From the Institute of Molecular Biology and the
§ Department of Biochemistry, The University of Hong
Kong, Hong Kong, China
Received for publication, December 17, 2002, and in revised form, January 20, 2003
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ABSTRACT |
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The anaphase-promoting complex activated by CDC20
and CDH1 is a major ubiquitination system that controls the destruction of cell cycle regulators. Exactly how ubiquitination is
regulated in time and space is incompletely understood. Here we report
on the cell cycle-dependent localization of CDH1 and its
regulation by phosphorylation. CDH1 localizes dynamically to the
nucleus during interphase and to the centrosome during metaphase
and anaphase. The nuclear accumulation of CDH1 correlates with a
reduction in the steady-state amount of cyclin A, but not of cyclin E. A nuclear localization signal conserved in various species was
identified in CDH1, and it sufficiently targets green fluorescent
protein to the nucleus. Interestingly, a CDH1-4D mutant mimicking the hyperphosphorylated form was constitutively found in the cytoplasm. In
further support of the notion that phosphorylation inhibits nuclear
import, the nuclear localization signal of CDH1 with two phospho-accepting serine/threonine residues changed into aspartates was
unable to drive heterologous protein into the nucleus. On the other
hand, abolition of the cyclin-binding ability of CDH1 has no influence
on its nuclear localization. Taken together, our findings
document the phosphorylation-dependent localization of CDH1
in vertebrate cells.
The timely and orderly progression of eukaryotic cell division
cycle is precisely controlled through ubiquitination-induced proteolysis. The anaphase-promoting complex
(APC)1 functions as a major
ubiquitin ligase in the cell, and it governs anaphase onset, mitotic
exit, and G1 events (1-4). APC is a large complex of
multiple subunits. It catalyzes the formation of polyubiquitin chains
on cyclins and other cell cycle regulators and thereby targets them for
degradation by proteosome. The components of APC are highly conserved
in organisms ranging from yeast to humans. The rise and fall of APC
activity is stringently regulated in a cell cycle-dependent
manner. It plateaus at the transition from metaphase to anaphase,
remains high throughout G1, and drops in S/G2
until early mitosis (5). One important mechanism for the regulation of
APC is through phosphorylation of its subunits (6-9). In addition,
CDC20 and CDH1 can act as activators and specificity factors of APC
(10-15).
CDC20 and CDH1 are WD40 repeat proteins that associate transiently with
and activate APC in a substrate-specific fashion. Many substrate
proteins targeted by the CDC20- and CDH1-activated APC share a sequence
motif known as destruction box (5, 16). Another motif termed the KEN
box is recognized by CDH1-activated APC and has been found in
substrates including CDC20, NEK2, HSL1, mitotic cyclins, and securin
(17-20). In addition, a distinct recognition domain called the A box
has been shown to be required for CDH1-dependent ubiquitination and degradation of the Aurora-A kinase (21). Exactly how
CDC20 and CDH1 activate APC remains to be fully understood. Existing
evidence suggests that they mediate the temporal and spatial activation
of APC through direct interaction with different sets of substrate
proteins (19, 22, 23).
CDC20 and CDH1 are tightly regulated during the cell cycle. The
steady-state amounts of CDC20 and CDH1 transcripts peak in G2-M and fall dramatically at the entry into G1
(5, 8, 24-26). A similar expression pattern has also been described
for CDC20 protein (5, 24). In contrast, the abundance of CDH1 protein was shown to be constant in two earlier studies (5, 25). However,
experiments conducted with a highly specific antibody against CDH1 have
demonstrated the oscillation of CDH1 protein as cells enter and exit
mitosis (8). CDH1 is ubiquitously expressed in differentiated tissues
including postmitotic neurons (27). Interestingly, a recent genetic
screen in Caenorhabditis elegans has revealed a role for
CDH1 in controlling cell proliferation (28). In line with this, a
significant reduction of CDH1 has been documented during the malignant
progression of a B-lymphoma cell line (29).
The phosphorylation of CDC20 and CDH1 has been well documented (22,
30), but it remains unclear as to whether and how phosphorylation of
CDC20 may influence the APC activity (7, 8). In contrast, the
phosphorylation of CDH1 is known to abolish its ability to activate APC
(7, 31-34). CDH1 is phosphorylated by cyclin-dependent
kinases (CDKs) and dephosphorylated by CDC14 phosphatase (32-34). A
coordinated action of CDC20 and CDH1 in time and space is crucial for
the orderly destruction of cell cycle regulators (11, 12, 17, 19,
34).
Emerging evidence suggests that the mitotic checkpoint targets APC
through CDC20 and CDH1 (35-37). In particular, mitotic checkpoint proteins MAD2 and BUBR1 interact with CDC20 and inhibit its
APC-activating activity, thereby inducing a mitotic arrest (38-44). In
addition, a separate arm of the mitotic checkpoint is defined by BUB2,
which targets CDH1 to inhibit cytokinesis and DNA replication (45-48). Interestingly, an MAD2 isoform termed MAD2B or MAD2L2, which is paralogous to MAD2 but does not bind to MAD1, also associates with and
inhibits CDH1 (49, 50).
We have previously identified and characterized human mitotic
checkpoint proteins MAD1 and MAD2 (51-53). Given that CDH1 is a
downstream effector of the mitotic checkpoint, we set out to characterize human CDH1 in cultured cells. In this study, we
demonstrate that human CDH1 localizes to the centrosome and the
nucleus. Moreover, the nuclear import of CDH1 is regulated by
phosphorylation in the vicinity of the nuclear localization signal
(NLS). Our findings implicate a complex cell
cycle-dependent regulation of human CDH1 activity in
temporal and spatial dimensions.
Antibodies--
Polyclonal anti-CDH1 antibody
Mouse monoclonal anti-NuMA (NA08) was purchased from Oncogene Research
Products. Mouse monoclonal anti- Plasmids--
Human CDH1 cDNA with a complete coding region
(GenBankTM AF083810) was assembled from expressed sequence
tags obtained from the American Type Culture Collection. Expression
vector for human CDH1 (pHACDH1) was derived from a previously described
plasmid pHA containing SV40 promoters and enhancers (54). pHA was also used to express the three CDH1 mutants CDH1-4A, CDH-4D, and CDH1-AAA. In CDH1-4A and CDH1-4D, serines 40, 151, 163, and threonine 121 were
substituted with alanines and aspartates, respectively through PCR-based site-directed mutagenesis. CDH1-AAA was constructed by
replacing the Arg-Val-Leu triplet (amino acids 445-447) with three
alanines. Sequences of all cDNAs coding for CDH1 mutants were
determined to verify that they have the desired substitutions and not
any undesired changes.
To express a chimeric green fluorescent protein (GFP) with the CDH1
nuclear localization signal (NLS) at the carboxyl terminus (GFPNLS), we
PCR-amplified a 104-bp DNA fragment coding for amino acids 151-178 of
human CDH1 and subcloned this fragment into plasmid pEGFP-C1 via
restriction sites XhoI and BamHI
(Clontech). The pEGFP-C1 vector was also used to
express GFP fusion proteins GFPCDH1, GFPNLS-2A, and GFPNLS-2D. In
GFPNLS-2A and GFPNLS-2D, serines 151 and 163 near the NLS of CDH1 had
been replaced by alanines or aspartates, respectively. Details for PCR
primers and for plasmid construction are available upon request.
Western Blot Analysis--
Extracts of HeLa cells were
solubilized directly in SDS gel loading buffer (60 mM Tris
base, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.1%
bromphenol blue). Proteins were separated on 12% SDS-PAGE and
transferred onto Immobilon-P membrane (Millipore) using a semidry
blotter (Hoefer). Blots were visualized by chemiluminescence (ECL,
Amersham Biosciences).
Confocal Microscopy--
Laser scanning confocal microscopy was
performed on a Zeiss Axiophot microscope as detailed elsewhere (51,
55). Dual immunofluorescent detection was achieved with primary
antibodies from different species and pre-adsorbed species-specific
secondary antibodies conjugated to different fluorochromes:
Cy5-conjugated goat anti-mouse immunoglobulin G (Zymax) and
fluorescein-conjugated goat anti-rabbit immunoglobulin G (Zymax). GFP
experiments were carried out as previously described (56).
Cell Cycle-dependent Localization of CDH1--
The
spatial regulation of APC activity is an integral part of cell cycle
control. The APC components and substrates, including cyclins and CDKs,
perform their specific functions at restricted subcellular locales (57,
58). To shed light on the spatial control of CDH1 activity, we
investigated the subcellular localization of endogenous CDH1 protein.
We raised two specific antisera against CDH1 (
To assess the dynamics of CDH1 localization during the cell cycle, we
co-stained CDH1 and the cell cycle marker Nuclear Accumulation of CDH1 Correlates with Degradation of Cyclin
A--
To further investigate the localization of CDH1 and the
functional consequence of CDH1 overexpression in cultured cells, we transiently transfected HeLa cells with pHACDH1, an expression plasmid
for hemagglutinin (HA)-tagged CDH1. The transfected cells were stained
with
The nuclear localization of CDH1 predicts that it might mediate the
APC-dependent proteolysis of nuclear regulators of the cell
cycle. Based on this reasoning, we asked how CDH1 might influence APC
activity in the nucleus. We overexpressed HA-CDH1 in HeLa cells and
co-stained for CDH1 and two nuclear cyclins, cyclin A and cyclin E
(Fig. 2B). In this regard, cyclin A is a known substrate of
the CDH1-activated APC (59-61), whereas cyclin E is degraded primarily
through the SCF complex (62, 63), but not through APC.
Consistent with previous studies (57, 64), ambient cyclin A and cyclin
E are predominantly nuclear (Fig. 2B, panels 2 and 5). As expected, CDH1 co-localized with cyclin A and
cyclin E to the nucleus (Fig. 2B, panels 3 and
6). If nuclear CDH1 is functional, it should induce
APC-mediated destruction of cyclin A. In contrast, nuclear CDH1 might
not affect the steady-state level of cyclin E, which is normally
degraded by SCF and not by APC (62, 63). Indeed, the forced
overexpression of CDH1 correlated with the diminution of nuclear cyclin
A (Fig. 2B, panel 2, compare cell with an
arrow with cells without arrows), but not cyclin E (panel 5). Thus, human CDH1 functions as an APC activator
in the nucleus.
Identification of a Nuclear Localization Signal in CDH1--
In
the process of identifying the NLS in CDH1, we noted that a CDH1
protein lacking the amino acid sequences encoded by exons 6 and 7 of
human CDH1 gene localized predominantly to the
cytoplasm.2 This raises the
possibility that exons 6 and 7 of the CDH1 gene could encode
an NLS. Indeed, a closer examination of the amino acid sequences
encoded by exons 6 and 7 identified a 28-residue stretch (amino acids
151-178) rich in arginines and lysines (Fig. 3A, 9 out of 28 residues are
positively charged as highlighted by asterisks). This
putative NLS is highly conserved from yeast to human and it also
contains two conserved CDK phosphorylation sites (serines 151 and 163;
Fig. 3A, highlighted by #).
To determine whether this is a functional NLS, we expressed in HeLa
cells a GFPNLS fusion protein with the putative NLS from CDH1 at the
carboxyl terminus. As a control, the GFP protein overexpressed in HeLa
cells was diffusely found in both the nucleus and the cytoplasm (Fig.
3B, panel 1 and Fig. 3C, panels
1-3; compare transfected cells with an arrow to
untransfected cells without arrows). In contrast, the GFPNLS
protein concentrated into the nucleus (Fig. 3, B
(panel 2) and C (panels 4-6);
co-localization of GFPNLS and the nuclear marker NuMA are in
yellow), suggesting that the NLS from CDH1 sufficiently
drove the heterologous GFP protein into the nuclear compartment. Thus,
amino acids 151-178 of CDH1 harbor a functional NLS.
Nuclear Import of CDH1 Is Regulated by Phosphorylation--
The
phosphorylation of CDH1 by CDKs leads to inhibition of its
APC-activating activity in cells (7, 31-34). In this regard, CDH1
mutants CDH1-4A and CDH-4D with the serine or threonine residues at the
four CDK phosphorylation sites replaced by alanines and aspartates,
respectively, are very useful for the study of CDH1 because they mimic
the constitutive hypo- and hyperphosphorylation states (33, 60).
Considered together with the fact that CDH1 and its substrates such as
cyclin A and cyclin B1 are all in the nucleus, it would be of interest
to see whether phosphorylation of CDH1 might influence its subcellular
localization. Importantly, a more recent study in budding yeast has
suggested that phosphorylation-dependent nuclear export of
CDH1 contributes to efficient inactivation of APC (65). In light of
this new finding, we asked whether phosphorylation of CDH1 might also
regulate its nuclear import or export in cultured human cells.
We expressed HA-tagged CDH1-4A and CDH1-4D proteins in HeLa cells and
assessed their localization by immunofluorescence microscopy with an
Consistent with the previous finding that the expression of CDH1-4A but
not of CDH1-4D in U2-OS cells resulted in the persistent degradation of
cyclin B1 (33), we observed that the accumulation of CDH1-4A correlated
with reduction of cyclin A level in the nucleus (Fig. 4B,
panels 1-3; steady-state amounts of cyclin A declined in
93% of the transfected cells). This effect is more drastic than that
induced by CDH1 wild-type (Fig. 2B, panels 1-3; 68% of the transfected cells had the phenotype). On the contrary, the
increase of CDH1-4D in the cytoplasm did not lead to destruction of
nuclear cyclin A (Fig. 4B, panels 4-6). Our
interpretation to these data is that the cytoplasmic retention of
CDH1-4D is causally linked to the loss of its APC-stimulatory activity.
This is generally in line with the notion that the constitutively
nuclear CDH1-4A is dominantly active whereas constitutively cytoplasmic CDH1-4D is a dominant negative form.
We noted that two of four conserved CDK phosphorylation sites targeted
in the CDH1-4A and CDH1-4D mutants are located in the vicinity of the
NLS of CDH1 (Fig. 3A). Thus, one likely mechanism for the
regulation of CDH1 localization is through phosphorylation on serines
151 and 163 near the positively charged residues in NLS. To test this
hypothesis, we replaced both serines in the GFPNLS with either alanines
or aspartates. Then we separately expressed GFP, GFPNLS as well as the
two GFPNLS-2A and GFPNLS-2D mutants in HeLa cells. The GFP-expressing
cells gave a bright and homogenous fluorescent signal throughout the
cells, suggesting that GFP is evenly distributed in both the cytoplasm
and the nucleus (see Fig. 5A,
panel 1 for one example). Thus the ratio of GFP fluorescence
in the nuclear versus in the cytoplasm was close to 1 (Fig.
5B). Consistent with data shown in Fig. 3B, the
ratio of GFPNLS-specific fluorescent signals in the nuclear
versus in the cytoplasm was greater than 4 (Fig.
5B), suggesting that GFPNLS targeted the GFP, which was
otherwise found in both the nucleus and the cytoplasm, into the
nucleus. Notably, CDH1-4D is constitutively cytoplasmic (Fig. 4,
panels 4-6). As such, GFPNLS-2D should also localize to the
cytoplasm if phosphorylation near the positively charged residues in
the NLS would affect the nuclear localization of CDH1. Indeed,
GFPNLS-2D was sufficiently retained in the nucleus-excluded region of
the cells (see Fig. 5A, panels 3 and 4
for examples). The ratio of GFPNLS-2D-specific fluorescent signals in
the nuclear versus in the cytoplasm is therefore lower than
0.5 (Fig. 5B). In line of this, the nuclear accumulation of
GFPNLS-2A was even more dramatic than the wild-type GFPNLS (Fig. 5,
A, panel 2 and B). These results
consistently support the notion that phosphorylation on serines 151 and
163 adjacent to the positively charged residues in NLS sufficiently
impedes the nuclear import of CDH1.
Influence of Cyclin Binding on Nuclear Localization of
CDH1--
The phosphorylation of CDH1 by cyclin A/CDK2 has an
important regulatory role in orchestrating S-phase progression with
mitosis driven by stabilization of cyclin B1 (33). This functional
interplay between cyclin A/CDK2 and CDH1-activated APC is determined by a conserved cyclin-binding domain in CDH1 (60). Above we have shown
that CDH1 co-localizes with cyclin A to the nucleus (Fig. 2B). In this scenario, one interesting question remains
unanswered as to whether the interaction with cyclin A might influence
the nuclear localization of CDH1.
To address this issue, we employed one CDH1 mutant called CDH1-AAA, in
which the Arg-Val-Leu triplet in the most conserved cyclin A-binding
motif has been substituted with three alanines. We expressed HA-tagged
CDH1-AAA in HeLa cells and co-stained for both CDH1 and cyclin A (Fig.
6). Interestingly, this CDH1-AAA mutant,
which is unable to interact with cyclin A as previously described (60),
retained the ability to localize to the nucleus (Fig. 6, panels
1 and 4, cells with arrows). In this
setting, a significant co-localization of CDH1-AAA and cyclin was
observed (Fig. 6, panels 3 and 6). In contrast to
the nuclear accumulation of wild-type CDH1, which led to
destabilization of cyclin A (Fig. 2B), the abundant CDH1-AAA
in the nucleus did not cause increased degradation of nuclear cyclin A
(Fig. 6, panels 2, 3, 5, and
6). In agreement with previous findings (60), these results
indicate that binding to cyclin A is crucial for CDH1 stimulation of
APC-mediated proteolysis of cyclin A itself. However, the cyclin
binding did not influence the subcellular localization of CDH1.
Here we documented the phosphorylation-dependent
subcellular localization of human APC regulator CDH1. CDH1 localized to
nuclei during interphase and to centrosomes at mitosis (Fig. 1). The nuclear accumulation of CDH1 induced the APC-mediated degradation of
cyclin A, but not cyclin E, in cultured cells (Fig. 2). A functional NLS rich in positively charged residues was identified in CDH1, and it
sufficiently targeted heterologous GFP protein into the nucleus (Fig.
3). Phosphorylation in the vicinity of the NLS led to constitutive
cytoplasmic retention of CDH1 protein (Figs. 4 and 5). However, the
disruption of cyclin-binding motif in CDH1 had no influence on nuclear
targeting (Fig. 6). Our work suggests an additional level of regulation
for APC activity in the spatial dimension.
Nuclear and Centrosomal Functions of CDH1--
The subcellular
localization pattern (Fig. 1) implicates CDH1 as a nuclear and
centrosomal activator of APC. In line with this, the constitutive
expression of CDH1 in the nucleus correlates with increased
destabilization of cyclin A (Fig. 2B). In order to function
as a substrate-specific activator of APC, CDH1 has to co-localize with
APC components, APC substrates as well as regulators of CDH1. The
nuclear localization of APC subunits has been documented (58, 66).
Meanwhile, the nuclear trafficking of CDKs and cyclins, including
cyclin A and cyclin B1, has important roles in cell cycle regulation
(57, 64). In addition, the CDH1 phosphatase CDC14, which is sequestered
in the nucleolus in budding yeast (32), is a nuclear protein in human
cells (67). Thus, the nucleus appears to be one of the major
subcellular compartments in which CDH1 performs its APC-stimulatory functions.
The centrosomal localization of CDH1 during metaphase and anaphase
(Fig. 1) also has important implications in the regulation of APC. The
centrosome is a major microtubule-organizing center that governs
spindle assembly, spindle bipolarity, chromosome segregation, and
mitotic progression (68, 69). It duplicates only once and undergoes
characteristic changes during the cell cycle. As yet the signaling
pathway that regulates centrosome duplication and links the centrosome
cycle to mitotic progression remains to be elucidated. Notably, many of
the substrates, regulators or subunit proteins of the APC localize to
the centrosome. These include cyclin B1 (57), CDC2/CDK1 (70),
CDC16/CDC27 (71), CDC20 (5, 40, 72), EMI1 (73), MAD1/MAD2 (40, 51), BUB2 (47, 48), Aurora-A kinase (21, 74), and NEK2 (75). Among them
CDC20, NEK2, cyclin B, and Aurora-A have been characterized as
substrates of the CDH1-activated APC (17, 21, 76). Results from this
and other studies suggest that the centrosome performs a role as a
relay point for signal integration, amplification, and distribution in
the regulation of APC activation.
Inhibition of CDH1 Nuclear Import by Phosphorylation--
Protein
trafficking between the nucleus and the cytoplasm is fundamentally
important to cell regulation (77). As such, nuclear import and export
are pivotal in orchestrating the activities of the key regulators of
the cell cycle (57). One mechanism for spatial control of cell cycle is
through the retention of particular proteins in the cytoplasm or in the
nucleus, thereby preventing them from physical contact with their
substrates or partners. In another perspective, some proteins, such as
cyclin A and cyclin E, shuttle continuously between the nucleus and the cytoplasm (64). Their apparent steady-state localization as shown by
immunofluorescence microscopy reflects the relative rates of nuclear
import and export.
We showed that CDH1 is apparently a nuclear protein (Figs. 1 and 2) and
that it harbors an NLS (Fig. 3), whose function is regulated by
phosphorylation adjacent to the positively charged amino acid residues
(Figs. 4 and 5). The co-localization of CDH1 with its substrates,
partners and regulators in the nucleus and the centrosome suggests that
it likely functions as a nuclear and centrosomal activator of APC. Our
findings that show that constitutive expression of CDH1 correlate with
the destabilization of nuclear cyclin A (Fig. 2) lend some support to
this notion. However, our work does not exclude the possibility that
CDH1 is constantly shuttling between the nucleus and the cytoplasm. One recent study in budding yeast suggests a dynamic localization of CDH1
in both the nucleus and the cytoplasm. Interestingly, in that study the
phosphorylation of yeast CDH1 has been shown to promote its nuclear
export (65). It remains to be seen whether phosphorylation of human
CDH1 enhances its export from the nucleus. We showed that the CDH1-4D
mutant mimicking the hyperphosphorylated form is constitutively
cytoplasmic in cultured human cells (Fig. 4). These results per
se are also compatible with a role of phosphorylation on the
nuclear export of CDH1. The inhibition of nuclear import or re-import
as shown in our study (Fig. 5) and the facilitation of nuclear export
as reported for yeast CDH1 (65) are not mutually exclusive. While the
phosphorylation near the positively charged residues blocks the
activity of the NLS of CDH1, the phosphorylation of CDH1 on other sites
plausibly mediates the enhancement of nuclear export. It is noteworthy
that the NLS we identified in human CDH1 is highly conserved in other
organisms including budding yeast (Fig. 3). In addition, the CDK
phosphorylation sites in the vicinity of the NLS of CDH1 are also
conserved from yeast to humans (Fig. 3). This raises the possibility
that budding yeast CDH1 might use the same NLS and phosphorylation
sites for nuclear import and for regulation of nuclear import, respectively.
The regulation of nuclear import by phosphorylation in the vicinity of
the NLS is not unique to CDH1, but has been reported for other proteins
including SV40 large T antigen (78) and adenomatous polyposis coli
protein (79). Furthermore, the dual regulation of nuclear import and
export by phosphorylation also represents a common mechanism for
inactivation of nuclear factors such as Pho4, NF-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-CDH1N was
raised in rabbits against a keyhole limpet hemocyanin-conjugated
peptide, which corresponds to the amino-terminal 20 residues (amino
acids 2-21) of human CDH1. Polyclonal anti-CDH1 antibody
-CDH1C was
raised in rabbits against a keyhole limpet hemocyanin-conjugated
synthetic peptide corresponding to amino acid residues 472-493 of
human CDH1.
-CDH1N and
-CDH1C antibodies were purified through
HiTrap N-hydroxysuccinimide-activated Sepharose columns
(Amersham Biosciences) coupled with the immunizing peptides.
-tubulin (B-5-1-2) was from Sigma.
Mouse monoclonal anti-HA (F-7), rabbit polyclonal anti-HA (Y-11),
rabbit polyclonal anti-cyclin A (H-432), and rabbit polyclonal
anti-cyclin E (C-19) antibodies were from Santa Cruz Biotechnology.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-CDH1N and
-CDH1C)
in rabbits and stained HeLa cells for CDH1 using affinity-purified
antibodies. As a first step, we verified the specificity of the
anti-CDH1 antibodies by immunoblotting. An example of this experiment
is shown in Fig. 1A, in which
HeLa cell extracts were immunoblotted with either
-CDH1N or
-CDH1N preincubated with excessive amount of immunizing peptide. A
discrete protein band of 55 kDa reactive to
-CDH1N was observed
(Fig. 1A, lane 1), and this band disappeared if
the antibodies were pre-absorbed with the peptide (lane 2).
Next HeLa cells were stained with either
-CDH1N or pre-absorbed
-CDH1N (Fig. 1B). The endogenous CDH1 in HeLa cells was
predominantly in the nucleus (Fig. 1B, panel 1),
and the peptide blocking experiment corroborated the specificity of
this staining pattern (panel 2).
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Fig. 1.
Subcellular localization of CDH1 protein in
HeLa cells. A, Western blotting. Extracts of HeLa cells
(~12 µg) were resolved by SDS-PAGE. Immunoblotting was performed
using purified -CDH1N serum. The position of CDH1a (55 kDa) is
highlighted. The
-CDH1N antibody was purified through a HiTrap
NHS-activated affinity column (Amersham Biosciences) coupling to the
immunizing peptide. A duplicate blot was separately probed with
-CDH1N preincubated with 3 µg of immunizing peptide (
-CDH1N
w/pep., lane 2). Similar results were obtained using another
antibody
-CDH1C raised against the carboxyl-terminal sequences
shared by CDH1a and CDH1b (data not shown). B, indirect
immunofluorescence microscopy. HeLa cells were fixed and stained with
purified
-CDH1N (panel 1) or with
-CDH1N pre-incubated
with 3 µg of immunizing peptide (
-CDH1N w/pep., panel
2). Bar, 20 µm. C, cell
cycle-dependent localization of CDH1. Asynchronized HeLa
cells were fixed and co-stained with purified rabbit
-CDH1N
(panels 1, 4, and 7) and mouse
anti-
-tubulin (panels 2, 5, and 8).
The CDH1 (red) and
-tubulin (green)
fluorescent signals were overlaid by computer assistance (panels
3, 6, and 9). Co-localizations are shown in
yellow. The same fields are shown in panels 1-3,
4-6, and 7-9. Arrows indicate cells
in metaphase (panels 4-6) or anaphase (panels
7-9). Bar, 20 µm. Similar results were
obtained with another purified anti-CDH1 antibody
-CDH1C (data not
shown).
-tubulin (Fig.
1C). During the interphase, the endogenous CDH1 in HeLa cells localized to the nucleus in a nucleoli-excluded pattern. During
this time CDH1 and
-tubulin resided in distinct subcellular compartments with minimal co-localization (Fig. 1C,
panels 1-3). Notably, as the cell entered metaphase, CDH1
concentrated into the centrosomes (Fig. 1C, panels
4-6). Thus a significant co-localization of CDH1 and
-tubulin
was noted. CDH1 remained in the centrosomes as the cell committed into
anaphase (Fig. 1C, panels 7-9). The subcellular
localizations of human CDH1 are highly reproducible and are based on
two different anti-CDH1 antibodies
-CDH1N and
-CDH1C (data not
shown). The dynamic changes in the localization of CDH1 provide
opportunities for a precise temporal and spatial regulation of APC
activity during the cell cycle.
-CDH1C (Fig. 2A,
panel 1) or anti-HA (
-HA, panel 2) antibodies.
-CDH1C reacted with both native (Fig. 2A, panel
1, cells without arrow) and exogenously introduced
(cell with an arrow) CDH1 protein, while
-HA recognized
the introduced CDH1 only (Fig. 2A, panel 2, cell
with an arrow). In addition, we also examined the
localization of CDH1 protein fused to GFP (GFPCDH1) in transiently
transfected cells (Fig. 2A, panel 3). Notably, a
nuclear localization pattern of CDH1 was consistently seen with
-CDH1C,
-HA, or GFPCDH1. This lends further support to the notion
that human CDH1 is a nuclear protein.
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Fig. 2.
Nuclear accumulation of CDH1 correlates with
cyclin A degradation. A, subcellular localization of
exogenously expressed CDH1 and GFPCDH1 in HeLa cells. Cells were
transfected respectively with expression vectors pHACDH1 (panels
1 and 2) and pEGFPCDH1 (panel 3).
pHACDH1-transfected cells (panels 1 and 2) were
fixed 36 h after transfection and stained with -CDH1C
(panel 1) or
-HA (panel 2) antibodies.
pEGFPCDH1-transfected cells (panel 3) were observed directly
under the confocal microscope 32 h after transfection.
Arrows indicate transfected cells. The patterns shown in all
three panels are representative of at least 72% of 200 transfected
cells. Bar, 20 µm. B, CDH1 expression
correlates with degradation of cyclin A, but not cyclin E. HeLa cells
were transfected with pHACDH1. Cells were co-stained with mouse
-HA
(panels 1 and 4) and rabbit
-cyclin A
(panel 2) or
-cyclin E (panel 5). In
panels 3 and 6, the CDH1 (green,
probed with
-HA) and cyclin A/cyclin E (red) fluorescent
signals were overlaid by computer assistance. Co-localizations are in
yellow. The same fields are shown in panels 1-3
and 4-6. Arrows indicate transfected cells.
Bar, 20 µm.
View larger version (28K):
[in a new window]
Fig. 3.
Identification of a functional NLS in human
CDH1. A, amino acid sequence alignment of the putative
NLS in CDH1 proteins from various species. Positively charged residues
(arginine and lysine) are indicated by asterisks (*), and CDK
phosphorylation sites are highlighted by #. A dash ( ) denotes an
amino acid residue identical to that in human CDH1.
yeast-f., fission yeast; yeast-b., budding yeast.
B, NLS of human CDH1 sufficiently targets heterologous GFP
protein to the nucleus. HeLa cells were transiently transfected with
either an empty vector pEGFP-C1 (panel 1) or pEGFPNLS
(panel 2). Unfixed cells were monitored directly under a
confocal fluorescence microscope 32 h after transfection. Also
shown are the same fields of the cells under the light microscope
(panels 1' and 2'). Transfected cells are
highlighted by arrows. The patterns shown in panels
1 and 2 represent 85 and 68% of 200 transfected cells.
The relative intensities of the fluorescent signals in the nucleus
versus in the cytoplasm of 200 transfected cells expressing
GFP and GFPNLS (N/C ratio) were 1.2 ± 0.20 and 4.18 ± 1.27, respectively. Bar, 20 µm. C, co-staining for
GFP/GFPNLS and the nuclear marker NuMA. HeLa cells were transfected as
in B, fixed 36 h after transfection, monitored for
GFP/GFPNLS fluorescence (panels 1 and 4) and
stained with mouse anti-NuMA to reveal the nuclear morphology
(panels 2 and 5, Ref. 51). In panels 3 and 6, the GFP/GFPNLS (green) and NuMA
(red) fluorescent signals were overlaid by computer assistance. Co-localizations are shown in yellow. The
same fields are shown in panels 1-3 and 4-6.
Arrows indicate transfected cells. Bar, 20 µm.
Partial co-localization of GFP and NuMA (panel 3)
demonstrates that GFP resided in both the nucleus and the cytoplasm. By
contrast, GFPNLS and NuMA co-localized substantially to the nucleus
(panel 6).
-HA antibody. Notably, CDH1-4A mimicking the hypophosphorylated form
was almost constitutively nuclear as shown in its precise co-localization with the nuclear marker NuMA (see Fig.
4A, panels 1-3 for
an example representative of 79% of the transfected cells). In sharp
contrast, the majority of CDH1-4D protein, which mimics the
hyperphosphorylated form, was found primarily in the cytoplasm, and its
staining pattern overlaps minimally with that of NuMA (see Fig.
4A, panels 4-6 for an example representative of
74% of the transfected cells). Thus, phosphorylation of CDH1
correlates with cytoplasmic retention of the protein. Plausibly
cytoplasmic sequestration of CDH1 could be a net result caused by
enhancement of nuclear export and/or blockade of nuclear import.
View larger version (46K):
[in a new window]
Fig. 4.
Subcellular localizations and activity of
CDH1-4A and CDH1-4D mutants. A, subcellular
localizations. HeLa cells were transfected respectively with expression
vectors pHACDH1-4A (panels 1-3) and pHACDH1-4D
(panels 4-6). Cells were fixed 36 h after transfection
and co-stained with rabbit -HA (panels 1 and
4) and mouse
-NuMA (panels 2 and 5)
antibodies. In panels 3 and 6, the CDH1
(green, probed with
-HA) and NuMA (red)
fluorescent signals were overlaid by computer assistance.
Co-localizations are in yellow. The same fields are shown in
panels 1-3 and 4-6. Arrows indicate
transfected cells. The patterns shown in panels 1 and
4 represent 79 and 74%, respectively, of 200 transfected
cells. Bar, 20 µm. B, correlation with cyclin A
degradation. HeLa cells were transfected as in A and
co-stained for CDH1 and cyclin A as in Fig. 2B. The patterns
shown in panels 2 and 5 represent 93 and 88%,
respectively, of 200 transfected cells. Notably, the overexpression of
either CDH1-4A or CDH1-4D had no influence on the steady-state levels
of nucleus cyclin E (data not shown).
View larger version (51K):
[in a new window]
Fig. 5.
Phosphorylation-dependent
activity of the NLS of human CDH1. A, subcellular
localizations of GFPNLS-2A and GFPNLS-2D proteins. HeLa cells were
transfected separately with pEGFP-C1 (panel 1), pEGFPNLS-2A
(panel 2), or pEGFPNLS-2D (panels 3 and
4). Unfixed cells were monitored directly under a confocal
fluorescence microscope 32 h after transfection. Transfected cells
in the fields are highlighted by arrows. The patterns shown
in panels 1-3 represent, respectively, 85, 90, and 72% of
200 transfected cells. Bar, 20 µm. B, graphic
quantitation of the nuclear versus cytoplasmic fluorescence
in GFPNLS-2A- and GFPNLS-2D-expressing cells. The relative intensities
of the fluorescent signals in the nucleus versus in the
cytoplasm of 200 transfected cells expressing the indicated proteins
(N/C ratio) were calculated. Results represent the average of three
experiments, and error bars indicate the S.E.
View larger version (52K):
[in a new window]
Fig. 6.
Disruption of cyclin-binding motif did not
influence nuclear localization of CDH1. HeLa cells were
transfected with plasmid pHACDH1-AAA. Cells were fixed 36 h after
transfection and co-stained for CDH1 (with -HA, panels 1 and 4) and cyclin A (panels 2 and 5).
In panels 3 and 6, the CDH1 (green,
probed with
-HA) and cyclin A (red) fluorescent signals
were overlaid by computer assistance. Co-localizations are shown in
yellow. The same fields are shown in panels 1-3
and 4-6. Arrows indicate transfected cells.
Bar, 20 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, and NF-AT (77).
Thus, it is not surprising that phosphorylation of CDH1 might have dual
effects on its nuclear import and export, both of which led to
cytoplasmic retention of the protein. It has been well documented that
phosphorylation of CDH1 inhibits its APC-activating activity (7,
31-34). In particular, the CDH1-4D mutant behaves as a dominant
negative form which is unable to stimulate the
APC-dependent proteolysis (Fig. 4B and Refs. 33
and 60). Our demonstration of the constitutive cytoplasmic localization
of CDH1-4D (Fig. 4) is compatible with the model that cytoplasmic
sequestration of CDH1 contributes to its inactivation. Nevertheless,
further investigations are required to elucidate the nucleocytoplasmic
shuttling of human CDH1 and the influence of CDH1 phosphorylation
on nuclear export.
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ACKNOWLEDGEMENTS |
---|
We thank H.-J. Zhou, K.H. Kok, and C.-M. Wong for technical assistance, and K.-T. Chin, K. H. Kok, and D. C. H. Ng for critical reading of manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Research Grant D43 TW06186 (to D.-Y. J.) funded by the Fogarty International Center.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.
¶ Leukemia and Lymphoma Society Scholar. To whom correspondence should be addressed: Dept. of Biochemistry, The University of Hong Kong, 3rd Floor, Laboratory Block, Faculty of Medicine Bldg., 21 Sassoon Rd., Hong Kong. Tel.: 852-2819-9491; Fax: 852-2855-1254; E-mail: dyjin@hkucc.hku.hk.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212853200
2 Y. Zhou, Y.-P. Ching, R. W. M. Ng, and D.-Y. Jin, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: APC, anaphase-promoting complex; CDK, cyclin-dependent kinase; HA, hemagglutinin; GFP, green fluorescent protein; NLS, nuclear localization signal.
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