(Received for publication, February 20, 1997, and in revised form, March 17, 1997)
From the Department of Molecular Pathobiochemistry, Mutations in Aurora of Drosophila and
related Saccharomyces cerevisiae Ipl1 kinase are known to
cause abnormal chromosome segregation. We have isolated a cDNA
encoding a novel human protein kinase of 402 amino acids with a
predicted molecular mass of 45.9 kDa, which shares high amino acid
identities with the Aurora/Ipl1 protein kinase family; hence the
cDNA is designated as aik
(aurora/IPL1-related kinase). Amino
acid sequence of C-terminal kinase domain of Aik shares 86, 86, 72, 59, and 49% identity with those of Xenopus XLP46APK and
XLP46BPK, mouse STK-1, Aurora of Drosophila, and yeast
Ipl1, respectively, whereas N-terminal domain of Aik shares high
homology only with those of XLP46APK and XLP46BPK. Northern and Western
blotting analyses revealed that Aik is expressed highly in testis and
various proliferating cells including HeLa cells. In HeLa cells, the
endogenous levels of aik mRNA and protein contents are
tightly regulated during cell cycle progression. Both of these levels
are low in G1/S, accumulate during G2/M, and
reduce rapidly after mitosis. Its protein kinase activity is also
enhanced at mitosis as inferred by exogenous casein phosphorylation.
Immunofluorescence studies using a specific antibody have shown that
Aik is localized to the spindle pole during mitosis, especially from
prophase through anaphase. These results strongly suggest that Aik is a
novel member of a protein kinase family possibly involved in a
centrosome function(s) such as chromosome segregation or spindle
formation.
Accurate segregation of chromosomes during mitosis is an important
event in the progression of cell cycle. Chromosome segregation is a
highly complex process requiring microtubule assembly and disassembly.
Centrosome and pericentriolar materials play important roles in
organizing the microtubules at interphase and mitotic phase. At the
G1 phase of cell cycle, duplicated centrioles closely associate and function as a single unit, and then separate into two
units at mitotic phase. Centrosome separation is initiated either
before or after nuclear envelope breakdown, and then microtubules are
arranged and the separated centrosome organizes a mitotic spindle. In
accordance with chromosome movement toward the spindle pole, the
spindle microtubules shorten. However, details of molecular mechanisms
by which centrosomal cycle or microtubule nucleation is regulated have
not been elucidated.
Multiple lines of evidence suggest that protein phosphorylation plays a
pivotal role in controlling spindle assembly and its functioning.
Several centrosomal components are known to be phosphorylated in
Drosophila (1) and vertebrates (2-6). Phosphorylation of centrosomal proteins has been shown to control both the rate of the
microtubule nucleation and its dynamic behavior (7). The reversible
phosphorylation may control the function of microtubule-based motor
proteins such as a kinesin-like protein (8). Several genetic studies on
fungi and fruit flies have identified various protein kinases and
phosphatases that play intriguing roles in chromosome segregation
(9-11). In mammalian cells, centrosomal localization of several
protein kinases has been reported on Cdc2 (12), Plk1 (13), Fyn (14),
Src (15), and A kinase (16).
Among the protein kinases that have been implicated in the control of
spindle function, the role of Cdc2/cyclin B complex is pivotal and has
been well characterized (17). This kinase is activated during mitosis
and inactivated immediately afterward. The Cdc2 kinase partly localized
in the mitotic spindle is known to regulate the dynamics and length of
microtubules (12). Ectopic expression of mutant cyclin B2 in HeLa cells
causes cell division arrest with multiple mitotic spindle poles (17).
In a recent study, a kinesin-like motor protein was identified as a
substrate for Cdc2 kinase, which regulates the centrosomal localization (8).
Other kinases that control mitotic spindle formation have also been
identified: budding yeast CDC5 (18), fission yeast Plo1 (19),
Drosophila Polo (20), mammalian Polo-like kinase (Plk1) (13,
21-26), and Xenopus Plx1 (27). The plo1 and
polo mutants show abnormal formation of monopolar spindle or
unequally separated bipolar spindle in which one pole is unusually
broad. Plk1 kinase is induced and activated at G2 phase and
disappears at the end of mitosis. The subcellular localization of the
Plk1 protein is in both centrosome and mitotic spindle at M phase. Plk1
is phosphorylated and activated by Cdc2 kinase (28). One of the
substrates of Plk1 was identified as CHO1/MKLP-1 kinesin-like protein,
and it was suggested that Plk1 is a regulator of the motor protein
(26).
Ipl1 (29) and Aurora (11) gene products have conserved serine/threonine
kinase domains, which are highly homologous to each other, and are
suggested to be profoundly involved in normal chromosome segregation.
Embryos derived from aurora mothers inappropriately display
closely paired centrosomes at mitotic stages. Loss of function of
aurora leads to a failure of the centrosomes to separate and
form a bipolar spindle (11). Conditional ipl1ts
mutants severely missegregate chromosomes at restrictive temperature (10). The substrate and the regulator of these kinases have not been
identified, but type 1 protein phosphatase was shown to act in
opposition to Ipl1 protein kinase in yeast (29). Other members of
Aurora/Ipl1 family kinases have been isolated from Xenopus
(XLP46APK, XLP46BPK) or mouse (30), but the functions of these proteins
have not been well studied.
We have cloned a cDNA encoding a human novel serine/threonine
kinase, Aik, which has a high homology with Aurora/Ipl1 family protein
kinases. Accumulation and activation at M phase as well as spindle pole
localization of Aik kinase suggest a possible role of the protein in
centrosome function.
cDNAs from a human B cell plasmid library
(CLONTECH) were sequenced randomly, and a novel
kinase (Aurora/Ipl1-related kinase, Aik) was identified. The obtained cDNA was a partial clone encoding only kinase domain, and a HeLa cells (obtained from
RIKEN Cell Bank) were cultured in MEM medium supplemented with 10%
fetal calf serum. Megakaryoblastic leukemia cell line CMK, Epstein-Barr
virus-transformed B cell line 1G8, Raji, and HL60 were cultured in RPMI
1640 medium supplemented with 10% fetal calf serum, and neuroblastoma
cell line NB69 (from RIKEN Cell Bank) was cultured with RPMI 1640 medium supplemented with 15% fetal calf serum.
For S phase synchronization, HeLa cells were synchronized by double
thymidine treatment (32). Cells were treated with 2.5 mM
thymidine for 20-24 h, followed by a 10-h release in fresh medium and
successive retreatment with the drug for 16 h. For M phase
synchronization, cells were synchronized by double thymidine treatment
as above, followed by 400 ng/ml nocodazole treatment for 12 h.
Cells to be released from the drug treatment were washed with
PBS1 twice and refed with fresh medium. At
appropriate time points, the cells were harvested for Northern or
Western blotting.
Blotted filters with 2 µg of
poly(A)+ RNA/lane from various human tissues were purchased
from CLONTECH. Total RNAs from synchronized HeLa
cells were isolated with ISOGEN according to the manufacturer's procedure (Nippon Gene). Twenty-five µg of RNA was loaded on each lane and subjected to electrophoresis on denaturing agarose gel, followed by transfer to GeneScreen Plus membrane (DuPont NEN) (31). The
blot was probed with 32P-labeled random-primed DNA
corresponding to the kinase domain of Aik cDNA (nucleotides
375-1884) or full-length human Polyclonal antisera against Aik were
generated against glutathione S-transferase-Aik fusion
protein produced in Escherichia coli. The obtained immune
sera were affinity-purified with Aik protein cross-linked to Affi-Gel
10 according to the manufacturer's protocol (Bio-Rad). Antibody
against cyclin B was purchased from Transduction Laboratories.
For cell cycle analysis, the synchronized cells were washed with
ice-cold PBS and harvested by scraping, then lysed in lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 10 mM
sodium diphosphate, 10 mM NaF, 5 mM EGTA, 1 mM EDTA, 1% (w/v) Nonidet P-40). Cell lysates were
subjected to SDS-polyacrylamide gel electrophoresis (33) and
transferred to Immobilon (Millipore) by standard methods. Membranes
were blocked for 2 h in TBST (0.1% Tween 20, 137 mM NaCl, 20 mM Tris, pH 7.6) with 3% nonfat dry milk
(blocking buffer). Probing with the specific antibody against Aik
(1:200) was carried out for 1 h at room temperature in the
blocking buffer containing 0.02% sodium azide. After washing four
times in TBST, the membranes were probed with 200 ng/ml
peroxidase-labeled goat anti-rabbit IgG for 1 h in TBST. After
extensive washing in TBST, the membranes were processed for enhanced
chemiluminescence using ECL reagents according to the manufacturer's
instructions (Amersham). ECL exposures were carried out using Kodak XAR
film.
All procedures during lysis,
clarification, and immunoprecipitation were performed at 4 °C. Cells
were lysed for 20 min in lysis buffer and clarified with protein
A-Sepharose. Supernatant (100 µg of protein) was incubated with Aik
antisera prebound to protein A-Sepharose for 2 h on a rotator,
then the immunocomplex was washed 3 times with lysis buffer and an
additional three times with kinase buffer (100 mM Tris, pH
7.5, 1 mM dithiothreitol, 2 mM EDTA, 20 mM MgCl2, 10 mM MnCl2).
In vitro Aik kinase reactions were performed for 20 min at
37 °C in the kinase buffer containing 20 µg of dephosphorylated
For indirect
immunofluorescence experiments, HeLa cells cultured on slide glasses
were fixed by placing them directly into absolute methanol for 10 s. Extraction was done by incubating the cells with PBS for 10 min at
room temperature. Cells were incubated with the antibody of interest
for 1 h at 37 °C, washed extensively in PBS, followed by
incubation with the appropriate secondary antibodies conjugated with
either fluorescein (Cappel Laboratories) or Cy3 (Pharmacia Biotech
Inc.). DNA was visualized by adding DAPI at a concentration of 0.1 µg/ml.
Homology search of randomly sequenced cDNAs revealed that
one of these clones encoded a putative protein kinase. Since this clone
did not contain initiation methionine, a
A computer search of the protein sequence data base (GenBankTM)
revealed that the kinase domain of the isolated kinase shares 86, 86, 72, 59, and 49% identity with those of XLP46APK, XLP46BPK, STK-1 (30),
Aurora (11), and Ipl1 (29), respectively.2
Therefore, we designated this novel kinase as an Aurora/Ipl1-related kinase, Aik.3 Multiple alignment of the
kinase domains is shown in Fig. 2. Amino acids that are
the same as Aik are indicated by asterisks. Since Aik shares
little amino acid sequence identity with kinases other than these five,
it is reasonable to assume that these kinases constitute a subfamily of
serine/threonine kinases. Although the N-terminal domain of Aik shares
little homology with Aurora and Ipl1, two closely related protein
kinases from Xenopus laevis XLP46APK and XLP46BPK exhibited
high homology with Aik throughout the entire sequence (approximately
62%). These results indicate that Aik is a novel member of the
Aurora/Ipl1-related protein kinase family.
The tissue distribution of aik was examined by
Northern hybridization. 32P-Labeled random-primed cDNA
corresponding to the human Aik kinase domain (nucleotides 373-1285)
hybridized with a single transcript of 2.4 kb. Expression was very high
in testis (Fig. 3), and weak bands were detected in
skeletal muscle, thymus, and spleen by long exposure of the film (data
not shown). Thus, aik is predominantly expressed in tissues
with proliferating cells.
To learn whether Aik is expressed only in testis or in various
proliferating cells, its expression at protein levels was examined in
some cell lines by Western blotting analysis. The affinity-purified antibody against Aik protein recognized a single band of 46-kDa Aik
protein (Fig. 4). HeLa cells showed the highest
expression level among the cell lines examined. Aik was also expressed
in other examined cell lines at lower level than HeLa cells.
Despite
intensive investigation of the phenotypes of ipl1 and
aurora mutants, their protein expression profiles or kinase activities have not been well characterized. HeLa cells have been used
in many investigations as a suitable line to analyze the biological
phenomena during cell cycle progression, because the procedure for cell
cycle synchronization is established and mitotic indexes were examined
in several studies (34). In addition, HeLa cells were found to express
a high content of Aik protein as shown in Fig. 4. To examine the cell
cycle dependence of aik expression, we performed Northern
hybridization on HeLa cells synchronized by double thymidine block
(Fig. 5A). 32P-Labeled human
aik was hybridized with a band of 2.4-kb transcript using
RNAs extracted from HeLa cells synchronized at and released from S
phase. The aik mRNA increased after the release, peaked at 8-12 h, and then decreased. Since mitotic index of HeLa cells peaks
at 12-13 h after the release from S phase block (34), high expression
of aik mRNA was observed at G2/M phase, but
expression was low in G1 and S phase (Fig.
5A).
Aik protein expression level was examined by Western blotting to see
whether it is also dependent on cell cycle progression of HeLa cells
synchronized at S phase with thymidine or at M phase with nocodazole
(Fig. 5). Aik protein level peaked by 4-fold at 12 h after S phase
release and decreased to the basal level at 16 h (Fig.
5B), showing a delay compared with the changes in mRNA expression. Aik protein level decreased dramatically in M phase release, and it was only 20% of the initial M phase level (Fig. 5C).
Expression of cyclin B was also studied to monitor the cell cycle
progression, and the level changed in a similar pattern to Aik,
indicating predominant expression of Aik at M phase. However, the Erk1
protein level did not change significantly during the cell cycle
progression (data not shown).
To confirm the kinase activity of Aik,
immunoprecipitates were used as enzyme sources from synchronized HeLa
cells, and dephosphorylated
Clues to the function of Aik kinase were sought by
examining subcellular localization in HeLa cells using indirect
immunofluorescent microscopy (Fig. 7). Anti-
We have identified a novel human gene encoding a serine/threonine
kinase designated as aik. The deduced Aik amino acid
sequence contains all of the amino acids conserved in serine/threonine kinases and is most closely related to members of the Aurora/Ipl1 family protein kinases, which are known to be required for normal chromosome segregation in Drosophila (11) and yeast (29). Temperature-sensitive mutants of IPL1 cause chromosome
number alteration (10). Temperature-sensitive mutants of
aurora form monopolar spindle and cannot segregate the
chromosomes (11). The conservation of amino acid sequences through
entire sequences between Aik and two kinases in Xenopus
suggests that closely related kinases to Aik must exist in vertebrates.
The sequence homology strongly suggests that Aik is a member of the
Aurora/Ipl1 family protein kinases conserved from yeast to human and
that it might play a role in chromosome segregation as do Aurora and
Ipl1.
The tissue distribution of aik examined by Northern blotting
showed that expression was very high in testis (Fig. 3) and weak bands
were detected in skeletal muscle, thymus, and spleen. Expression of Aik
at protein levels was examined in some cell lines by Western blotting
analysis. All of the proliferating cells examined expressed Aik protein
with HeLa cells showing the highest level (Fig. 4). A computer search
of protein sequence data base (GenBankTM) revealed the existence of Aik
transcripts in several infant tissues such as brain (accession no.
Z43879[GenBank]) or liver spleen (accession no. N64029[GenBank]). These results indicate
the expression of Aik in some proliferating cells, suggesting its
possible involvement in cell proliferation. The expression of mouse
STK-1 is high in the proliferating tissues of testis, spleen, small
intestine, and colon (30). The difference of the expression pattern
between Aik and STK-1 may suggest some functional difference between
them.
Since the phenotypes of IPL1 and aurora mutants
are abnormalities in chromosome segregation, it is conceivable that the
homologous kinase Aik may also function at M phase. The present
investigation provided evidence supporting this notion. Aik is
predominantly expressed in a subset of proliferating tissues or cells.
Cell cycle-dependent expression of Aik was observed in HeLa
cells by Northern and Western blotting. The levels of both are low in
G1/S but high in G2/M (Fig. 5). Since the
reduction of Aik was observed after mitosis, a sequence similar to the
destruction box was searched on a computer. Several destruction
box-like sequences were found in the Aik kinase domain: RTTLCGTLDY,
amino acids 285-294; RPMLREVLE, amino acids 370-378; and REVLEHPWI,
amino acids 374-382. One or more of these sequences might function as
a destruction box of Aik to be degraded after M phase.
The kinase activity of Aik also peaked at M phase (Fig. 6). Since this
activity appeared to be correlated with the protein levels, it is
conceivable that it is at least partly regulated by the changes in
protein content. These results suggest that Aik plays a role in cell
cycle regulation at M phase in relation to the function of the
centrosome/spindle pole region. As the N-terminal portion of Aik has
little homology with Aurora, Ipl1, or STK-1, it might also be possible
that the N-terminal region acts to regulate the kinase activity.
Aik is localized to centrosome and spindle pole from prophase to
anaphase (Fig. 7). This subcellular localization as well as the
expression profile during cell cycle suggests that Aik has a role(s) in
centrosome or spindle organization at M phase. Although the subcellular
localization of Aurora and Ipl1 has not been well studied, the
phenotypes of these mutants suggest that the localization of these
proteins may also be at centrosome or spindle pole body and may be
involved in spindle organization. Thus, the localization of Aik
suggests that it may function in spindle formation like Aurora.
Various centrosomally localized protein kinases have been identified in
vertebrate cells, including Cdc2 (12), Plk1 (13), Fyn (14), Src (15),
and A kinase (16). Some of the kinases were suggested to function as
regulators of microtubule organization and spindle formation. Ectopic
expression of mutant cyclin B2 in HeLa cells causes cell division
arrest with multiple mitotic spindle poles (17). In a recent study, one
of the substrates of Cdc2 kinase was identified to be a kinesin-like
motor protein and to regulate the centrosomal localization (8). Plk1 is
phosphorylated and activated by Cdc2 kinase (28). Aik was
phosphorylated in M phase (data not shown), suggesting that the protein
kinase cascade containing Aik may play a part in centrosomal
function.
Complementation experiments were performed to examine whether Aik is a
functional homolog of Ipl1. Low copy number plasmid harboring Aik
coding region was transformed to Saccharomyces cerevisiae ipl1 mutant, but Aik failed to complement the mutant phenotype (data not shown). Although the sequence homology and the subcellular localization of Aik suggested that these two kinases might have similar
functions, this was not the case. It is conceivable that because of the
difference in amino acid sequence between Aik and Ipl1, a substrate or
a regulatory protein cannot interact with Aik in yeast cells. Some
human homologs of yeast proteins are known to lack the possibility to
complement the yeast mutants, so that we cannot exclude the possibility
that Aik is not a functional human homolog of Ipl1. It remains to be
elucidated whether Aik plays the same role as Ipl1 in human.
Since Aik has protein kinase activity, the question arises what the
physiological substrate(s) is for it. Several centrosomal components
have been shown to be phosphorylated and to control the rate of
microtubule dynamic behavior at the centrosome. Furthermore, phosphorylation regulates the function of a microtubule-based motor
protein such as kinesin-like protein (8). The substrate of Aik has not
been identified, nor has that for Aurora or Ipl1. Our experiments
showed a high molecular weight phosphorylated band in the in
vitro kinase assay, suggesting that a putative Aik substrate was
co-immunoprecipitated and phosphorylated with the enzyme. The candidate
substrate protein was co-immunoprecipitated only when it was extracted
from M phase-blocked cells (data not shown). The substrate of Aik
kinase may be a protein that participates in centrosome function.
We are now investigating the kinetics of Aik kinase and identifying
substrates to elucidate the function of the protein.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D84212[GenBank]. We are grateful to Dr. M. Saitoh (Hokkaido
University) for providing CMK cells, Dr. H. Wakasugi (Japan National
Cancer Center) for 1G8, Dr. T. Okazaki (Osaka Medical College) for
HL60, and Dr. C. S. Chan (University of Texas, Austin) for providing us with unpublished results on complementation assay. We thank Dr. Y. Kitajima (Department of Dermatology, Gifu University) for allowing us
to use a fluorescent microscope, and Dr. Y. Banno (Department of
Biochemistry, Gifu University) for kind help in preparation of specific
antibody. We are indebted to Dr. Y. Nozawa (Department of Biochemistry,
Gifu University) for helpful discussion.
Tsukuba Life Science Center,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Cloning and Sequencing of a Human Aurora/Ipl1-related Kinase
cDNA
gt11 human B cell cDNA library
(Epstein-Barr virus-transformed) (CLONTECH) was
screened using this (aik) cDNA fragment as a probe.
Hybridization conditions were as described (31). Positive clones were
isolated, and the insert DNA was subcloned into pBluescript. The
nucleotide sequences of both strands of the isolated clone were
determined by the dideoxy chain termination method.
-actin cDNA. Hybridization was
performed as described (31). The blots were washed at 55 °C for
1 h in a buffer containing 2 × SSC and 0.5% SDS, and
signals were visualized by autoradiography using Kodak XAR film.
-casein, 5 mM cold ATP, and 20 µCi of
[
-32P]ATP (3000 Ci/mmol). The reaction was terminated
by adding sample buffer, and the samples were subjected to analysis by
SDS-polyacrylamide gel electrophoresis on 12% gel, followed by
autoradiography with Kodak XAR film.
Cloning of a cDNA Encoding a Human Aurora/Ipl1-related Kinase,
Aik
gt11 library was rescreened
with the obtained clone. The nucleotide and deduced amino acid sequence
of the longest clone obtained have been deposited in
DDBJ/EMBL/GenBankTM (accession number D84212[GenBank]). This cDNA encodes
402 amino acids with a predicted molecular mass of 45.9 kDa. The
deduced amino acid sequence contains 11 conserved regions
characteristic of protein kinase catalytic domain at the C terminus of
the protein. Eleven conserved subdomains known in various protein
kinases are indicated by Roman numerals (Fig. 1).
Fig. 1.
Nucleotide and deduced amino acid sequences
of human aik cDNA. The putative translation
initiation methionine and the termination codons are
underlined. The upstream in-frame stop-codon is
boxed. The conceptual translation yields a 402-amino acid
protein. Nucleotide and amino acid residue numbers are shown at the
right of each line.
[View Larger Version of this Image (86K GIF file)]
Fig. 2.
Amino acid alignment of sequences of human
Aik, Xenopus XLP46APK, mouse STK-1, Drosophila
Aurora, and S. cerevisiae Ipl1. The protein sequences
are presented in single-letter code. The identical amino
acids with Aik are indicated as asterisks. Gaps were
introduced into sequences to optimize alignments. Amino acid residue
numbers are shown at the right of each line. Eleven
conserved subdomains of protein kinases are indicated by Roman
numerals.
[View Larger Version of this Image (65K GIF file)]
Fig. 3.
aik mRNA level in human
tissues. Northern hybridization was performed with
poly(A)+ RNAs from human tissues. Blotted filters with 2 µg of poly(A)+ RNA from various human tissues were
hybridized with 32P-labeled aik cDNA or
-actin cDNA probes.
[View Larger Version of this Image (82K GIF file)]
Fig. 4.
Aik protein level in various human cell
lines. Fifty µg of lysed protein from various cell lines were
loaded on each lane and subjected to Western blotting. HeLa cells,
megakaryoblastic leukemia cell line CMK, Epstein-Barr virus-transformed
B cell line 1G8, Raji, HL60, and neuroblastoma cell line NB69 exhibited a 46-kDa band of Aik.
[View Larger Version of this Image (85K GIF file)]
Fig. 5.
Cell cycle-dependent expression
of Aik. Total RNA was isolated from synchronized HeLa cells at the
indicated time points. Twenty five µg of total RNA was loaded on each
lane (A). A full-length coding region of Aik cDNA was
used for a probe. The size of the Aik transcript was estimated as 2.4 kb. 28 S ribosomal RNA was shown as a control. Mitotically synchronized
HeLa cells at S phase (B) or M phase (C) were
prepared, and 50 µg of total protein was loaded on each lane and
subjected to Western blotting using anti-Aik or anti-cyclin B
(Cyc B) antibodies.
[View Larger Version of this Image (58K GIF file)]
-casein was included as an exogenous
substrate. Both S and M phase-released HeLa cell extracts were used for
the enzyme source to study the changes in Aik kinase activity during
cell cycle progression. In S phase-released cells, the phosphorylation
of
-casein increased to a peak at 12 h, when the cells are at M phase (34), and then rapidly decreased after cell division (Fig. 6). This was confirmed with M phase-released samples in
which phosphorylated
-casein rapidly decreased 120 min after release (Fig. 6). No kinase activity was detected in the precipitates with
preimmune antisera. Considering the results of protein expression shown
in Fig. 5, Aik kinase activity showed a good correlation with the
protein contents.
Fig. 6.
Cell cycle-dependent activation
of Aik kinase. Mitotically synchronized HeLa cells at S or M phase
were collected, and total protein was isolated at the indicated time
points. The panels indicate the times after the release from S phase
(h) and M phase (min). The supernatant was immunoprecipitated with
preimmune (Pre) or Aik antisera prebound to protein
A-Sepharose. In vitro Aik kinase reactions were performed in
a buffer containing -casein and [
-32P]ATP.
[View Larger Version of this Image (32K GIF file)]
-tubulin
antibody and DAPI staining revealed limited localization of Aik in
centrosome of mitotic cells. Further careful analysis of these cells
from prophase to anaphase showed Aik localized not only in centrosome
but also spreading to the spindle pole region. However, it was not
detected in this region in telophase and interphase HeLa cells at all. In COS7 cells and melanoma cell line MMG1, similar centrosomal localization of Aik was also observed (data not shown).
Fig. 7.
Localization of Aik kinase to spindle pole
region. Localization of Aik during mitosis was examined by
indirect immunofluorescent microscopy. HeLa cells were triple-stained
with affinity-purified polyclonal anti-Aik antibody, monoclonal
anti--tubulin antibody, and DAPI. The cell cycle stages of HeLa
cells are shown at left.
[View Larger Version of this Image (79K GIF file)]
*
This work was supported in part by a grant-in-aid from the
Ministry of Education, Science, Sports, and Culture of Japan, and grants from the Life Science Research Project of RIKEN and the Science
and Technology Agency of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 81-58-267-2367;
Fax: 81-58-267-2950; E-mail: bunbyo{at}cc.gifu-u.ac.jp.
1
The abbreviations used are: PBS,
phosphate-buffered saline; kb, kilobase pair(s); TBST, Tris-buffered
saline plus Tween 20; DAPI, 4,6-diamidino-2-phenylindole
olihydrochloride.
2
An unpublished sequence of mouse Ayk1 (GenBankTM
accession no. U80932[GenBank]) has recently been deposited that has high
homology with the kinase.
3
A gene symbol of STK6 for Aik was
obtained from the HUGO Nomenclature Committee of GDB.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.