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INTRODUCTION |
Mitotic spindle assembly is a complex and essential event in the
cell division process because it is a prerequisite for chromosome segregation (1). During this process, motor proteins and the microtubules of the bipolar spindle ensure even distribution of the
genomic information present in daughter cells (2). In order to assemble
the bipolar mitotic spindle in metaphase, cells re-organize their
entire microtubule network upon entering in mitosis primarily by means
of phosphorylation reactions (3, 4).
In the case of Xenopus laevis, a number of different
activities that are required for the assembly of a bipolar spindle were isolated, that is to say, motor proteins such as kinesin-related proteins (XlEg51 (5), Xklp1
(6), Xklp2 (7), XCTK2 (8), CENP-E (9), XKCM1 (10)), cytoplasmic dynein
(11), dynactin (12), NuMA (13), protein kinases like
p34cdc2 (3), Plx (14, 15), pEg2 (16), and
protein phosphatases PP1 and PP2A (17).
Motor protein activities such as association with microtubules,
interaction with other proteins, and subcellular localization are
regulated by phosphorylation reactions (18-21).
The X. laevis pEg2 centrosome protein kinase was first
isolated through a differential screening undertaken during early
development (22). pEg2 mRNA belongs to the Eg family of mRNA,
which is adenylated, recruited in polysomes, and translated in
unfertilized eggs and deadenylated after fertilization (23). The kinase
was also recently isolated during a screen designed to isolate early
players in the progesterone-induced maturation pathway of the
Xenopus oocyte. In the oocyte, the overexpression of active
pEg2 accelerates the appearance of the germinal vesicle breakdown after
contact with progesterone (24).
In Xenopus egg extracts, pEg2 kinase activity is required
for mitotic spindle assembly (16). pEg2 is also a
microtubule-associated protein (16). In vitro pEg2 binds to
paclitaxel-stabilized microtubules independently of its kinase activity
(25), whereas in vivo pEg2 binds to mitotic microtubule
structure and not to interphasic microtubule networks (25).
pEg2 belongs to a family of protein kinases related to
Drosophila Aurora (26) and Saccharomyces
cerevisiae Ipl1 (increase in ploidy) (27).
Aurora gene mutation is characterized by a centrosome separation
defect, a mechanism that needs to be completed in prophase to allow
bipolar spindle assembly to occur (26). The mutation manifestations in
the Ipl1 gene show a chromosome segregation defect (27). Three
different Aurora-related kinases were found in mammalian cells (mouse
and human) (28-31). Two of the human Aurora-related kinases were found
to be overexpressed in several human cancer types (30-33), and one was
shown to be transforming when overexpressed (30, 31).
The X. laevis XlEg5 microtubule-based motor protein is a
kinesin-related protein that was isolated through the same differential screening as pEg2 (5), and its mRNA behaves in the same manner of
pEg2 mRNA during early development (23). Like pEg2, XlEg5 activity
is required for mitotic spindle assembly in Xenopus egg extract (34). The XlEg5 sequence ranked it in the bimC
family of kinesin-related protein, which includes yeast, fungus,
Drosophila, and human proteins (35-39). All members of the
bimC family have a highly preserved motor domain (N-terminal
domain) and a small preserved domain in the tail domain (C-terminal
domain) containing a threonine residue in a phosphorylation consensus
sequence for p34cdc2. The mutations of the
potential phosphorylation site for p34cdc2 in
XlEg5 inhibited its binding to the mitotic spindle (18). In addition,
p34cdc2 phosphorylation of the human Eg5 (HsEg5)
threonine 1067 was shown to control any association with the spindle
in vivo (19) and interaction with the dynactin component
p150Glued (20). XlEg5 was suspected to be
required for centrosome separation because the phenotypes obtained
after XlEg5 inhibition are reminiscent of those obtained after
cytoplasmic dynein inhibition, which is involved in centrosome
migration (19, 40).
In Drosophila, mutations in both Aurora (related to pEg2)
and KLP61F (related to XlEg5) genes are characterized by improper centrosome positioning, leading to the formation of monopolar mitotic
spindles (26, 39, 41). In Xenopus egg extracts, both the
inhibition of XlEg5 through the addition of antibodies (34) and the
inhibition of pEg2 through the addition of an inactive dominant
negative pEg2 form (16) provoke an inhibition of the mitotic spindle
assembly. These results suggest that pEg2 may act on a substrate
involved in centrosome separation, and one of the obvious candidates is
the kinesin-related protein XlEg5.
In Xenopus cultured cells, pEg2 associates with the
centrosome in G2 and binds to microtubules at the mitotic
spindle poles (16). XlEg5 also binds to the microtubules over the
entire spindle (18). In interphase XlEg5 staining has been described as
a "weak cytoplasmic staining" and was not detected specifically
around centrosomes (18). Here, we re-examined XlEg5 subcellular
localization in Xenopus XL2 cultured cells and showed that
the protein started to accumulate between the duplicated centrosomes at
the end of S phase and associated with the centrosome in prophase where
pEg2 was localized.
In this study, we looked for evidence of direct interaction between
pEg2 and XlEg5 and demonstrated that pEg2 associates with XlEg5. In
addition, we demonstrated that pEg2 is capable of phosphorylating a
serine in the stalk domain of XlEg5. Our results definitely suggest
that XlEg5 is phosphorylated in vivo on two residues with two different kinases; a threonine residue is phosphorylated by p34cdc2, and a serine is phosphorylated by pEg2.
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EXPERIMENTAL PROCEDURES |
Xenopus Eggs and Cultured Cells--
X. laevis
oocytes and eggs were obtained from laboratory-reared females. The
embryonic X. laevis cells XL2 (42) were grown at 25 °C,
without CO2 in L15 Leibovitz medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Bio Times) and
antibiotic-antimycotic (Life Technologies, Inc.).
Purification of Recombinant Proteins--
All recombinant
proteins were prepared from Escherichia coli strain
BL21(DE3)pLysS induced to overexpress the protein.
pEg2-(His)6 and pEg2-K/R-(His)6 were prepared
as described by Roghi et al. (16) and
Sup35-(His)6 as described by Frolova et al.
(43). Histidine-tagged proteins were purified on a nickel-NTA-agarose column (Qiagen). All the histidine-tagged proteins proved soluble. Recombinant XlEg5 (GST-Eg5HS, GST-Eg5ST, and GST-Eg5T) proteins were
prepared as described by Sawin et al. (34). Because all three proteins were highly insoluble, they were affixed and used on
glutathione-agarose beads (Amersham Pharmacia Biotech).
Antibodies--
Anti-pEg2 1C1 antibody is a mouse monoclonal
antibody described by Roghi et al. (16). Anti-XlEg5
antibodies are rabbit polyclonal antibodies described by Sawin et
al. (34), with an anti-stalk/tail domain and an anti-tail domain.
The antibodies raised against the C-terminal end of XlEg5 were purified
on a nitrocellulose membrane containing the tail domain of XlEg5 fused
to glutathione S-transferase. Briefly, 5 µg of GST-XlEg5T
protein were run on an SDS-polyacrylamide gel and transferred onto
nitrocellulose. The proteins were subsequently stained with Red
Ponceau, and the bands corresponding to GST-XlEg5T protein were cut
out. The various pieces of the membrane (10 cm2) were first
incubated at 4 °C in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 5% dry skim milk for
2 h and then incubated overnight at 4 °C in 10 ml of TBST
containing 2.5% milk and 1 ml of anti-tail domain serum. The pieces of
the membrane were next washed with TBST, and the anti-tail antibodies were eluted with 2 ml of 100 mM glycine-HCl, pH 2.9, and
immediately neutralized in 200 µl of 1 M Tris-HCl, pH 8. The purified antibodies were concentrated and washed with PBS in a
Centricon 30 (Amicon) following the manufacturer's instructions.
Indirect Immunofluorescence Microscopy--
X. laevis
XL2 cells were grown on round coverslips in 12-well plates (Corning
Inc.) for 48 h, washed with phosphate-buffered saline (PBS: 136 mM NaCl, 26 mM KCl, 2 mM
Na2HPO4, 2 mM
KH2PO4, pH 7.2) and fixed by immersion in cold
(
20 °C) methanol for 6 min. Following successive washes in PBS,
the cells were blocked in PBS containing 3% BSA for 30 min and then
incubated with both mouse anti-pEg2 monoclonal antibody 1C1 (20 µg/ml) and rabbit anti-XlEg5 purified polyclonal antibody (anti-tail)
(dilution 1/60). The antibodies were sequentially revealed by
fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (dilution
1/75) (Interchim) and Texas Red-conjugated goat anti-mouse IgG
(dilution 1/35) (Interchim). All antibody reagents were diluted in PBS
containing 1% BSA. Incubations were performed at room temperature for
60 min. The cells were rinsed in PBS containing 1% BSA between each incubation. The coverslips were rinsed in PBS and mounted in a Mowiol
containing antifade. All samples were observed using a Zeiss
fluorescent microscope (Axiovert 35) and photographed using a Nikon 601 camera.
Immunoprecipitation--
The egg extracts were prepared as
described by Lohka and Maller (44) and diluted 10 times in a TBS-IP
buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl,
complemented with an IP buffer composed as followed: 0.5% Nonidet
P-40, 5 mM EDTA, 3 mM EGTA, 5 mM
glycerophosphate, 0.5 mM sodium vanadate, 0.5 µg/ml each
leupeptin, pepstatin, and chymostatin). The XL2 cells were lysed in a
PBS-IP buffer at 4 °C for 30 min. After centrifugation at
13,000 × g at 4 °C for 15 min, the supernatant was
used for immunoprecipitation. Anti-pEg2 1C1 monoclonal antibodies were
conjugated to protein G-Sepharose (Amersham Pharmacia Biotech), whereas
anti-XlEg5 polyclonal antibodies were conjugated to protein A-Sepharose
(Amersham Pharmacia Biotech). In both cases, the beads were saturated
by incubation at 4 °C for 1 h in PBS containing an excess of
antibodies and extensively washed with PBS. For the immunoprecipitation
of XlEg5 from Xenopus XL2 cells, 50 µl of purified XlEg5
anti-tail domain antibodies (10 µg) were covalently bound to 50 µl
of CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) following
the manufacturer's instructions.
Regarding immunoprecipitations, 10 µl of beads was added both to 50 µl of diluted egg extract and to 1.5 ml of cell lysate (2 × 106 cells) and incubated at 4 °C for 1 h on
rotating wheel. After centrifugation, the beads were washed three times
with 1 ml of a corresponding buffer (TBS-IP buffer or PBS-IP buffer).
An additional 30-min wash on a rotating wheel was performed when
labeled proteins were immunoprecipitated. The beads were then
heat-denatured by incubation at 90 °C for 10 min in 20 µl of
Laemmli sample buffer, and immunoprecipitated proteins were analyzed on
SDS-polyacrylamide gels, followed by electrotransfer onto
nitrocellulose membranes and then immunodetection (45, 46).
If the beads were to be used for phosphorylation reactions, they were
further washed twice with 1 ml of kinase buffer: 50 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol.
Detection of pEg2/XlEg5 Interaction through the Two-hybrid
System--
The HpaI/XhoI pEg2 fragment cloned
in pET21 (16) was subcloned in pGBT11. A cDNA encoding the stalk
and the tail domains of XlEg5 was PCR-amplified using a primer
containing a BamHI restriction site
(5'-CGCGGATCCTATGAGCACTTTGG-3') and a primer containing an XhoI restriction site (5'-CAGAACTCGAGTTGTACATTC-3'). The PCR
fragment was digested by BamHI and XhoI and
subcloned in a pGADGH vector in the BamHI/SalI
sites. The same strategy was used to clone a cDNA encoding the same
part of XlEg5 in the pGBT9 vector except for the 5' primer used for PCR
(5'-CGCGGATCCGTATGAGCACTTTGG-3'). Yeast strain SFY 526 (MATa, ura3-52, his 3-200, ade2-101, lys2-801, trp1-901,
leu2-3, 112, canr, gal4-54, gal80-538,
URA3::GAL1-lacZ) (47) was transformed by means of
pairwise combination of both two-hybrid vectors and grown on a medium
without leucine or tryptophan.
-Galactosidase activities were
assayed using 5-bromo-4-chloro-3-indolyl
b-D-galactopyranoside staining on filter replicates
according to Breeden and Nasmyth (48). Both pGADGH and pGBT9 vectors
without insert were used as negative control, whereas pGADGH-XlEg5ST
and pGBT9-XlEg5ST constructs were used as positive control.
Affinity Chromatography on a Nickel-NTA-Agarose Column--
10
µg of purified histidine-tagged protein (pEg2-(His)6 or
Sup35-(His)6) were incubated at 4 °C with 10 µl of
dried Ni-NTA-agarose beads in IMAC20 (20 mM Tris-HCl, pH
7.5, 500 mM NaCl, 10% glycerol, 20 mM
imidazole) for 30 min. After extensive washing with IMAC20 buffer, the
beads were incubated at 4 °C for 30 min in 100 µl of interaction
buffer, which was IB (50 mM Tris-pH 8, 100 mM
KCl, 5 mM MgCl2, 0.1% Triton X-100, 20%
glycerol) containing 1 µl of rabbit reticulocyte lysate previously
programmed with XlEg5. The beads were then washed three times with 1 ml
of IB containing 20 mM imidazole. Histidine-tagged proteins
and proteins bound to histidine-tagged proteins were eluted with 15 µl of IB containing 250 mM imidazole. After heat
denaturation in a Laemmli sample buffer (45) for 10 min at 90 °C,
the proteins were analyzed by SDS-polyacrylamide gel electrophoresis.
Western Blot Analysis--
Electrophoresis on SDS-polyacrylamide
gel was performed according to Laemmli (45) and gels transferred onto
nitrocellulose membranes as described by Towbin et al. (46).
The membranes were blocked in TBST containing 5% skim milk for 2 h at 4 °C and incubated at 4 °C for 1 h with antibodies
diluted in TBST containing 2.5% skim milk. Immunocomplexes were
revealed with antibodies coupled with alkaline phosphatase (Sigma)
using either nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate as substrates or a chemiluminescence according to the
manufacturer's instructions (NEN Life Science Products).
In Vitro Transcription and Translation--
1 µg of
pBluescript containing XlEg5 (minus 39 nucleotides encoding 13 missing
amino acids in the N-terminal end; Refs. 5 and 34) was transcribed and
translated in 25 µl of reaction mix containing 20 µCi of
[35S]methionine (Amersham Pharmacia Biotech) using a
TNT-T7 quick coupled transcription translation system following the
manufacturer's instructions (Promega).
Protein Kinase Assay--
The assays were performed in 10 µl
of 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl2, 10 µM ATP containing 3000 Ci/mmol [
-32P]ATP
(Amersham Pharmacia Biotech). The reactions were incubated at 37 °C
for 15 min, stopped by addition of 10 µl of 2 × Laemmli SDS
sample buffer and denatured at 90 °C for 10 min. The proteins were
then separated by SDS-polyacrylamide gel electrophoresis, electrotransferred onto nitrocellulose membranes and analyzed by autoradiography.
Metabolic Labeling of XL2 Cells--
After serum starvation, the
XL2 cells were blocked in the G1/S border by aphidicolin as
described by Uzbekov et al. (49). They were then released in
the cycle and cultured for 2 h in a phosphate-free Leibowitz L-15
medium (Life Technologies, Inc.) containing 10% fetal calf serum
dialyzed at 4 °C overnight against TBS. The cells were then cultured
for 5 h in 1 ml of the same medium containing 2 mCi of
[32P]orthophosphorus (Amersham Pharmacia Biotech). When
the cells reached the end of G2 or the beginning of M, they
were washed three times with cold PBS and lysed on ice in 1 ml of 20 mM phosphate buffer, pH 7.5, 1% Nonidet P-40, 500 mM NaCl, 0.25% SDS, 2 mM EDTA, 5 mM MgCl2, 0.5 mM sodium vanadate,
0.5 µg/ml each leupeptin, pepstatin, and chymostatin. After
centrifugation at 13,000 × g at a temperature of
4 °C for 15 min, the supernatant was submitted to immunoprecipitation.
Phosphoamino Acid Analysis--
The immunoprecipitated proteins
were separated on SDS-polyacrylamide gels and transferred onto
Immobilon membranes (Applied Biosystems). A piece (0.4 cm2)
of membrane containing the radioactive protein was cut and incubated at
110 °C for 1 h in 200 µl of 6 N HCl under
nitrogen atmosphere for amino acid hydrolysis. The HCl was evaporated
in a Speed-Vac. The pellet was washed three times with water and
dissolved in 10 µl of 2.5% v/v formic acid, 7.8% v/v acetic acid,
pH 1.9. The amino acids were loaded together with 2.5 µg of
nonradioactive phosphoserine, phosphothreonine, and phosphotyrosine
(Sigma) on a thin layer cellulose (TLC) plate (Sigma) and submitted to
high voltage electrophoresis (50) using a Hunter thin layer peptide mapping system (model HTLE-7000). Non radioactive amino acids were
revealed with ninhydrin (Sigma) and radioactive amino acids by autoradiography.
Phosphopeptide Mapping--
The immunoprecipitated proteins were
separated on SDS-polyacrylamide gels and transferred onto
nitrocellulose membranes (Amersham Pharmacia Biotech). A piece of
membrane (0.4 cm2) containing the radioactive protein was
cut out and incubated in 0.5% polyvinylpyrrolidone and 10 mM acetic acid at 37 °C for 30 min, then washed twice
with water followed by the addition of 50 mM ammonium
bicarbonate. The protein was digested by incubating the piece of
membrane in 400 µl of 50 mM ammonium bicarbonate containing 10 µg of trypsin (Roche Molecular Biochemicals) for 2 h, followed by an overnight incubation at 37 °C after the addition of another 10 µg of trypsin. The tryptic peptides released from the
membrane were washed twice in 100 µl of water and lyophilized. The
peptides were then oxidized at 0 °C for 1 h in 50 µl of
performic acid (10% v/v 30% H2O2 and 90% v/v
formic acid). The tryptic peptides were washed three times in water and
lyophilized again. Next they were dissolved in 5 µl of water and
analyzed by two-dimensional separation on a TLC plate (20 cm × 20-cm polyester silica gel, 100 µm thick; Sigma). The tryptic
peptides were then first submitted to high voltage electrophoresis at
pH 8.6 in a Hunter thin layer peptide mapping system (model HTLE-7000)
(1000 V for 20 min) and then submitted to ascendant chromatography in a
buffer containing 37.5% butanol, 25% pyridine, and 7.5% acetic acid
(51). The plate was dried and the phosphotryptic peptides detected in a PhosphorImager (Molecular Dynamics).
 |
RESULTS |
pEg2 and XlEg5 Localize in the Centrosome and at the Poles of the
Mitotic Spindle--
The first evidence that pEg2 might interact with
XlEg5 came from the indirect immunodetection of the proteins in
Xenopus XL2 cultured cells. We used the specific monoclonal
antibody 1C1 directed against pEg2 (16) and an affinity-purified
polyclonal antibody directed against the specific C-terminal domain of
XlEg5 (34). Both antibodies are highly specific. In XL2 cell lysate,
anti-Eg2 antibody detected a single protein with a relative molecular
mass of 46 kDa (Fig. 1A,
lane 1), while anti-XlEg5 antibodies detected a
doublet at 130 kDa (Fig. 1B, lane
1).

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Fig. 1.
Specificity of pEg2 and XlEg5
antibodies. Proteins in X. laevis XL2 cell lysate
(2 × 105 cells) were separated onto two
SDS-polyacrylamide gels (A, 17.5%; B, 10%),
transferred onto nitrocellulose membranes and immunodetected with
anti-pEg2 1C1 mouse monoclonal antibody (A, lane
1) and anti-XlEg5 purified rabbit polyclonal antibodies
(B, lane 1) revealed with phosphatase
conjugated antibodies directed against mouse IgG (A) and
rabbit IgG (B). Lanes 2 in
A and B are controls without primary
antibodies.
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In XL2 cells, no protein is apparent prior to the end of S phase. pEg2
appears as two bright spots on the duplicated centrosomes at the end of
S phase (Fig. 2B) (16). XlEg5
can only be detected between the two duplicated centrosomes after pEg2
appears on the centrosome (Fig. 2C). At this stage, the two
proteins localize differentially (Fig. 2D).

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Fig. 2.
XlEg5 starts to aggregate between duplicated
centrosome just before centrosome separation. X. laevis XL2 cultured cells were grown and fixed as described
under "Experimental Procedures." Cells were then labeled for double
indirect immunofluorescence microscopy with anti-pEg2 1C1 monoclonal
antibody (B and D) and with anti-XlEg5 polyclonal
antibody (C and D); an enlargement of the double
staining is shown in D, upper right
corner. The cell in A was observed using phase
contrast microscopy. The bar is 10 µm.
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XlEg5 and pEg2 were both detected among the centrosomes in prophase,
when they had reached their position at opposite side of the nucleus
(Fig. 3, B and C).
However, XlEg5 seemed to occupy a larger area than pEg2 in the
centrosome (Fig. 3D, upper left) as
revealed by double staining with a yellow dot in
the centrosome and a green halo around each
centrosome (Fig. 3D). Indirect electron microscopy showed
that both proteins are localized around the pericentriolar material
(data not shown). From that time on, both proteins localized on same
structures throughout mitosis. During prometaphase and metaphase, both
proteins slipped from the centrosome position to invade the spindle
microtubules. pEg2 appeared as "cup shape" staining only at the
spindle poles (Fig. 3, F and J), whereas XlEg5
was also found along the spindle microtubules (Fig. 3, G and
K). In anaphase, XlEg5 appeared to move back to the pole of
the spindle and was characterized by a weaker staining of spindle
microtubules (Fig. 3O). From prometaphase to anaphase, both
pEg2 and XlEg5 localized on the microtubules that form the poles region
of the spindle. A proportion of pEg2 remained associated with the
centrosome, while XlEg5 was rather found only on the spindle
microtubules. Double staining always showed a red
dot at the centrosome, a yellow pole, and
green microtubules (Fig. 3, H, L, and
P). In telophase, both proteins still localized in the
centrosome with a dramatic decrease in XlEg5 staining. Moreover, XlEg5
relocated on the microtubules in the midzone without any association
with pEg2 (Fig. 3, R, S, and T). To
summarize, both proteins are present around the centrosome in prophase
and on microtubules during metaphase and anaphase.

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Fig. 3.
During mitosis, XlEg5 localizes with pEg2 in
separated centrosomes and at the poles of the mitotic spindle.
X. laevis XL2 cultured cells were grown and fixed
as described under "Experimental Procedures." The cells were then
processed for double immunofluorescence staining with antibodies
specific for pEg2 (panels B, F,
J, N, and R) and antibodies specific
for XlEg5 (panels C, G, K,
O, and S). Double staining is shown in
panels D, H, L,
P, and T with an enlargement of the centrosome in
D, upper left corner. Cells
were observed by phase contrast microscopy (panels
A, E, I, M, and
Q) to identify the different stages of mitotic progression:
prophase (A-D), prometaphase (E-H), metaphase
(I-L), anaphase (M-P), and telophase
(Q-T). The bar is 10 µm.
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pEg2 and XlEg5 Co-immunoprecipitate--
Given the fact that both
proteins localized on the same structures in the cell during mitosis,
we searched for evidence of direct interaction between the two proteins
in immunoprecipitation. pEg2 and XlEg5 were immunoprecipitated from
protein extracts prepared from Xenopus unfertilized eggs
using the same antibodies intended for indirect immunofluorescence. The
precipitates were subjected to Western blot analysis. Control
immunoprecipitations did not reveal any unspecific interactions (Fig.
4, lanes 1 and
3). XlEg5 was found among pEg2 immunoprecipitates and pEg2
among XlEg5 immunoprecipitates (Fig. 4, lanes 2).
In the case of both XlEg5 and pEg2 precipitates, XlEg5 appeared as a
doublet, as had already been reported for HsEg5 (19), suggesting that
different forms of XlEg5 associate with pEg2. The comprehensive
immunodepletion of pEg2 from Xenopus extracts revealed that
5-10% of total Xenopus egg XlEg5 molecules was present in
the pEg2 immunoprecipitate (data not shown). pEg2 and XlEg5 were also
co-immunoprecipitated from unsynchronized Xenopus XL2 cell
lysate, but the signals were very weak (data not shown), which would
suggest that the association process depends on the cell cycle. This
would tend to agree with and confirm the previous immunofluorescence
studies, which have indicated that both proteins interact only in
mitosis.

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Fig. 4.
pEg2 and XlEg5 co-immunoprecipitates.
pEg2 and XlEg5 were immunoprecipitated from Xenopus egg
extract. Immunoprecipitations were performed using either anti-pEg2 1C1
monoclonal antibody affixed to protein G-Sepharose (lane
2, left panel) or anti-XlEg5 purified
polyclonal antibodies affixed to protein A-Sepharose (lane
2, right panel). Control
immunoprecipitations were done either without antibody
(lanes 1) or without extract (lanes
3). The presence of both proteins in different
immunoprecipitates were detected using specific antibodies for XlEg5
(upper panels) or pEg2 (lower
panels) and revealed by chemiluminescence.
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pEg2 Interacts Directly with XlEg5--
Although pEg2 and XlEg5
turned out to be co-immunoprecipitated, we wished to demonstrate that
pEg2 interacted directly with XlEg5. Purified bacterially expressed
pEg2-(His)6 was affinity-bound to nickel beads
(Ni-NTA-agarose) and mixed with reticulocyte lysate-expressed [35S]methionine labeled XlEg5. Reticulocyte lysate was
used because XlEg5 is known to be highly insoluble when produced in
bacteria. The various controls were performed with beads alone (Fig.
5, lane 2) or beads
containing another histidine-tagged protein: Sup35-(His)6
(the eukaryotic release factor eRF3) (43) (data not shown). After
incubation at a temperature of 4 °C for 60 min, the histidine-tagged
proteins together with associated proteins were eluted with 250 mM imidazole. [35S]Methionine-labeled XlEg5
was detected only in the fraction eluted from the nickel column that
contained pEg2-(His)6 (Fig. 5, lane 1). Although 90% of [35S]methionine-labeled
XlEg5 loaded onto the column was recovered in the flow-through, 10%
was specifically retained by pEg2-(His)6, demonstrating
that XlEg5 produced in reticulocyte lysate can bind to pEg2. Such poor
interaction may be explained by the fact that pEg2-(His)6
produced in bacteria might not be appropriately folded. Although the
purified kinase is active, its activity is much lower than that of
immunoprecipitated pEg2 (data not shown).

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Fig. 5.
pEg2 directly interacts in vitro
with XlEg5. In vitro translated
[35S]methionine XlEg5 was incubated with nickel-agarose
beads containing pEg2-(His)6 (lane 1)
or no protein (lane 2). After extensive washes
the proteins bound to the beads were eluted with 250 mM
imidazole and separated on a 15% SDS-polyacrylamide gel. The gel was
stained with Coomassie Blue (lower panel) and
autoradiographed (upper panel).
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pEg2 Interacts with XlEg5 in the Two-hybrid
System--
Interaction between pEg2 and XlEg5 was assayed using the
two-hybrid system. Because it had been previously reported that
kinesin-related proteins could form dimers (52), we used XlEg5
dimerization as a positive test for the assay. Two XlEg5 molecules
associated through their stalk domain to form homodimers. Then, we
inserted an XlEg5 cDNA encoding the stalk (S) and the tail domain
(T) in both vector (pGADGH and pGBT9) used for the two-hybrid. The
XlEg5 motor domain was not present in any of the construct because it has been previously reported that production of full-length HsEg5 in
yeast was lethal (20). Strong
-galactosidase activity was obtained
when homodimer formation was assayed and when the XlEg5ST construct
proved to be present in both two-hybrid vectors (Fig. 6, lane 1). When
pEg2 interaction with XlEg5ST was assayed, significant
-galactosidase activity was detected, indicating that both proteins interacted (Fig. 6, lane 2). This activity,
however, was much lower than that of XlEg5ST/XlEg5ST interaction. This
significant difference in
-galactosidase activities may be explained
by the generally low affinity of protein kinases for their substrates, which is much lower than the affinity of proteins that form stable dimers. However, the assay clearly showed that pEg2 interacts with
XlEg5. Interestingly enough, this experiment also indicates that the
motor domain of XlEg5 is not necessary for pEg2 interaction.

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Fig. 6.
pEg2 interact with XlEg5 in the two-hybrid
system. Yeast strain SFY 52 (MATa, ura3-52, his
3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, canr, gal4-54, gal80-538,
URA3::GAL1-lacZ) (47) was transformed with a pair-wise
combination of both two-hybrid vectors and grown on media without
leucine or tryptophan. Filter replicates containing
5-bromo-4-chloro-3-indolyl b-D-galactopyranoside were used
to assay the -galactosidase activity of yeast colonies
co-transfected with both two-hybrid vectors (in duplicate):
lane 1, pGADGH-XlEg5ST and pGBT9-XlEg5ST;
lane 2, pGADGH-XlEg5ST and pGBT11-pEg2,
lane 3, pGADGH and pGBT9.
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pEg2 Phosphorylates a 130-kDa Protein in the Immunoprecipitated
Complex--
Because the 1C1 monoclonal antibody is not an inhibitory
antibody, immunoprecipitated pEg2 remains active and can be assayed for
kinase activity. In this undertaking, pEg2 was immunoprecipitated from
Xenopus egg extracts, and the precipitate was incubated with [
-32P]ATP. The radioactive proteins were analyzed by
means of SDS-polyacrylamide gel electrophoresis (Fig.
7A). At least four
phosphorylated proteins were detected in the immunoprecipitate: a
46-kDa protein that was proved to be autophosphorylated pEg2, a 90-kDa
protein, a 130-kDa protein that was highly phosphorylated, and a very
high molecular mass protein that migrated above the 200-kDa marker (Fig. 7A, lane 3). The highly
phosphorylated 130 kDa protein migrated at the same position as the
XlEg5 identified by Western blot analysis and present only in the
immunoprecipitate containing pEg2 (Fig. 7B, lane 3, top panel).

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Fig. 7.
pEg2 associates to and phosphorylates XlEg5
within immunoprecipitates. A and B, the
Xenopus egg extracts (lanes 1) were
incubated either with protein G-Sepharose (lanes
2) or with anti-pEg2 1C1 monoclonal antibody affixed to
protein G-Sepharose (lanes 3). Immunoprecipitated
proteins were then incubated for 30 min at 37 °C in the presence of
[ -32P]ATP as described under "Experimental
Procedures." After heat denaturation at 90 °C for 10 min in
Laemmli sample buffer, phosphorylation reaction products were separated
on a SDS-polyacrylamide gel, transferred onto nitrocellulose membrane,
and autoradiographed (panel A). The membrane was
subsequently cut for XlEg5 (upper panel
B) and pEg2 (lower panel
B). immunodetections. C, the specificity of XlEg5
phosphorylation by pEg2 was controlled by adding an excess of inactive
recombinant pEg2-K/R-(His)6 to the immunoprecipitation
prior to the phosphorylation reaction. Phosphorylation of XlEg5 was
assayed in immunoprecipitates performed either with protein G-Sepharose
alone (lane 1) or with anti-pEg2 antibody affixed
to protein G-Sepharose (lanes 2-4). The
phosphorylation reactions were performed without any previous addition
(lane 2) or in the presence of 8 µg of BSA
(lane 3) or 5 µg of pEg2-K/R-(His)6
(lane 4).
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In order to confirm that the phosphorylation observed in the
immunoprecipitate was due to pEg2 kinase activity, we added to the
reaction an excess of the bacterially expressed dominant negative mutant of pEg2, pEg2-K/R-(His)6 in which the lysine 169 was
replaced by an arginine residue (16). The addition of 5 µg of
pEg2-K/R-(His)6 inhibited 90% of XlEg5 phosphorylation
(Fig. 7C, lane 4), while the addition
of 5 µg of BSA exerted no effect (Fig. 7C, lane
3). The addition of pEg2-K/R-(His)6 specifically inhibited
pEg2 activity, presumably by titrating its substrates. This result
demonstrated that pEg2 phosphorylated a 130-kDa protein in the
immunoprecipitate that migrated at the same position as
XlEg5.
pEg2 Phosphorylates XlEg5 in Vitro in the Stalk Domain--
Like
the other kinesin-related proteins from the bimC family,
XlEg5 is composed of three distinct domains, an N-terminal motor domain
or head (H), a stalk domain (S), and a C-terminal tail (T) (Fig.
8A). Three different
bacterially expressed XlEg5 truncated proteins were expressed in
bacteria as GST fusion proteins, purified, and used in vitro
as substrates for recombinant pEg2-(His)6 protein kinase
(Fig. 8B). The HS construct contained the head
and stalk domain of XlEg5. The ST construct contained the
stalk and tail domain, and the T construct
contained only the tail domain. Because the GST-XlEg5
proteins were highly insoluble, the phosphorylation reactions were
performed directly on GST proteins fixed to glutathione-agarose beads. Western blot analysis with anti-GST antibodies (Sigma) was
performed to ensure that equal amounts of the three proteins were
present in the reactions (data not shown). The two XlEg5 fusion
proteins, HS and ST, which had the stalk domain in common, were
phosphorylated only when pEg2 was present in the reaction (Fig.
8C, compare lanes 1 and 3 with lanes 4 and 6). The third T
protein, which contained only the tail domain, was not found to be
phosphorylated even in the presence of pEg2, indicating that the tail
is not the substrate (Fig. 8C, lanes 2 and 5). In order to determine if the label was located in
the stalk domain or in the motor domain, two-dimensional mapping of the
in vitro phosphorylated tryptic peptides of both proteins was
performed. Both maps were identical. One major and one minor common
phosphorylated tryptic peptide were detected, which demonstrated that
only the stalk domain contained the in vitro phosphorylation
site for pEg2 (data not shown). The XlEg5 tail domain contained a
phosphorylation site for p34cdc2 (18, 19). Our
results indicated that in vitro pEg2 phosphorylated XlEg5 in
the stalk domain. In addition, phosphoamino acid analysis by high
voltage electrophoresis revealed that in vitro pEg2
phosphorylated XlEg5 on a serine residue (Fig. 8D).

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Fig. 8.
In vitro recombinant pEg2-(His)6
phosphorylates the stalk domain of recombinant GST-XlEg5.
A, diagram of XlEg5 recombinant proteins fused with
glutathione S-transferase (5). B, overexpression
of XlEg5 recombinant protein in E. coli. Crude extracts of
bacteria induced to express the GST fusion protein were separated by
SDS-polyacrylamide gel electrophoresis and analyzed by Coomassie
staining. Lane 1, molecular size markers;
lanes 2-4, overexpression of recombinant protein
containing different domains of XlEg5; lane 2,
the head and the stalk domain (HS); lane
3, the tail domain (T); and lane
4, the stalk and the tail domain (ST).
C, GST proteins shown in B and affixed to the
beads were phosphorylated in vitro by recombinant
pEg2-(His)6 as described under "Experimental
Procedures" and analyzed by SDS-polyacrylamide gel electrophoresis
and autoradiography. Lanes 1-3, control
reactions without pEg2 kinase added; lanes 4-6,
with pEg2-(His)6. D, phosphoamino acid analysis
of XlEg5 phosphorylated in vitro by pEg2-(His)6.
Amino acids obtained after hydrochloric acid hydrolysis were separated
on TLC plate and autoradiographed.
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Phosphorylation State of XlEg5 in Vivo--
The human protein
HsEg5 was found to be phosphorylated in vivo on two distinct
tryptic peptides, with one phosphorylated on a serine residue and the
other on a threonine residue (19). p34cdc2
phosphorylated the threonine residue located in the tail domain that is
conserved within the bimC family of kinesin-related protein. The kinase that phosphorylated the serine, which was the major phosphorylated site in HsEg5 in vivo, has not been
identified yet (19).
In order to investigate the phosphorylation state of XlEg5 in
vivo, the Xenopus XL2 cells were metabolically labeled
with [32P]orthophosphorus. Because
p34cdc2 is a mitotic kinase and because pEg2 and
XlEg5 localized on the same structure only after duplication of the
centrosomes in G2 and M, the XL2 cells were labeled and
harvested in the course of G2 and M phases (49).
32P-Labeled XlEg5 was then immunoprecipitated with
affinity-purified polyclonal antibodies and digested with
trypsin. The resulting phosphotryptic peptides were resolved
with thin layer electrophoresis, followed by ascendant chromatography.
Like HsEg5, XlEg5 was phosphorylated in vivo on two tryptic
peptides (Fig. 9B), one of
which migrated to the same location as the peptide phosphorylated
in vitro by p34cdc2 (Fig.
9A, black arrow), thereby
demonstrating that XlEg5 was indeed phosphorylated in vivo
by p34cdc2 as had been suggested by Sawin and
Mitchison (18). The second phosphotryptic peptides migrated to the same
location as a peptide phosphorylated in vitro by recombinant
pEg2 (Fig. 9, C and D), which strongly suggested
that pEg2 phosphorylates XlEg5 in vivo.

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Fig. 9.
Two-dimensional analysis of XlEg5 tryptic
phosphopeptides after phosphorylation in vivo and
in vitro. GST-XlEg5ST (Stalk-Tail) phosphorylated
in vitro by p34cdc2 (A),
endogenous XlEg5 labeled in vivo (B), GST-XlEg5ST
phosphorylated in vitro by pEg2-(His)6
(C), or a mixture of GST-XlEg5ST phosphorylated in
vitro by pEg2 and endogenous XlEg5 labeled in vivo
(D) was digested with trypsin, and the peptides were
separated by high voltage electrophoresis followed by ascendant
chromatography. Phosphopeptides were visualized using an instant
imager. Black arrow, peptide phosphorylated by
p34cdc2; open arrow,
peptide phosphorylated by pEg2.
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DISCUSSION |
pEg2 and XlEg5 are two proteins that have been reported to be
necessary for bipolar mitotic spindle assembly in Xenopus
egg extracts (16, 34). The mutation of the genes encoding the Drosophila orthologues of Xenopus pEg2 and XlEg5,
Aurora and KLP61F, respectively, causes a centrosome separation defect
(26, 39, 41). These observations have led us to posit two questions: (1) is there a physical association between pEg2 and XlEg5, and (2)
does pEg2 phosphorylate XlEg5?
We first investigated whether XlEg5 and pEg2 could be found on the same
subcellular localization in Xenopus XL2 cells during the
cell cycle. First, pEg2 appeared in the centrosome at the end of S
phase. Then, 2 h later XlEg5 started to accumulate between the two
duplicated centrosomes. In prophase, when both centrosomes had reached
their position on each side of the nucleus, both pEg2 and XlEg5 were
present in the separated centrosomes. During metaphase and anaphase,
the area shared by pEg2 and XlEg5 moved to the poles of the spindle. In
metaphase, while pEg2 was localized in the centrosome and on the
microtubules present in the spindle poles, XlEg5 was found only on the
spindle microtubules. In telophase, both proteins were found together
in the centrosome, but XlEg5 was also found in the midzone in the
absence of detectable pEg2. The localization of XlEg5 between the
duplicated centrosome during the S phase and on the centrosome during
prophase was in compliance with its presumptive role in centrosome
separation (18, 19). Its localization on the spindle microtubules was
in compliance with its presumptive role in regulating the stability of
the spindle (34). The localization of XlEg5 on the microtubule in the
midzone may indicate a role in cytokinesis that remains to be demonstrated.
Although pEg2 and XlEg5 showed a shared area of localization on the
mitotic spindle during the prophase-to-anaphase stages of the cell
cycle, the localization process proved temporally different, suggesting
that the molecular mechanisms of localization are different for the two proteins.
Furthermore, the physical association of the two proteins was detected
by immunoprecipitation from metaphase-arrested Xenopus egg
extract. Only partial co-immunoprecipitation was detected, indicating
that the localization of pEg2 may depend on the localization of XlEg5
but also on another mechanism. When the same experiment was performed
with lysates prepared from unsynchronized Xenopus XL2 cells,
interaction was very weak. This phenomenon suggests that the
interaction only occurs at a specific point in the cell cycle.
Recombinant pEg2 protein can directly interact with in vitro
translated XlEg5. Accordingly, one may deduce that there is a direct
physical interaction of the two proteins. It cannot be ruled out that a
protein in the reticulocyte lysate may have helped physical
interaction, although this is very unlikely because in vitro
purified recombinant pEg2 phosphorylates purified recombinant XlEg5
which indicates direct interaction. Furthermore, both proteins interact
through the two-hybrid system, demonstrating that pEg2 interacts with
XlEg5. The XlEg5 motor domain is not necessary for interaction
purposes. XlEg5 phosphorylation through pEg2 kinases is a physiological
reaction, because the same phosphopeptide is found to be phosphorylated
both in vivo and in vitro as a result of pEg2.
The site phosphorylated by pEg2 was found to be located in the stalk
domain, which is the domain also involved in protein-protein association and dimerization. The kinesin-related protein members of
the bimC family can form homotetramers (52, 53), with the dimer formed head-to-head via the stalk domain, and two dimers associated head-to-tail to form the tetramer. The alignments of the
various kinesin-related proteins stalk domains related to XlEg5 did not
reveal any conserved phosphorylation domain that might be the target of
pEg2, thereby suggesting that the recognition motif of the substrate
depends largely upon its secondary and tertiary structure.
We also showed that, like HsEg5, XlEg5 was phosphorylated by
p34cdc2 in vivo. The
p34cdc2 phosphorylation site located in the tail
domain of XlEg5 was shown to be preserved within the bimC
family of kinesin-related proteins (20). Although the conservation of
this site throughout the bimC family is real in terms of
sequence, it is not necessarily a p34cdc2
target. In Cut7 from Schizosaccharomyces pombe (36, 54) the mutation of the threonine 1011 (the potential
p34cdc2 phosphorylation site) to an alanine
remained ineffectual on spindle assembly (55). Conversely, mutation of
the threonine 927 in HsEg5 eliminated spindle association (19) and any
binding to the dynactin subunit p150 (20). Mutation of threonine 937 in XlEg5 also disrupted mitotic spindle association (18).
Potential orthologues of XlEg5 were also found in fission yeast (36,
54), and in Drosophila (39, 41, 52, 53). Candidate proteins
that could be orthologues of pEg2 were pinpointed in rat (56), in mouse
(57- 60), in Drosophila (26), and in human (28-30, 60-62).
It was obvious that both the Aurora-related kinases and the
bimC kinesin-related proteins were conserved throughout evolution. Accordingly, we showed that pEg2 kinase associated with and
phosphorylated the kinesin-related protein XlEg5, which suggests that
the Aurora-related kinase family might be involved in the regulation of
kinesin-related protein activities. To our knowledge, XlEg5 proved to
be the first reported physiological substrate for an Aurora-related kinase.