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INTRODUCTION |
Survivin was first identified as a novel transcript in the
opposite orientation to the effector cell protease receptor-1 (EPR-1) gene. The functions of these two genes are unrelated, and survivin has
quickly attracted much attention because it is overexpressed in most
cancer cells but not in adjacent normal tissues (1). Sequence analysis
suggests that survivin contains a domain shared by members of the
inhibitor of apoptosis protein
(IAP)1 gene family. The IAP
family proteins were first identified in baculovirus and all of them
contain variable number of baculoviral IAP repeat (BIR) domains (2).
Several human and Drosophila IAPs inhibit apoptosis by
directly binding and potently inhibiting caspases (2). However, the
evidence for survivin directly inhibiting caspases remains
controversial (3, 4). Nevertheless, overexpression of survivin provides
a survival advantage for cells undergoing interleukin-3
withdrawal, treatment with Fas, or treatment with chemotherapeutics (1, 4).
In normal cells, the expression of survivin is limited to the mitotic
stage of the cell cycle. Recent studies suggest that survivin plays an
essential role in cytokinesis (5). Down-regulation of survivin in HeLa
cells by antisense oligonucleotides leads to cytokinesis defects (6,
7). Both immunostaining of endogenous survivin and ectopic expression
of green fluorescent protein-tagged survivin have shown that survivin
is sequentially colocalized with Aurora-B and the inner centromere
protein, INCENP, to the centromeres, the spindle midzone, and then to
the cleavage furrow during mitosis, a typical behavior of chromosomal
passenger proteins (7-9).
The genetic evidence for the functional interaction among survivin,
Aurora-B kinase, and INCENP comes from gene knock-out studies (10, 11).
Survivin knock-out embryos are polyploid and show disrupted microtubule
formation resulting in lethality at embryonic day 4.5. This phenotype
was remarkably similar to that seen in INCENP knock-out mice (11),
consistent with the idea that survivin and INCENP act together in the
same pathway. In budding yeast, mutants of the orthologs of survivin
(Bir1p), Aurora-B (Ipl1p), and INCENP (Sli15) shown similar defects in chromosome segregation (12, 13). Moreover, overexpression of Ark1p, a
fission yeast ortholog of Aurora-B, can rescue the defects associated
with a Bir1p mutant (14), indicating that yeast ortholog of Aurora-B
and survivin can functionally complement each other. Similar phenomena
have also been observed in Caenorhabditis elegans. Ablation
of C. elegans ortholog of Aurora-B, AIR-2, by RNAi also
results in cytokinesis defects, which are indistinguishable from the
down-regulation of the ortholog of survivin, BIR-1 by RNAi (15)
Biochemically, yeast and Xenopus laevis Aurora-B
and INCENP interact with each other, and their interaction is required
for the completion of cytokinesis (13, 16). A recent study has also
shown that human survivin interacts directly with Aurora-B and INCENP
(9). The migration of these three chromosomal passenger proteins is
intriguing. RNAi-mediated reduction of BIR-1 in C. elegans
results in displacement of the Aurora-B ortholog AIR-2 from chromosomes
(17), suggesting that survivin is required to target Aurora-B to the
chromosomes. Depletion of Drosophila INCENP by RNAi
displaces Drosophila Aurora-B from the chromosome and the
spindle midzone, while RNAi-mediated reduction of Drosophila Aurora-B prevents INCENP from being transferred to the spindle midzone,
indicating that the relocation of these proteins is interdependent (18).
Among chromosome passenger proteins, only Aurora-B is a mitotic
serine/threonine kinase. It has been implicated to play key roles in
chromosome segregation, cytokinesis, and cancer development (8, 19,
20). C. elegans Aurora-B is required for recruiting ZEN-4/CeMKLP, a key kinesin-related protein essential for completion of
cytokinesis (21). Aurora-B phosphorylates histone H3, a requirement for
premitotic chromosome condensation (8, 22). The substrates of Aurora-B
kinase also include the kinesin-related protein Eg5 (23), other
chromosomal passenger proteins CENP-A and INCENP (24, 25), and the
myosin II regulatory light chain, a key component of the actomyosin
ring (26). Finally, two recent studies have shown that Aurora-B kinase
activity is required for kinetochore-microtubule interactions and
microtubule dynamics during mitosis (27, 28).
Taken together, the data to date suggest that survivin interacts
with Aurora-B physically, genetically, and functionally. Current
evidence suggested that these proteins together with INCENP might form
a complex (8); however, the role that survivin plays in the complex is
not fully understood. Although survivin is overexpressed in most cancer
cells, the mechanism that links survivin to tumor development is poorly
understood. In this paper we have focused on the role survivin plays
with Aurora-B in this complex. We describe that survivin binds to the
catalytic domain of Aurora-B, enhancing its kinase activity both
in vitro and in vivo, and targets Aurora-B to its substrates.
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EXPERIMENTAL PROCEDURES |
Molecular Cloning--
An Incyte (Incyte is a registered
trademark of Incyte Genomics, Inc.) clone (ID 1272707) containing the
complete DNA sequence for human Aurora-B protein was identified from
the LifeSeq data base (LifeSeq®) using a BLAST homology search. The
following primers were designed for PCR amplification and cloning into
the pET-30 Ek/LIC vector: 5'-gacgacgacaagatggcccagaaggagaactcctacc-3'
and 5'-gaggagaagcccggttcaggcgacagattgaagg-3'. PCR products were cloned using the Ek/LIC vector kit (Novagen) per the manufacturer's
instructions. pET-30Aurora-BK106R was generated using
QuikChange Mutagenesis Kit (Stratagene). The mutagenesis primers are:
5'-ttcatcgtggcgctcagggtcctcttcaagtcc-3' and
5'-gacttgaagaggaccctgagcgccacgatgaa-3'. Primers used for constructing Aurora-B66-344 in pET28 were:
5'-ggggatgccatatggacatcttaacgcggcacttc-3' and
5'-cgcggatcctcaggcgacagattgaagggcag-3'. Human survivin (residues 1-120) was cloned into a modified pET14b (Novagen) using primers: 5'-acgtgtccatgggtgccccgacgttgc-3'and
5'-acgtgtctcgagcttattgttggtttcctttg-3'. GST-survivin constructs were
generated as described previously (3, 29). All clones were subsequently
confirmed by sequencing analysis.
Protein Expression, Purification, and
Biotinylation--
Exponentially growing BL21 (DE3) cells transformed
with pET30Aurora-B, or pET30Aurora-BK106R, were induced
with 1 mM
isopropyl-1-thio-
-D-galactopyranoside at 30 °C
for 3 h. The induction condition for His-survivin or GST-survivin
is 40 µM
isopropyl-1-thio-
-D-galactopyranoside at 23 °C
for 16 h. Cell lysates were incubated with Ni+ ProBond
beads (Invitrogen) for 1 h at 4 °C. The beads were washed with
buffer A (50 mM Tris, pH 8.0, 500 mM NaCl, 20 mM imidazole). The His-Aurora-B and His-survivin were
eluted with 250 mM imidazole, dialyzed against buffer B (50 mM Tris, pH 8.0, 100 mM NaCl) with 50 units of
thrombin in the dialysis bag at 4 °C for 16 h. GST-survivin was
purified using GST purification kit (Amersham Biosciences) according to manufacturer's instructions. Purified survivin was first
incubated with EZ-Link Sulfo-NHS-LC-Biotin solution (Pierce) at 1:3
molar ratio for 45 min at room temperature and then dialyzed against
PBS with 1 mM DTT for 16 h. The average biotin
molecule per survivin is 1.7 determined by HABA solution (Pierce). The proper folding of this biotin-survivin was confirmed later by NMR.
In Vitro Binding Assay--
Full-length Aurora-B and
Aurora-B66-344 were translated in the TNT T7-coupled
reticulocyte system (Promega) according to the manufacturer's
instructions. Five microliters of each
[35S]methionine-labeled Aurora-B or p27kip1 was
incubated with 10 µg of survivin plus 30 µl of streptavidin beads
or with streptavidin beads alone for 1 h at 25 °C. The beads were washed with PBS plus 0.5 M NaCl five times before
resolving on SDS gels and analyzing them with a PhosphorImager
(Amersham Biosciences).
Cells and Transfections--
HEK 293 cells (ATCC) transfected
with pCDNAhis-survivin or pCDNAhis vector were selected in
Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal
bovine serum and 0.5 mg/ml G418 (Invitrogen). Single cell clones
overexpressing survivin or neomycin control were confirmed by survivin
immunobloting. HeLa cells (ATCC) were maintained in Dulbecco's
modified Eagle's medium (Invitrogen) with 10% fetal bovine serum.
Transfections were performed using LipofectAMINE Plus
(Invitrogen) according to manufacturer's instructions. Survivin
missense and antisense oligonucleotides were obtained from ISIS
Pharmaceuticals and used as described previously (7).
Immunoblotting--
50 µg of cell lysate or 1 × 105 cells lysed in Laemmli sample buffer (Sigma) were
resolved on SDS gels. Immunoblotting were performed as described
previously (7). The rabbit polyclonal antibody against phosphorylated
Ser-10 of histone H3 was purchased from Santa Cruz Biotechnology Inc.
The rabbit polyclonal antibody against survivin was purchased from R&D System.
Aurora-B Kinase Assay--
The kinase reactions were performed
in kinase buffer (20 mM Hepes, pH 7.5, 10 mM
MgCl2, 1 mM DTT, 10 mM KCl)
containing 10 µg histone H3 (Sigma), 5 µCi of
[
-33P]ATP, 5 µM ATP, 140 ng of Aurora-B
or Aurora-BK106R, and 2 µg of survivin. The reactions
were incubated at 30 °C for 30 min before resolving on SDS gels,
transferring to polyvinylidene difluoride membranes, and analyzing them
with a PhosphorImager.
Immunoprecipitation and Kinase Assay--
24 h after
transfection, cells were incubated with 0.4 µg ml
1
nocodazole (Sigma) overnight. The next day cells were washed with PBS
and lysed in buffer C (20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 10 mM NaF, 1 mM NaVO4, 0.5% Nonidet
P-40, protease inhibitor mixture (Roche Molecular Biochemicals)
and phosphatase inhibitor mixture (Sigma)). Lysates (1 mg) were
incubated with 2.5 µg of anti-Aim-1 (Aurora-B) antibody (BD
Biosciences) at 4 °C for 1 h, and the immune complexes were
precipitated with protein G-agarose beads (Invitrogen) for 2 h at
4 °C. Immunoprecipitates were washed four times with buffer C and
one time with buffer D (10 mM Hepes, pH 7.5, 10 mM MgCl2, 1 mM DTT, 10 mM KCl). Each sample was resuspended in 25 µl of kinase
mixture (20 mM Hepes, pH 7.5, 10 mM
MgCl2, 1 mM DTT, 10 mM KCl, 10 µg
of histone H3, 5 µCi of [
-33P]ATP, and 5 µM ATP). The kinase reactions were performed and analyzed
as described above.
Immunohistochemistry and Confocal Microscopy--
Cells grown
and treated on eight-chamber slides were fixed with 10%
phosphate-buffered formalin for 10 min, washed two times with PBS, and
blocked with 2% bovine serum albumin, 0.2% nonfat dry milk, 0.4%
Triton-X-100 in PBS for 1 h at room temperature. Cells were
incubated with either rabbit anti-survivin IgG (R&D Systems) or mouse
anti-Aim-1 (BD Biosciences), both diluted 1:200 in PBS, for 1 h at
37 °C. Cells were washed three times in PBS containing 0.2% Tween
20 and incubated with Alexa 546-conjugated goat anti-rabbit IgG
(Molecular Probes) or Alexa 488-conjugated anti-mouse IgG, both diluted
1:1000 in PBS for 30 min at 37 °C. Cells were washed once as
described above, stained for microtubules with a fluorescein
isothiocyanate-conjugated
-antitubulin IgG (Sigma) diluted 1:75 in
PBS for 1 h at 37 °C, and then washed one time as described
above. The DNA was counterstained with Hoechst (15 µg
ml
1) for 10 s, and the cells were washed two more
times. Cells were mounted with Citifluor and imaged with a Bio-Rad
MRC-1000 confocal system attached to an inverted Nikon microscope
fitted with epifluorescence optics and a ×60 (n.a. = 1.4) objective.
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RESULTS |
Survivin Interacts Directly with the Catalytic Domain of
Aurora-B--
To characterize the interaction between survivin and
Aurora-B, we conducted pull-down experiments using biotin-labeled
survivin1-120 and [35S]methionine-labeled
Aurora-B. As demonstrated by a previous study (9), we observed the same
interaction between survivin and Aurora-B (Fig.
1A). Similar results were
obtained using GST-survivin instead of biotin-labeled survivin (data
not show). The interaction between survivin and Aurora-B was also
confirmed by anti-Aurora-B antibody immunoprecipitation followed by
Western blot analysis with anti-survivin antibody (data not shown).
Although a previous study reported that the localization of Aurora-A
kinase protein depends on the N-terminal non-catalytic domain of the
kinase in Xenopus laevis (30), we found that the
catalytic domain of Aurora-B was sufficient to bind to survivin-coated
streptavidin beads, but not streptavidin beads alone (Fig.
1B). A non-related [35S]methionine-labeled
protein, p27kip1, was used to confirm that the interaction
between survivin and Aurora-B was specific. Under the same conditions,
p27kip1 did not bind to survivin-coated streptavidin beads, nor
did it bind to streptavidin beads alone (Fig. 1C). These
results indicate that survivin specifically binds to the catalytic
domain of Aurora-B.

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Fig. 1.
Survivin interacts directly with the
catalytic domain of Aurora-B. Biotin-labeled survivin was bound to
streptavidin beads and incubated with in vitro translated
Aurora-B (A), Aurora-B66-344 (B), or
p27kip1 (C). Proteins bound to streptavidin beads
were resolved by SDS gels and analyzed using a PhosphorImager.
Lane 1, total input of Aurora-B; lane 2, Aurora-B
bound to streptavidin beads alone; lane 3, Aurora-B bound to
survivin streptavidin beads; lane 4, total input of
Aurora-B66-344; lane 5,
Aurora-B66-344 bound to streptavidin beads alone;
lane 6, Aurora-B66-344 bound to survivin
streptavidin beads; lane 7, total input of p27kip1;
lane 8, p27kip1 bound to streptavidin beads alone;
lane 9, p27kip1 bound to survivin streptavidin
beads.
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Survivin Enhances Aurora-B Kinase Activity in Vitro--
When a
protein binds to the catalytic domain of a kinase, it usually acts as a
substrate, a regulator, or an adaptor. Since a recent study claimed
that Aurora-B does not phosphorylate survivin (9), we hypothesized that
survivin might regulate Aurora-B activity. Indeed, we found that in the
presence of survivin, Aurora-B phosphorylated one of its physiological
substrates histone H3 much more strongly than in the absence of
survivin (Fig. 2, lanes 1 and
2). As survivin and histone H3 have similar molecular
weights, we included a control without histone H3 to rule out the
possibility that the increase of 33P labeling was due to
the phosphorylation of survivin. Under the same assay conditions we did
not observe the phosphorylation of survivin by Aurora-B (Fig. 2,
lane 3). As a negative control, we constructed a
kinase-inactive mutant Aurora-BK106R (31). Using
Aurora-BK106R, we did not observe any detectable kinase activity under the same conditions (Fig. 2, lanes 4-7).

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Fig. 2.
Survivin enhances Aurora-B kinase activity
in vitro. Bacterial expressed Aurora-B or
Aurora-BK106R mutant was incubated with
[33P]ATP and histone H3 in the presence or absence of
survivin. The reactions were resolved on the SDS gel and analyzed using
a PhosphorImager. WT, wild type.
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Overexpressing Survivin Enhances Aurora-B Kinase Activity in
Vivo--
To determine whether survivin stimulates Aurora-B kinase
activity in vivo, we first generated HEK 293 cells stably
transfected with N-terminal His-tagged survivin (Fig.
3A). Subsequently, we overexpressed Aurora-B in both HEK 293 cells that overexpress survivin
and vector control HEK 293 cells that do not overexpress survivin.
Although similar expression of Aurora-B in both transfected cells was
confirmed by immunoblot (Fig. 3B), we found that
immunoprecipitated Aurora-B kinase activity was higher in cells that
overexpress survivin using histone H3 as a substrate (Fig.
3C).

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Fig. 3.
Overexpressing survivin enhances Aurora-B
kinase activity in vivo. A, cell
lysates from HEK 293 permanently transfected with N-terminal His-tagged
survivin (His-Survivin) or vector control
(Control) were immunoblotted with anti-survivin antibody.
B, overexpressing survivin HEK 293 cells and vector control
cells were subsequently transfected with Aurora-B. Lysates from these
cells were normalized by total protein amount and immunoblotted with
anti-Aurora-B antibody. C, Aurora-B immunoprecipitated
kinase reactions of these transfected HEK 293 lysates were resolved on
the SDS gel and analyzed using a PhosphorImager.
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Down-regulation of Survivin Decreases Aurora-B Kinase Activity in
Vivo--
Next, we used survivin antisense oligonucleotides to
down-regulate endogenous survivin in HeLa cells (Fig.
4A). We discovered that the
immunoprecipitated endogenous Aurora-B activity in survivin antisense
oligonucleotide-treated cells was 9-fold lower than that in missense
oligonucleotide-treated cells (Fig. 4B), even though the
Aurora-B protein level was unaffected (Fig. 4C). This result
indicates that survivin regulates Aurora-B kinase activity rather than
its protein stability. We reasoned that regulating survivin levels
might have an impact on the phosphorylation of endogenous histone H3
phosphorylation, since a previous study showed that RNAi mediated AIR-2
reduction led to a defect in histone H3 phosphorylation by
immunostaining (17). Using an antibody against phosphorylated Ser-10 of
histone H3, we found that the phosphorylated histone H3 in survivin
antisense oligonucleotide-treated cells is much lower than that in
survivin missense oligonucleotide-treated cells (Fig. 4D).
These observations further confirmed that survivin enhances Aurora-B
kinase activity in vivo.

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Fig. 4.
Down-regulation of survivin decreases
Aurora-B kinase activity in vivo. A,
cell lysates from HeLa cells treated with survivin antisense
(AS) oligonucleotides or missense (MS) control
oligonucleotides were immunoblotted with anti-survivin antibody.
B, immunoprecipitated endogenous Aurora-B kinase activity
from survivin missense (MS) or antisense (AS)
oligonucleotide-treated HeLa cells was assayed using histone H3 as
substrate. C, same lysates were immunoblotted with
anti-Aurora-B antibody. D, HeLa cells treated with survivin
missense (MS) or antisense (AS) oligonucleotides
were immunoblotted with anti-phosphorylated histone H3 antibody.
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Survivin Is Required for Targeting of Aurora-B--
To test if
human survivin, like C. elegans BIR1, targets Aurora-B to
chromosomes, we used a commercially available monoclonal anti-Aurora-B
antibody to detect the localization of Aurora-B in HeLa cells that had
been treated with survivin antisense or missense oligonucleotides. In
survivin missense oligonucleotide-treated HeLa cells we observed that
survivin and Aurora-B colocalized throughout mitosis as described
previously (9). For example, both survivin (Fig.
5, A and B) and
Aurora-B (Fig. 5, C and D) colocalized to the
spindle midzone at metaphase. As reported previously by us and others,
we observed an obvious increase in the number of multinucleated cells
in survivin antisense oligonucleotide-treated cells (7). Survivin
staining in the multinucleated cells was below the level of detection
and was not observed in the mitotic apparatus (Fig. 5, E and
F, cell shown at metaphase). In addition, Aurora-B staining
was not localized to the mitotic apparatus in multinucleated cells
(Fig. 5, G and H, cell shown in metaphase). However, there was some diffuse staining of Aurora-B throughout the
cytoplasm (Fig. 5G). This diffuse staining is consistent
with the result that the Aurora-B protein level remains the same in both survivin antisense and missense oligonucleotide-treated cells (Fig. 4C). These findings suggest that human survivin is
required for targeting of Aurora-B in mitosis.

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Fig. 5.
Immunolocalization of survivin and Aurora-B
in fixed HeLa cells at metaphase 48 h following survivin missense
or antisense oligonucleotide treatment. Immunolocalization
of survivin following survivin missense (A, B) or
survivin antissense (E, F) oligonucleotide
treatment and colocalization with DNA and microtubules (B,
F) are shown. Immunolocalization of Aurora-B following
survivin missense (C, D) or survivin antissense
(G, H) oligonucleotide treatment and
colocalization with DNA and microtubules (D, H)
are shown. Red, survivin (A, B,
E, F) or Aurora-B (C, D,
G, H); green, microtubules;
blue, Hoechst-stained DNA.
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DISCUSSION |
In summary, we have found that survivin localizes Aurora-B to its
substrates and enhances its kinase activity in vitro and in vivo. These findings may provide a possible mechanism of
action of survivin in cells.
First, we speculate that the cytokinesis defects caused by depletion of
survivin are through mislocalization of Aurora-B and a decrease in its
kinase activity, since Aurora-B is known to recruit ZEN-4 and
phosphorylates the myosin II regulatory light chain, two essential
proteins for the completion of cytokinesis (21, 26). Results from
overexpression of the Aurora-BK106R mutant also indicate
that Aurora-B kinase activity is essential for normal cytokinesis (32).
Our evidence that ablation of survivin in HeLa cells causes
mislocalization of Aurora-B, a decrease of Aurora-B kinase activity,
and increased cytokinesis defects further supports our speculation.
Second, survivin has been implicated in the regulation of microtubule
stability (33). We, along with others, could not find evidence that
survivin directly binds to microtubules (34). Two recent studies have
shown that Aurora-B kinase activity is required for regulation of
microtubule dynamics (27, 28). Therefore, that survivin is a positive
regulator of Aurora-B may explain how survivin regulates microtubule stability.
Third, we postulate that survivin targets Aurora-B to the chromosome
where Aurora-B can phosphorylate substrates such as CENP-A and INCENP
(24, 25). Phosphorylated INCENP then moves to the central spindle
and brings survivin and Aurora-B with it. This may provide an
explanation of the following intriguing results: depletion of INCENP by
RNAi displaces Drosophila Aurora-B from the chromosome and
the spindle midzone, while RNAi mediated reduction of
Drosophila Aurora-B prevents INCENP from being transferred to the spindle midzone (18).
Finally, Aurora-B kinase has been implicated in human cancer
development and is known to be overexpressed in a variety of human
cancers (19). Positive regulation of Aurora-B by survivin may provide a
connection between survivin and tumorigenesis or perhaps tumor
maintenance. So far, two other kinases (CDK1, CDK4) have been reported
to interact with survivin (35, 36). A recent study has shown that CDK1
regulates BAD, a pro-apoptotic protein, via phosphorylation (37).
Whether survivin exerts its anti-apoptosis function through Aurora
kinase or other kinases is unknown. Further investigation is warranted.
In conclusion, our observations in this study show that survivin
directly stimulates the kinase activity of Aurora-B and properly localize Aurora-B, thus providing a mechanism as to how survivin exerts
its function in cells.