantitation by PhosphorImager
analysis. The amounts of [35S]methionine-labeled mutant
Xic1 proteins were added at a volume normalized to equal the same
relative amount of radioactively labeled Xic1-WT protein.
Dephosphorylation was performed using calf intestinal phosphatase as
described previously (32).
DNA Replication Assay--
DNA replication assays were conducted
as described previously with the following modifications (32, 36).
[35S]Methionine-labeled Xic1 (0.625 µl) was added to
LSS (10 µl) containing cycloheximide, an energy regenerating system,
ubiquitin (1.25 mg/ml), and demembranated sperm chromatin (10 ng/µl).
DNA replication was normalized to 100% of the control containing
unprogrammed rabbit reticulocyte lysate. Each sample was measured three
times, and the standard error of the mean for each sample was
calculated and displayed as error bars.
Ubiquitination and Degradation of Xic1 Occur in the
Nucleus--
To begin to understand the requirement for nuclei in Xic1
degradation, we first determined whether Xic1 is transported into nuclei and where ubiquitinated Xic1 is localized. Xenopus
interphase egg extracts derived from a low speed centrifugation of eggs
(low speed supernatant or LSS) support the formation of
transport-competent nuclei as well as a single round of initiation and
elongation phases of semi-conservative chromosomal DNA replication (36, 39, 40). Demembranated sperm chromatin was preincubated with LSS,
followed by the addition of [35S]methionine-labeled Xic1
and His6-ubiquitin and separation of the nuclear and
cytosolic fractions by centrifugation. Our results show that
polyubiquitinated Xic1 is found exclusively in the nuclear fraction,
whereas Xic1 in the cytosolic fraction remains unmodified (Fig.
1A). We next asked whether
Xic1 degradation also occurs in the nucleus. To do this, we incubated
[35S]methionine-labeled Xic1 with demembranated sperm
chromatin in LSS and then separated the nuclei and cytosol. The
isolated nuclei and cytosol were added back separately to fresh LSS and
incubated for 0 or 3 h. The sample containing nuclei in fresh LSS
was again centrifuged to separate the nuclear and cytosolic fractions,
and all the samples were analyzed by SDS-PAGE and PhosphorImager
analysis to quantitate the amount of Xic1 remaining. The results show
that the nuclear Xic1 pool is 90% degraded after 3 h (Fig.
1B, lanes 1, 2, 6, and
7), whereas Xic1 in the cytosolic fraction remains stable
under these conditions (Fig. 1B, lanes 4,
5, 9, and 10). A slower migrating form
of Xic1, which most likely represents a mono-ubiquitinated species, was
observed in the nucleus (Fig. 1B, lanes 1 and
2). To eliminate the possibility that Xic1 must be exported
to the cytoplasm to be degraded, we tested the degradation of Xic1 in
the presence of leptomycin B, a specific inhibitor of CRM1/exportin 1 nuclear export (41-43). We did not observe any inhibitory effect of
leptomycin B on the efficiency of Xic1 degradation (data not shown), an
observation consistent with a recent study of Xic1 ubiquitination (33).
Collectively, these results indicate that Xic1 is predominantly
ubiquitinated and degraded in the nucleus.
The Xic1 Primary Sequence Contains Two Potential Nuclear
Transportation Regulatory Domains--
Because Xic1 appears to be
ubiquitinated and degraded in the nucleus, it is important to identify
the sequences of Xic1 that regulate its nuclear localization. The Xic1
primary sequence contains two regions that are likely to contribute to
nuclear localization and/or nuclear retention. These regions are
comprised of the CDK-cyclin binding region within the N terminus and
several putative NLSs within the C terminus of Xic1 (30). The residues
of mammalian p27Kip1 critical for CDK-cyclin binding have
been identified by mutagenesis and by examination of the co-crystal
structure of human CDK2 and cyclin A with a fragment of
p27Kip1 (14, 16, 44). Because Xic1 exhibits significant
sequence homology with human p27Kip1 in the CDK-cyclin
binding domain, we identified and mutated residues in Xic1 that we
predicted would disrupt binding of Xic1 to CDK2 (Xic1k: F65A and F67A) and to
cyclin E (Xic1c: R33A and L35A)
(Fig. 2A). These Xic1 mutants
were in vitro translated with [35S]methionine,
added to LSS, and immunoprecipitated with either CDK2 or cyclin E
antibodies. The results indicate that both the WT Xic1 and
Xic1c mutant readily form trimeric complexes with
endogenous CDK2-cyclin E (Fig. 2B, lanes 1-4),
whereas the Xic1k mutant is decreased in its ability to
form a trimeric complex with CDK2-cyclin E (Fig. 2B,
lanes 1-4). Interestingly, Xic1k protein bands
in association with endogenous CDK2-cyclin E were shifted to slower
mobility species that could be converted to a faster migrating species
of Xic1 by treatment with calf intestinal phosphatase (Fig.
2B, lanes 5-7). These species may represent Xic1k phosphorylated by a catalytic CDK2-cyclin E that is
only loosely associated with the Xic1k mutant or by an
alternate kinase present in interphase extracts. When both c and
k mutations are combined in Xic1ck
There are three regions in the C terminus of Xic1 that are rich in
arginine and lysine residues (165RRKR, 180KRKK,
and 205PRKK), indicating they may be important for nuclear
localization of Xic1 through an importin pathway (45). To address how
these sequences influence the nuclear localization of Xic1, the
residues in each of these three putative NLSs were mutated, resulting
in the following mutants: Xic1-NLS1, R166A, K167A, and R168A; Xic1-NLS2 (ARAA), K180A, K182A, and K183A; Xic1-NLS2 (RRRR), K180R, K182R, and
K183R; and Xic1-NLS3, R206A, K207A, and K208A (Fig. 2A).
Xic1 mutants were also generated that combined all three individual NLS
mutations (Xic1-NLS1/2/3) as well as both CDK2-cyclin E binding mutations and all NLS mutations (Xic1ck Nuclear Localization of Xic1 Is Mediated by CDK2-Cyclin E Binding
and C-terminal NLS Sites--
To examine how either CDK2-cyclin E
binding to Xic1 or the putative NLSs contribute to the nuclear
localization of Xic1, we tested the Xic1 mutants described in Fig. 2
for their abilities to translocate to the nucleus. Xic1 WT and mutants
were [35S]methionine-labeled and individually incubated
in LSS containing preformed nuclei and supplemented with
methyl-ubiquitin. The addition of methyl-ubiquitin allows
mono-ubiquitination of lysine residues while preventing
polyubiquitination (37). Because polyubiquitination is largely blocked,
degradation by the proteasome is inhibited and mono-ubiquitinated
substrate species accumulate. The nuclei were separated from the
cytoplasm, and the amount of Xic1 localized to nuclei was determined as
a percentage of the total Xic1 in the nucleus and cytosol. The Xic1-WT
and mutants Xic1-NLS1, Xic1-NLS2 (ARAA), Xic1-NLS2 (RRRR), and
Xic1-NLS3 all translocated efficiently to the nucleus (Fig.
3, lanes 2-6). This indicates
that no single putative NLS site alone functions as a prototypic NLS.
By contrast, both the Xic1-NLS1/2/3 and Xic1ck A Stable Trimeric Complex between Xic1 and CDK2-Cyclin E Is Not
Required for Xic1 Ubiquitination or Degradation--
Previous studies
have shown that Xic1 degradation is sensitive to the purine analog
6-dimethylaminopurine, an inhibitor of CDKs and DNA replication
initiation events (32, 46, 47). This suggests that Xic1 degradation is
dependent on CDK activity or initiation events but does not address
whether Xic1 must form a stable trimeric complex with CDK2-cyclin E for
its ubiquitination and degradation. Studies show that mutations that
disrupt the binding of mammalian p27Kip1 to CDK2-cyclin E
eliminate ubiquitination of p27Kip1, indicating that a
stable trimeric complex between CDK2-cyclin E-p27Kip1 is
necessary for ubiquitination and degradation (14, 16). We studied the
ubiquitination and degradation of Xic1 point mutants defective for
binding to CDK2-cyclin E (Fig. 2). We used a GST-Xic1 fusion protein
for our ubiquitination studies because it facilitated the detection of
the ubiquitinated Xic1 species by allowing Xic1 to be labeled to a
higher specific activity (Xic1 contains 2 methionines and GST-Xic1
contains 11 methionines). GST-Xic1-WT is localized to both the nucleus
and cytosol but is predominantly ubiquitinated in the nucleus, whereas
GST is localized predominantly to the cytosol and is not ubiquitinated
(Fig. 4A). Equivalent amounts of [35S]methionine-labeled GST-Xic1-WT or
GST-Xic1ck
To fully understand how Xic1 binding to CDK2-cyclin E modulates its
turnover, the c, k, and ck Nuclear Localization of Xic1 Is Necessary but Not Sufficient for
Degradation--
We next asked how the C-terminal NLS sites contribute
to the degradation of Xic1 by examining the turnover of the Xic1 NLS mutants. To accomplish this, Xic1 single, double, and triple NLS mutants were tested for Xic1 degradation, and the results are shown in
Fig. 5A. The data from two
separate experiments were averaged and are shown graphically in Fig.
5B. The results show that greater than 85% of Xic1 WT is
degraded after 3 h (Fig. 5, A, lanes 1-3,
and B). Xic1-NLS1, Xic1-NLS3, and Xic1-NLS1/3 are also
degraded efficiently to levels comparable with the Xic1 WT (Fig. 5,
A, lanes 4-6, 10-12, and
16-18, and B). Interestingly, Xic1-NLS2 (ARAA)
exhibits a striking defect in degradation with only ~15% of the
Xic1-NLS2 (ARAA) degraded after 3 h. This indicates that even
though Xic1-NLS2 is transported to the nucleus as efficiently as WT
Xic1 (Fig. 3), it is not appreciably degraded (Fig. 5, A, lanes 7-9, and B). As expected, the degradation
of the Xic1ck Xic1-NLS2 Mutation Does Not Inhibit DNA Replication in the Xic1
Degradation Assay--
One trivial explanation for the stability of
Xic1-NLS2 (ARAA) is that this nondegradable mutant inhibits the events
of DNA replication initiation in the degradation assay, events that
appear to be required for Xic1 degradation (32). To test this
possibility, we measured DNA replication and Xic1 degradation in
parallel, using the same preparation of LSS and the conditions we use
for Xic1 degradation. Xic1-WT and Xic1-NLS2 (ARAA) were
[35S]methionine-labeled and were added to Xic1
degradation and DNA replication assays. Under the degradation assay
conditions, we found that the added amount of Xic1-WT and Xic1-NLS2
(ARAA) did not appreciably inhibit DNA replication compared with a
control sample containing unprogrammed rabbit reticulocyte lysate (Fig. 6). In parallel degradation assays,
Xic1-WT was degraded efficiently as expected, whereas Xic1-NLS2 (ARAA)
was stable (data not shown). Therefore, the degradation defect of
Xic1-NLS2 (ARAA) cannot be attributed to a block in DNA replication
initiation by Xic1-NLS2 (ARAA) in the degradation assay.
Basic Lysine Residues within NLS2 Are Essential for Xic1
Degradation, but Do Not Comprise the Only Critical Ubiquitination Sites
of Xic1--
It is possible that the region of Xic1-NLS2 (ARAA) may
comprise the only critical lysine residues that are ubiquitinated in Xic1 or, alternatively, that this region of Xic1 is important for
binding a cofactor protein required for its degradation. This putative
cofactor might be a component of the ubiquitin conjugation machinery or
a regulator required for post-translational modification of Xic1 prior
to ubiquitination. To test these possibilities, we engineered an
additional Xic1 mutant to carry conservative arginine mutations at the
NLS2 residues (K180R, K182R, and K183R). This mutant would prevent
ubiquitination at NLS2 residues but should not disrupt the interaction
of Xic1 with a regulator that is dependent upon the basic residues of
NLS2 (Fig. 2A) for binding. The Xic1-NLS2 (RRRR) mutant
localizes to the nucleus as efficiently as Xic1-WT (Fig. 3, lane
5). Equivalent amounts of [35S]methionine-labeled
GST-Xic1 proteins were incubated with LSS in the presence of nuclei and
methyl-ubiquitin. The results show that GST-Xic1-WT, GST-Xic1-NLS1,
GST-Xic1-NLS3, and GST-Xic1-NLS2 (RRRR) are all efficiently
mono-ubiquitinated on ~3-5 independent sites in the nucleus (Fig.
7, lanes 1, 2,
4, and 5). In contrast, GST-Xic1-NLS2 (ARAA) and
GST-Xic1-NLS1/2/3 are not appreciably ubiquitinated (Fig. 7,
lanes 3 and 6). None of the GST-Xic1 derivatives was appreciably ubiquitinated in the cytosol (Fig. 7, lanes
7-12). There was little difference in the ubiquitination pattern
of a specific NLS mutant when tested as a GST or a non-GST fusion
protein (data not shown). These results indicate that although
Xic1-NLS2 (ARAA) is not efficiently ubiquitinated, when the NLS2 lysine residues are converted to arginine residues, NLS2 (RRRR) is now efficiently ubiquitinated. NLS2 (RRRR), like NLS2 (ARAA) cannot be
ubiquitinated at lysine residues 180, 182, or 183, indicating that NLS2
(RRRR) must be ubiquitinated at alternative lysine residues. Because
NLS2 (RRRR) can be ubiquitinated, this demonstrates that lysines 180, 182, and 183 are not the only critical lysine residues that may be
ubiquitinated in Xic1.
To confirm and extend the ubiquitination results in Fig. 7, the
GST-Xic1 fusion proteins were tested for degradation. The results
indicate that Xic1-WT and GST-Xic1-WT are efficiently degraded,
although GST alone is not (Fig.
8A). GST-Xic1-NLS3, GST-Xic1-NLS1, and GST-Xic1-NLS2 (RRRR) are all degraded, whereas GST-Xic1-NLS2 (ARAA) and GST-Xic1-NLS1/2/3 are not degraded (Fig. 8A). Similarly, as non-GST fusion proteins, Xic1-NLS2 (RRRR)
is degraded, whereas Xic1-NLS2 (ARAA) is not appreciably degraded (Fig.
8B). Taken together, these results suggest that the basic lysine residues of Xic1 amino acids 180-183 are essential for the
efficient ubiquitination and degradation of Xic1. We postulate that
this region of Xic1 is critical for binding a mediator of Xic1
degradation.
Regulation of Xic1 Nuclear Transport and Additional Events Required
for Xic1 Nuclear Degradation--
Our work indicates that Xic1
degradation is dependent upon its transport to the nucleus, a
requirement mediated through CDK2-cyclin E binding and three C-terminal
NLS sites of Xic1. It is unclear from our studies whether Xic1 binding
to CDK2-cyclin E influences its active nuclear transport, nuclear
retention, or both. Our results also suggest that the binding of Xic1
to CDK2-cyclin E and importin may be sufficient for its efficient
nuclear localization. However, because we do not demonstrate a direct
interaction between the C terminus of Xic1 and importin, it is possible
that an alternative and as yet unidentified protein binds to the Xic1 C
terminus and mediates its nuclear import. Although nuclear transport is
necessary, it is not sufficient for Xic1 degradation. There is at least
one other nuclear event required for Xic1 degradation that is defective in the Xic1 mutant NLS2 (K180A, K182A, K183A). The ubiquitination and
degradation defects of Xic1-NLS2 (ARAA) are not attributable to
defective nuclear import or defective CDK2-cyclin E binding. Conservative arginine substitutions at lysine residues 180, 182, and
183 do not prevent ubiquitination of Xic1, indicating that these
residues do not comprise the only possible ubiquitination sites of
Xic1. However, because the degradation of Xic1-NLS2 (RRRR) is reduced
~2-fold compared with the Xic1-WT, this may indicate that the lysine
residues at amino acids 180-183 are important sites of Xic1
ubiquitination and that alternative lysine residues are not used quite
as efficiently. Nonetheless, despite the ubiquitination of alternative
sites in NLS2 (RRRR), these sites are not ubiquitinated in NLS2 (ARAA).
This indicates that the basic residues of Xic1 (amino acids 180-183)
more importantly define a region that is important for binding a
protein required for efficient nuclear ubiquitination and degradation.
This putative regulator is likely to be either a component of the
ubiquitination machinery such as an F-box protein or alternatively, a
regulator that must modify Xic1 prior to ubiquitination such as a
kinase. Substrates of SCF are thought to require specific
phosphorylation to bind their designated F-box proteins (reviewed in
Ref. 48). The binding of Xic1 to an F-box protein is predicted because
it has been shown that Xic1 is degraded in a
Cdc34-dependent manner, implying that it will be degraded
in a SCF- and F-box-dependent manner (32). However, to
date, a requirement for SCF has not been demonstrated, nor has a Xic1
F-box protein been identified and shown to be required for Xic1
degradation. Xic1 has also been shown to bind to PCNA within
unspecified C-terminal residues between Xic1 amino acids 97 to 210 (30). Based on weak homology to the PCNA binding regions of
p21Cip1 and p57Kip2, we predict that Xic1 may
bind to PCNA through amino acids flanking the NLS2 sequence (49, 50).
How NLS2 may influence the binding of Xic1 to PCNA is currently under investigation.
Binding of Xic1 to CDK2-Cyclin E Is Dispensable for Nuclear
Degradation--
We find that although Xic1 binding to CDK2-cyclin E
is required for efficient nuclear transport or nuclear retention of
Xic1, it is not required for Xic1 degradation. The Xic1ck Proteolysis of Vertebrate CKIs: Similarities and
Differences--
Although a stable trimeric complex of CDK2-cyclin E
with mammalian p27Kip1 has been shown to be required for
its ubiquitination and degradation, our results indicate that a stable
complex of Xic1 with CDK2-cyclin E is not essential for the degradation
of Xic1 (14, 16). Additionally, the degradation of p27Kip1
exhibits a strict dependence on CDK2-cyclin E phosphorylation of
Thr187, whereas phosphorylation of Thr204 (the
residue equivalent to Thr187 of p27Kip1) is not
required for Xic1 degradation (14, 16, 20). Two individual mutations of
Xic1 at this residue, T204A and T204E, are both degraded in a
nuclei-dependent manner with the exact kinetics as the
Xic1-WT.2 This is surprising
because Thr187 of p27Kip1 and
Thr204 of Xic1 are located within highly conserved
C-terminal sequences termed the QT domain. Moreover, although both Xic1
and Kix1 are efficiently degraded in a nuclei-dependent
manner in Xenopus interphase extracts, neither human nor
mouse p27Kip1 are degraded in Xenopus extracts
with or without nuclei.3
Considering the high homology of the vertebrate CKIs, why then does
Xic1 appear to be degraded so differently compared with
p27Kip1? The differences observed to date may be explained
by the structural differences that exist between the vertebrate CKI
molecules, their possible functional differences or experimental
differences. It is likely that Xic1 possesses additional functions
compared with p27Kip1 as suggested by the homology of the
C-terminal Xic1 and p21Cip1 sequences, as well as the
observed binding of Xic1 to PCNA (30, 31). The unique features of Xic1
may result in altered regulation by protein turnover for the
Xenopus CKI compared with the mammalian CKI. Additionally,
Xic1 may be degraded along more than one pathway as suggested by
Swanson et al. (33), one dependent upon and one independent
of phosphorylation by CDK2-cyclin E. These alternate degradation
pathways may be dictated by the binding status of Xic1 to CDK2-cyclin
E. p21Cip1 has been shown to be degraded by the proteasome
only in the nucleus, suggesting that the degradation of Xic1 may
resemble the degradation of p21Cip1, although
p21Cip1 degradation does not require ubiquitination (34).
With regard to the methodology used in mammalian versus
Xenopus degradation experiments, one chief difference exists
that may potentially account for some discrepancies observed between
p27Kip1 and Xic1 degradation. The Xenopus
extract measures S phase events that occur within early embryonic cell
cycles, whereas mammalian extracts measure S phase events that occur
largely in differentiated somatic cells. Further studies will be
required to clarify the differences observed in vertebrate CKI
degradation and to fully understand the mechanisms regulating the
events of DNA replication initiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerol phosphate, pH 7.4, 20 mM EGTA, and 15 mM MgCl2)
containing 0.1% Nonidet P-40, 10 µg/ml leupeptin, chymostatin,
pepstatin, and 1 mM phenylmethylsulfonyl fluoride, followed
by three washes with HBS (10 mM HEPES, pH 7.4, 150 mM NaCl) at 23 °C (31). The samples were then subjected to SDS-PAGE and quantitation by PhosphorImager analysis using ImageQuant software (Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
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Fig. 1.
Xic1 is ubiquitinated and degraded in the
nucleus. A, [35S]methionine-labeled Xic1
was incubated in LSS with demembranated sperm nuclei and then separated
into nuclear (NUC) and cytosolic (CYT) fractions
by centrifugation using a chromatin spin down assay. Results were
resolved by SDS-PAGE and analyzed by PhosphorImager analysis. Molecular
mass markers (lane M) are indicated in kDa.
Arrows denote [35S]methionine-labeled Xic1
(XIC1), mono-ubiquitinated Xic1 (MONO-Ub),
di-ubiquitinated Xic1 (DI-Ub), and polyubiquitinated Xic1
(POLY-Ub). B, left panel,
[35S]methionine-labeled Xic1 was added to LSS and
demembranated sperm chromatin, the nuclei and cytosol were separated by
nuclei spin down assay (SPIN), and the nuclei
(NUC) and cytosol (CYT) were re-incubated in
fresh LSS for 0 or 3 h. At 0 or 3 h, the samples were further
separated into nuclear (NUCLEI, lanes 1 and
2) and cytosolic (CYT, lane 3)
fractions. The samples were subjected to SDS-PAGE and autoradiography.
The Xic1 band is indicated by *, and the mono-ubiquitinated Xic1 is
represented by **. Right panel, quantitation of results by
PhosphorImager analysis where the 0 h time point is normalized to
100% of the Xic1 remaining at the 0 h time point. Black
bars represent the nuclear fraction (NUC), NUC
CYT represents the cytosol of the second nuclei incubation
(lane 8), and gray bars represent the cytosolic
fraction (CYT, lanes 9 and 10).
(R33A, L35A, F65A, and
F67A), all detectable binding to CDK2 and cyclin E is abolished (Fig.
2B, lanes 1-4). However, it remains possible
that a small amount of the Xic1ck
mutant remains very
loosely bound to CDK2-cyclin E in the extract and is not stable to
coimmunoprecipitation.
View larger version (40K):
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Fig. 2.
Xic1 contains two potential nuclear
transportation regulatory domains. A, Top
panel, schematic of Xic1 showing the CDK binding region (CDK
BINDING) and putative NLS sites (NLS). Bottom
panel, schematic of Xic1 indicating the sites of point mutation.
The numbers designate specific amino acid residues of Xic1 ().
The Xic1 mutations are as follows: c, 33RNL to ANA; k,
65FDF to ADA; ck , 33RNL to ANA and
65FDF to ADA; NLS1, 165RRKR to RAAA; NLS2,
180KRKK to ARAA or RRRR; NLS3, 205PRKK to PAAA.
B, [35S]methionine-labeled Xic1-WT and mutants
were added to LSS and immunoprecipitated with normal rabbit serum
(NRS), anti-CDK2 antibody (
-CDK2),
or anti-cyclin E antibody (
-CycE). One-tenth
of the total reaction is shown for each sample in lanes 1 and 8 (10% INPUT).
[35S]Methionine-labeled Xic1k was incubated
in LSS (+LSS) without (
) or with (+) calf intestinal
phosphatase (lanes 5-7).
&NLS1/2/3).
These mutants were tested for their ability to bind to endogenous CDK2
and cyclin E in LSS. The results show that Xic1-NLS2 (ARAA) and
Xic1-NLS1/2/3 both bind endogenous CDK2 and cyclin E efficiently,
whereas, as expected, the Xic1-ck-&NLS1/2/3 mutant is totally defective
for CDK2-cyclin E binding (Fig. 2B, lanes
8-11).
mutants
were reduced 2.5-3-fold in nuclear transport proficiency compared with
WT Xic1 (Fig. 3, lanes 7 and 8). This suggests
that the binding of Xic1 to CDK2-cyclin E and the NLS sequences of Xic1
independently contribute equally to efficient nuclear localization or
retention. Importantly, when both the CDK2-cyclin E binding sites and
NLS sites are mutated together, the ability of Xic1 to localize to the
nucleus is severely reduced to near background levels as determined by
the amount of the cytoplasmic protein GST found in the nuclear fraction
(Fig. 3, lanes 1 and 9). These results suggest
that the amount of Xic1 transported to or retained in the nucleus is
regulated through the interactions of Xic1 with CDK2-cyclin E and
importin.
View larger version (23K):
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Fig. 3.
Nuclear localization of Xic1 is mediated by
binding to CDK2-cyclin E and C-terminal NLSs.
[35S]Methionine-labeled GST, Xic1-WT, and Xic1 mutants
were added to LSS and preformed nuclei. After 45 min, the amount of
protein transported to nuclei was analyzed by SDS-PAGE and quantitated
by PhosphorImager analysis. The percent nuclear transport was
calculated as the amount of protein in the nucleus divided by the sum
of the total amount of protein in the nucleus and cytosol. The
percentage of nuclear transport for the Xic1 WT was set at 100%, and
the other samples were normalized relative to the WT. The
bars represent the mean of two to four experiments with
error bars representing the standard error of the
mean.
were added to LSS and nuclei in the presence
of methyl-ubiquitin to block polyubiquitination and subsequent
degradation and to allow the accumulation of mono-ubiquitinated Xic1
species (37). In the presence of methyl-ubiquitin, both GST-Xic1-WT and
GST-Xic1ck
efficiently accumulate mono-ubiquitinated Xic1
species in the nucleus (Fig. 4B). Based on the molecular
mass of ubiquitin (~8 kDa) and the estimated molecular masses
of the stabilized bands in Fig. 4B, ubiquitination appears
to occur at ~3-5 independent sites on Xic1. The amount of
ubiquitinated GST-Xic1ck
observed in the nucleus was
reduced by ~2-3-fold compared with the GST-Xic1 WT, correlating with
the ~2-3-fold reduction in nuclear transport proficiency observed
for Xic1ck
compared with Xic1-WT (Fig. 3, lanes
2 and 8). Thus, the inability of Xic1ck
to bind CDK2-cyclin E reduces the efficiency of its nuclear transport by ~2-fold but does not prevent the efficient ubiquitination of Xic1ck
once it is localized to the nucleus.
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Fig. 4.
Xic1 binding to CDK2-cyclin E is not required
for Xic1 ubiquitination or degradation. A,
[35S]methionine-labeled GST and GST-Xic1-WT were
incubated in LSS with preformed nuclei and methyl-ubiquitin, after
which the nuclear (NUC) and cytoplasmic (CYT)
fractions were separated by nuclei spin down assay and analyzed by
PhosphorImager. B, [35S]methionine-labeled
GST-Xic1-WT and GST-Xic1ck were incubated in LSS with
preformed nuclei and methyl-ubiquitin. The nuclear fractions were
recovered by nuclei spin down assay and analyzed by PhosphorImager.
Molecular mass markers are indicated in kDa (M).
Arrows denote [35S]methionine-labeled GST-Xic1
(GST-Xic1) and GST-Xic1 mono-ubiquitinated at one site
(1-MeUb), two different sites (2-MeUb), three
different sites (3-MeUb), or four or five different sites
(*). C, [35S]methionine-labeled Xic1 was
incubated in LSS with (+) or without (
) nuclei for 0, 1, and 3 h
and was resolved by SDS-PAGE. The results of two separate experiments
were quantitated, and the mean percentage of Xic1 remaining is shown
where the 0 h time point of Xic1 was normalized to 100% of Xic1
remaining for each sample.
mutants were analyzed for degradation in
LSS containing nuclei. Both the Xic1-WT and Xic1c, which
form stable trimeric complexes with CDK2-cyclin E, are efficiently
degraded (Fig. 4C, lanes 1-8). The
Xic1k mutant, which was reduced for CDK2-cyclin E binding,
is still degraded effectively (Fig. 4C, lanes
9-12). The Xic1ck
mutant, which was totally
defective for CDK2-cyclin E binding, is also degraded with only
slightly reduced efficiency (Fig. 4C, lanes
13-16). The slight reduction in degradation efficiency of Xic1ck
can be attributed to its reduced nuclear transport
proficiency. Degradation of all the mutants required the presence of
nuclei (Fig. 4C). These results suggest that binding of Xic1
to CDK2-cyclin E or the formation of a stable trimeric complex is
dispensable for nuclei-dependent degradation of Xic1.
&NLS1/2/3 mutant that is not efficiently
transported to the nucleus as well as other NLS mutant combinations
containing NLS2 (ARAA) are not degraded (data not shown and Fig. 5,
A, lanes 13-24, and B). The
destruction of the degradable NLS mutants occurred in a
nuclei-dependent manner (data not shown). These results
indicate that Xic1 degradation involves at least two functional
requirements, one being nuclear transport and the other involving at
least one nuclear event that is defective in the Xic1-NLS2 mutant.
Therefore, we conclude that nuclear transport is necessary but not
sufficient for Xic1 degradation.
View larger version (40K):
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Fig. 5.
Nuclear localization is necessary, but not
sufficient for Xic1 degradation. A,
[35S]methionine-labeled Xic1 was incubated in LSS with
nuclei for 0, 1, and 3 h and was resolved by SDS-PAGE.
B, the results of two separate experiments were quantitated
by PhosphorImager analysis, and the mean percentage of Xic1 remaining
is shown where the 0 h time point of Xic1 was normalized to 100%
of Xic1 remaining for each sample.
View larger version (17K):
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Fig. 6.
Xic1-NLS2 (ARAA) does not inhibit DNA
replication under conditions used for Xic1 degradation.
Semi-conservative DNA replication was measured in LSS using
demembranated sperm chromatin as a template and
[ -32P]dATP. The replication assay was conducted under
degradation assay conditions with either unprogrammed rabbit
reticulocyte lysate, [35S]methionine-labeled Xic1-WT, or
Xic1-NLS2 (ARAA). The results are the mean of three experiments, and
the error was calculated as the standard error of the mean. DNA
replication for the control rabbit reticulocyte lysate sample was
normalized to 100%.
View larger version (59K):
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Fig. 7.
Basic lysine residues 180, 182, and 183 of
NLS2 are not the only critical ubiquitination sites of Xic1.
[35S]Methionine-labeled GST-Xic1 proteins were incubated
in LSS with nuclei and methyl-ubiquitin. The nuclear
(NUCLEI) and cytoplasmic (CYTOSOL) fractions were
then separated and analyzed by PhosphorImager. Molecular mass markers
are indicated in kDa (M). Arrows denote
[35S]methionine-labeled GST-Xic1 (GST-Xic1)
and GST-Xic1 mono-ubiquitinated at one site (1-MeUb), two
different sites (2-MeUb), three different sites
(3-MeUb), or four or five different sites (*).
View larger version (51K):
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Fig. 8.
A Xic1 mutant with conservative arginine
substitutions at NLS2 (KRKK to RRRR) is degraded, while NLS2 (KRKK to
ARAA) is not degraded. A,
[35S]methionine-labeled proteins were added to LSS with
(+) or without ( ) nuclei and time points were analyzed for
degradation after 0, 1, and 3 h. The arrow indicates
the full-length product for GST-Xic1. The results from two separate
experiments were quantitated, and the averages of the results are shown
as the percentage of Xic1 remaining (% Xic1 Remaining)
where the 0 h time point was normalized to 100% of the Xic1
remaining for each sample. B,
[35S]methionine-labeled Xic1-WT and Xic1-NLS2 (RRRR or
ARAA) mutants were added to LSS with (+) or without (
) nuclei and
incubated for 0, 1, and 3 h. The results from two separate
experiments were quantitated and the averages of the results are shown
as the percentage of the Xic1 remaining (% Xic1 remaining).
The 0 h time point was normalized to 100% of the Xic1 remaining
for each sample.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant that retains no measurable binding to CDK2 or cyclin E, is still
ubiquitinated and degraded efficiently in the nucleus. This finding
also implies that the phosphorylation of Xic1 by CDK2-cyclin E may not
be required for its nuclear degradation. Our results conflict somewhat
with a recent study suggesting that an association with CDK2-cyclin E
is required for Xic1 destruction (33). This study demonstrates that
immunodepletion of cyclin E inhibits Xic1 destruction (33). Xic1
degradation has previously been shown to be sensitive to the CDK
inhibitor, 6-dimethylaminopurine, which inhibits DNA replication
initiation (32, 46, 47). In light of our current results, it is likely
that Xic1 degradation is dependent on initiation events mediated by
CDK2-cyclin E but is not directly dependent on CDK2-cyclin E binding or
phosphorylation. In vivo, it is unclear whether Xic1 is a
physiological substrate of CDK2-cyclin E or how phosphorylation of Xic1
by CDK2-cyclin E may regulate its function.
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ACKNOWLEDGEMENTS |
---|
We are grateful to M. W. Kirschner and T. G. Boyer for their continued support; T. G. Boyer, J. Roberts, and all past and present members of the laboratory for helpful discussions; K. Block for constructing pCS2+/GST and pCS2+/GST-Xic1-WT; P. K. Jackson and M. W. Kirschner for anti-CDK2 and anti-cyclin E antibodies; S. Hollenberg for the 9.5/10.5 days postcoital mouse embryo library; W. Dunphy for the Kix1 plasmid; J. Roberts and B. Amati for human and mouse p27Kip1 plasmids; F. Aparicio-Ting and C. Herrera for technical assistance; H. Yan, W. M. Michael, A. Philpott, T. Civco, and R. Davis for advice on sperm chromatin preparation and frog care; and T. G. Boyer, A. Tomkinson, and P. Sung for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by a Leukemia and Lymphoma Society Special Fellowship, a Howard Hughes Medical Institute New Faculty Start Up Award, an UTHSCSA Institutional Research Grant, and an award from the UTHSCSA Competitive Research Enhancement Fund for New Faculty (to P. R. Y.).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.
To whom correspondence should be addressed. E-mail:
yew@uthscsa.edu.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M008896200
2 K. Block and P. R. Yew, unpublished result.
3 P. R. Yew and M. W. Kirschner, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; LSS, low speed supernatant; NLS, nuclear localization sequence; PAGE, polyacrylamide gel electrophoresis; WT, wild type; GST, glutathione S-transferase; PCNA, proliferating cell nuclear antigen.
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