From the Department of Cell Biology and Neuroscience,
Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita,
Osaka 565-0871 and the § Institute for Molecular and
Cellular Biology, Osaka University, 1-3 Yamada-oka, Suita,
Osaka 565-0871, Japan
Received for publication, March 13, 2001
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ABSTRACT |
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In a previous study, we demonstrated that the
forkhead associated (FHA) domain of pKi-67 interacts with the novel
kinesin-like protein, Hklp2 (Sueishi, M., Takagi, M., and Yoneda, Y. (2000) J. Biol. Chem. 275, 28888-28892). In this
study, we report on the identification of a putative RNA-binding
protein of 293 residues as another binding partner of the FHA domain of
pKi-67 (referred to as NIFK for nucleolar protein
interacting with the FHA domain of
pKi-67). Human NIFK (hNIFK) interacted with the FHA domain of pKi-67 (Ki-FHA) efficiently in vitro when hNIFK was
derived from mitotically arrested cells. In addition, a moiety of hNIFK was co-localized with pKi-67 at the peripheral region of mitotic chromosomes. The hNIFK domain that interacts with Ki-FHA was mapped in
the yeast two-hybrid system to a portion encompassed by residues 226-269. In a binding assay utilizing Xenopus egg
extracts, it was found that the mitosis-specific environment and two
threonine residues within this portion of hNIFK (Thr-234 and Thr-238)
were crucial for the efficient interaction of hNIFK and Ki-FHA,
suggesting that hNIFK interacts with Ki-FHA in a mitosis-specific and
phosphorylation-dependent manner. These findings provide a
new clue to our understanding of the cellular function of
pKi-67.
The Ki-67 antigen (pKi-67), originally identified as the antigen
for a monoclonal antibody raised against the nuclear extract from a
Hodgkin's lymphoma-derived cell line, was characterized as a class of
proteins that localize around mitotic chromosomes (1). As a result, it
is assumed that pKi-67 is involved in mitotic chromosome organization.
pKi-67 is a convenient cell proliferation marker, since its expression
is restricted to growing cells (2). Although the recent identification
and characterization of a marsupial counterpart of pKi-67, which is
referred to as chmadrin, suggests that pKi-67 plays some type of role
in the organization of higher order chromatin structure (3), the actual
role of pKi-67 in the cell cycle progression remains unclear.
The N-terminal portion of pKi-67 is well conserved between human pKi-67
and chmadrin (62% identical) and contains a forkhead associated
(FHA)1 domain. It was
originally reported that the FHA domain constituted a region that has
been conserved in a subset of forkhead-type transcription factors (4).
The sequence profile has been reported for a variety of proteins
with diverse functions (transcription, DNA repair, cell cycle
progression, etc.). In several instances, the FHA domain preferentially
recognizes partner proteins when they are present in the phosphorylated
form (5-8). Moreover, the strong specificity of the FHA domain for
phosphopeptides has been clearly demonstrated by binding assays with
synthetic phosphopeptides (9). Therefore, it is currently thought that
the FHA domain is a general phosphopeptide recognition motif that is
involved in certain phosphopeptide-mediated signal transduction
pathways (10). A search for the interaction partner(s) of the FHA
domain of pKi-67, which could exist in the phosphorylated form, is an intriguing issue, since such interactions would constitute a
significant component of the regulation of the cell cycle progression.
In order to investigate this issue further, a two-hybrid screening from
a HeLa cDNA library was carried out using the N-terminal portion of
pKi-67 as bait. As a result, two novel molecules that bind to the FHA
domain of pKi-67 have been isolated. In our previous study, we revealed
that one of them was a novel kinesin-like protein, Hklp2 (8). In this
study, we report on the characterization of the other protein, which we
refer to as NIFK (nucleolar protein interacting
with the FHA domain of pKi-67). The interaction
between human NIFK (hNIFK) and the FHA domain of pKi-67 occurs in a
manner that is dependent on the mitosis-specific modification of hNIFK, which could include the phosphorylation of Thr-234 and Thr-238. Although the concrete function of hNIFK remains to be examined, the
interaction found here provides some clues to our understanding of the
relationship between the expression of pKi-67 and cell cycle
progression. Moreover, the findings herein represent an example of a
phosphopeptide recognition of the FHA domain and will contribute to a
better understanding of the mode of action of the FHA domain.
Molecular Cloning of NIFK--
One of the clones obtained by the
two-hybrid screen using the FHA domain of pKi-67 as bait (8) contained
a 293-residue open reading frame (ORF) of hNIFK. Extensive trials of
5'-rapid amplification of cDNA ends using SuperScript II (Life
Technologies, Inc.) or BcaPLUS RTase (Takara) did not result in any
additional upstream sequences. By using the amino acid sequence of
hNIFK as a query, the mouse expressed sequence tag (EST) data base was searched by TBLSTN, resulting in finding AA260128, AI048665, and
AI851818 as probable cDNA fragments coding for the mouse NIFK
(mNIFK). The mNIFK-specific primers P1 (5'- ATGGCTGGGTTAGCAGGCCC-3') and P2 (5'- TCACTGCTTGCTCTTCCTTTTTCTCGG-3') were designed with respect
to these sequences. The cDNA of mNIFK was recovered by reverse
transcriptase-polymerase chain reaction on polyadenylated RNA of mouse
Ehrlich tumor cells (a gift of Dr. Takuya Shimamoto, Osaka University)
using P1 and P2, subcloned directly into pGEM-T easy (Promega), and
analyzed using an ABI310 Genetic Analyzer (Applied Biosystems, Inc.).
The nucleotide sequence of mNIFK was identical to that of the clone
2310021G21 reported by the RIKEN group (11), except that the latter
contained two additional nucleotides (cytosine and guanine after
cytosine 914 and guanine 920 of the former, respectively). According to
the RIKEN sequence, the amino acid similarity with hNIFK was lost at
their C-terminal regions. Moreover, the same sequences as ours
at the position of disagreement could be found from the mouse EST data
base. The sequence described here was determined with great care, and
we are certain that it is correct. Since the RIKEN clone was a
cap-trapper selected cDNA (12), the sequence of the mNIFK contains
a full-length ORF.
Recombinant Proteins--
Plasmids coding for GST fusions of
Ki-FHA (residues 1-168 of pKi-67) and the N-terminal half of hNIFK
(residues 1-146) were created by cloning the appropriate inserts into
pGEX (Amersham Pharmacia Biotech). A plasmid coding for the
maltose-binding protein fused to hNIFK-(1-146) was created by cloning
the appropriate insert into pMALc2 (New England Biolabs, Inc.). BL21
(DE3), transformed with these plasmids, was grown at 37 °C to a
density of ~0.6 (A600), cooled to
20 °C, and cultured in the presence of 0.1 mM
isopropyl- Antibody Production--
Antisera were prepared by immunizing
two rabbits (kbs:JW) (purchased from Kitayama Labes Co., Ltd.) with
purified GST-hNIFK-(1-146). The antibodies were affinity purified
against maltose-binding protein fused to hNIFK-(1-146), which had been
immobilized on a nitrocellulose membrane using a procedure described
previously (13).
Cell Culture and Synchronization--
HeLa cells were grown at
37 °C in a 5% CO2 atmosphere in RPMI 1640 supplemented
with 10% heat-inactivated fetal bovine serum and
penicillin/streptomycin (100 IU/ml and 100 µg/ml, respectively). Prometaphase-arrested HeLa cells were obtained as described previously (8). S-phase-arrested HeLa cells were obtained by treating ~75%
confluent cultures with 1 µg/ml aphidicolin for 17 h.
Preparation of Cell Extracts and Pull-down Assay--
Cell
extracts were prepared from asynchronous or synchronized HeLa cells
essentially as described previously (8) with minor modifications in the
buffer composition. The extraction buffer (EB150) was supplemented with
RNase A and okadaic acid at 50 µg/ml and 0.5 µM,
respectively. For the preparation of the cell extracts shown in
lanes 2 and 4 of Fig. 2B, phosphatase
inhibitors (NaF, Immunofluorescence--
HeLa cells, grown on coverslips, were
washed once with ice-cold PBS(+) (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4, and 2 mM
MgCl2), pre-extracted with 0.1% Triton X-100 in PBS(+) for
2 min on ice, and fixed with 4% formaldehyde in PBS(+) for 10 min at
room temperature. In the experiment shown in Fig. 3A (d-f), cells were treated with 50 µg/ml RNase A in PBS(+)
at 37 °C for 15 min before fixation. Procedures after fixation were the same as those described previously (3). Anti-hNIFK antibodies were
used at a level of ~1 µg/ml. MIB-1 (Immunotech), the monoclonal antibody against pKi-67, was used at 1:100. The photographs shown in
Fig. 3, A and B, were acquired with Axiophot2
(Zeiss) and an LSM510 confocal microscope (Zeiss), respectively.
Mapping and Dissection of the FHA Interaction Domain of hNIFK in
Yeast--
Full-length hNIFK and its deletion derivatives (depicted in
Fig. 4A) were subcloned into pGAD GH
(CLONTECH) using standard methods. Starting from
pGAD GH-hNIFK-(226-269), T234A, T238A, T240A, and S247A mutants
were prepared using the QuickChange site-directed mutagenesis system
(CLONTECH) and were verified by sequencing. All
proteins were tested for their interaction with the FHA domain of
pKi-67 (residues 1-99), which was subcloned into pGAD424
(CLONTECH), in strain Y190 via the expression of
reporter genes.
In Vitro Transcription-Translation and in Vitro Association
Experiments--
Full-length hNIFK was subcloned into pcDNA3.1(+)
(Invitrogen) and pRSETc (Invitrogen) using standard methods to generate
pcDNA-hNIFK and pRSETc-hNIFK, respectively. Starting from
pRSETc-hNIFK, T234A and T238A mutants were prepared as described above.
Cloned proteins (hNIFK, His-tagged hNIFK(T234A), and His-tagged
hNIFK(T238A)) were transcribed and translated using the TNT T7 Quick
Transcription/Translation System (Promega) in the presence of
[35S]methionine (PerkinElmer Life Sciences) for 2 h
at 30 °C in an 11-µl system and incubated with or without mitotic
Xenopus egg extracts, which contained an ATP-regeneration
system (16.2 µl) for 30 min at 30 °C in a final volume of 60 µl.
A portion of each reactant (6 µl) was separated on 12.5%
SDS-polyacrylamide gel electrophoresis (PAGE). Other portions of each
reactant (25 µl) were separated on the same gel after affinity
purification with GST or GST-Ki-FHA-(1-168) beads. Labeled
proteins were visualized by autoradiography. Xenopus egg
extracts were prepared from unfertilized eggs as described previously
(14), and their reliability was checked by the retardation of the
electrophoretic mobility of hamster Cdc25C, expressed from
pET3a-hamCdc25C (15) (a gift from Drs. Hideo Nishitani and Takeharu
Nishimoto, Kyusyu University), after incubation, as described
previously (16).
Cloning of Human and Mouse NIFK--
By using the two-hybrid
screening from HeLa cDNA library using the FHA domain of pKi-67
(named "Ki-FHA" in this report) as bait, we were able to obtain
five positive clones. Two of these encoded certain portions of a novel
kinesin-like protein (termed Hklp2), and the interaction between pKi-67
and Hklp2 occurred preferentially in a cell extract obtained from
nocodazole-arrested cells (8). Here we analyzed the remainder of the
positive clones. All clones contained overlapped nucleotide sequences,
suggesting that they were derived from the same gene. The longest clone
contained a 293-residue ORF (Fig.
1A) and the 3'-untranslated
region of ~500 bases. The deduced protein had a calculated molecular
mass of 34 kDa and contained a putative RNA binding domain at residues 42-122, which was characterized as comprising two ribonucleoprotein motifs (17) (Fig. 1, A and B). We refer to the
protein as hNIFK (human nucleolar protein interacting with the FHA
domain of Ki-67 antigen).
By using the protein sequence of hNIFK as a query, a mouse EST data
base was searched by TBLSTN. Several clones were likely to encode
certain portions of the mouse counterpart, mNIFK. By linking the
sequences of these clones, the plausible full-length sequence for mNIFK
could be obtained. To confirm the existence and identity of this ORF,
the cDNA was amplified from mouse Ehrlich tumor cells by reverse
transcriptase-polymerase chain reaction and sequenced. The confirmed
ORF in mouse codes for a 317-amino acid protein with a basic insertion
at the residues 228-249. On the amino acid level, the NIFK of human
and mouse showed 54 and 75% identity, respectively, in overall
sequence and within the RNA binding domain (Fig. 1, B and
C). Although a full-length mouse cDNA collection
prepared by the RIKEN group (11) contains essentially the same
cDNA, there are several minor conflicts resulting in differences in
the amino acid sequences at their C-terminal portions (see
"Experimental Procedures" for detail).
Mitotically Modified hNIFK Interacts Efficiently with the FHA
Domain of pKi-67--
The FHA domain appears to be a phosphopeptide
recognition domain (10). In our previous report, we revealed that the
FHA domain of pKi-67 (Ki-FHA) actually recognized mitotically modified, probably phosphorylated, Hklp2 (8). To examine the issue of whether
Ki-FHA also recognizes phosphorylated hNIFK, we first prepared specific
antibodies against hNIFK. The antibodies recognized a protein that had
an apparent molecular mass of 38 kDa (band III) from the cell extracts
of asynchronous and S-phase-arrested HeLa cells (Fig.
2A, lanes 1 and 2,
and Fig. 2B, lane 1). In contrast, the antibodies mainly
recognized 44- and 40-kDa proteins (bands I and II) in mitotic HeLa
cells (Fig. 2A, lane 3 and Fig. 2B, lane 3). When
mitotic HeLa extracts were prepared in the absence of phosphatase
inhibitors, the band I disappeared and was replaced by a smeared signal
between bands II and III (Fig. 2B, lane 2). After incubation
of the phosphatase inhibitor-free extract for 30 min at 30 °C, the
band III became prominent (Fig. 2B, lane 4). These
observations suggest that hNIFK is differently modified (having at
least two variations) in mitosis and that the modifications include
phosphorylation(s) that is the origin of the mobility shifts in
SDS-PAGE. The phosphorylation(s) of hNIFK was counteracted by a
cellular phosphatase(s). From the cell extracts of differently synchronized HeLa cells, proteins that interacted with Ki-FHA were
pulled down with a GST-Ki-FHA fusion protein and analyzed by Western
blotting with the anti-NIFK antibodies. As shown in Fig. 2A,
the mitotic forms of hNIFK (bands I and II) were efficiently pulled
down (lane 3"). A small quantity of ~39-kDa protein was pulled down from the asynchronous and S-phase HeLa extracts
(lanes 1" and 2"). This band is not identical to
band II or band III in its electrophoretic mobility and, therefore, is
unlikely to be derived from the mitotic cells that existed somewhat in
the asynchronous and S-phase-arrested cultures.
Comparison of Cellular Behaviors of hNIFK and pKi-67 during the
Cell Cycle--
The cellular localization of hNIFK was examined in
HeLa cells using the affinity-purified antibodies against hNIFK. hNIFK was found mainly in nucleoli but also in other (nucleoplasmic and
cytoplasmic) spaces (Fig. 3A,
b). Essentially the same observation was obtained as for the
localization of GFP fusion protein of hNIFK (not shown). The staining
of hNIFK was greatly reduced by treatment of cells with RNase A prior
to fixation (Fig. 3A, e), supporting the conclusion that
hNIFK is an authentic RNA-binding protein, as deduced from its primary
structure. The remaining population of hNIFK, after RNase extraction,
was found at the center of nucleoli (Fig. 3A, e), whereas
pKi-67 was mainly localized at the outer region of nucleoli (possibly
the region known as "dense fibrillar component") before and after
RNase extraction (Fig. 3A, c and f). In mitosis,
hNIFK would be likely to co-localize with pKi-67 at the surface of
mitotic chromosomes (Fig. 3B). In most cases, an hNIFK
moiety was preferentially associated with certain regions of the
mitotic chromosomes (Fig. 3B, arrowheads). The issue of
which regions of the chromosomes are preferred by hNIFK remains to be
examined.
The Residues 226-269 of hNIFK Are Sufficient for Its Interaction
with the FHA Domain of pKi-67 in Yeast--
To map the FHA
domain-binding region of hNIFK, a deletion analysis of hNIFK was
performed using the yeast two-hybrid system. Various deletion mutants
(Fig. 4A), expressed as fusion
proteins with the GAL4 activation domain (denoted GAL4 AD), were tested for interaction with the FHA domain of pKi-67 fused to the DNA binding domain (DBD) of GAL4 in yeast. As summarized in Fig.
4A, the region corresponding to the residues 226-269 of
hNIFK was found to be sufficient for interaction with the FHA domain of pKi-67. Alignment of this region with the corresponding region of mouse
NIFK reveals that this region is well conserved and contains conserved
threonine (Thr-234, Thr-238, and Thr-240) and serine (Ser-247) residues
(Fig. 4B). Since the FHA domains are thought to interact
directly with phosphorylated targets (10), it is likely that some of
these residues represent targets of phosphorylation and subsequent
binding of the FHA domain of pKi-67. We therefore constructed hNIFK
mutants that contained point mutations at the above four residues and
tested their interaction with Ki-FHA in the yeast two-hybrid system
(Fig. 4C). A substitution of Thr-240 or Ser-247 with alanine
had no effect on the interaction. In contrast, a single alanine point
mutation at either Thr-234 or Thr-238 abolished the interaction.
Threonine 234 and 238 of hNIFK and Their Phosphorylations Appear to
be Crucial for Interaction with the FHA Domain of pKi-67--
To test
whether hNIFK binding to pKi-67 is dependent on the mitotic
phosphorylation of hNIFK at Thr-234 and Thr-238, we applied an
experimental system, originally developed by Kirschner and co-workers
(18), to identify mitotic phosphoproteins systematically. In our
experiment, in vitro translated hNIFK and its mutants (T234A and T238A) were incubated with mitotic extracts of Xenopus
eggs (CSF extracts) and then separated on SDS-PAGE directly or after affinity purification with GST-Ki-FHA beads. After incubation with the
CSF extract, certain population of in vitro translated hNIFK
migrated with a reduced mobility (Fig.
5A, lane 2), the origin of
which was phosphorylation(s) since the shifts were abolished by
treatment with alkaline phosphatase (not shown). hNIFK was pulled down
efficiently with GST-Ki-FHA beads, not with GST beads, only after
incubation with the CSF extract (Fig. 5A). In contrast, T234A and T238A mutants of hNIFK, which had been treated with the CSF
extract, were pulled down less efficiently with GST-Ki-FHA beads (Fig.
5C). These observations indicate that hNIFK binds specifically to Ki-FHA in a mitosis-specific manner and is dependent on
the presence of Thr-234 and Thr-238 in hNIFK, suggesting that Thr-234
and/or Thr-238 of hNIFK are phosphorylated and recognized by Ki-FHA
in a mitosis-specific manner.
A novel protein, hNIFK, has been identified as a binding partner
of the FHA domain of pKi-67. We also cloned the probable mouse
counterpart, mNIFK. NIFK-related proteins from other organisms exist
and include those from Saccharomyces cerevisiae
(Z71386), Schizosaccharomyces pombe (AL023777),
Caenorhabditis elegans (Z35663), Drosophila
melanogaster (AE003739), and Arabidopsis thaliana
(T48456). All these proteins show considerable similarity with respect
to the RNA binding domains and moderate similarity in the flanking
regions. It should be noted that Z71386, AL023777, and T48456
lack the regions that correspond to the Ki-FHA binding region of hNIFK.
Given the fundamental function of hNIFK, pKi-67 might have no function
at all. The issue what is the "fundamental" function of hNIFK could
be addressed by the analyses in genetically amenable organisms. From
recent functional genomic analyses carried out after genome sequencing
projects, aspects of the functions of hNIFK might be deduced. In fact,
the systematic disruption of each gene in S. cerevisiae (19)
and the systematic RNA-mediated interference of each gene on chromosome
III of C. elegans (20) showed that NIFK-related molecules of
both organisms were essential for viability. The detailed cellular
function, however, remains to be determined. Considering the existence
of the putative RNA binding domain and the nucleolar localization in
interphase, hNIFK is thought to function in rRNA metabolism. Our
preliminary observation indicates that hNIFK forms protein complexes,
both in interphase and mitotic cell extracts. The identification of the
components of such a complex might allow for a better understanding of
the fundamental function of hNIFK.
The findings here show that hNIFK interacts with Ki-FHA efficiently in
mitosis, although a small population of hNIFK, which was
uncharacterized, also interacts with Ki-FHA in other phases of the cell
cycle. It is most likely that the Thr-234 and Thr-238 residues of hNIFK
and their phosphorylation(s) are crucial for the interactions during
mitosis. Since the interaction with pKi-67, as discussed above, appears
to be dispensable for the fundamental function of NIFK-related
proteins, NIFK-related proteins might have begun to utilize pKi-67 for
some regulatory reasons or luxurious functions, as evolution
progressed. Since pKi-67 harbors a potent activity for driving
chromatin compaction at its C-terminal portion (LR domain) (3), the
putative activity of hNIFK might be regulated via chromatin compaction
mediated by the LR domain of pKi-67. In vertebrate cells, entry into
mitosis is accompanied by a global inhibition of transcription.
Ribosomal genes (rDNA) are also silenced during mitosis, which is
mainly under the control of Cdc2-cyclin B kinase (21-23). It is
tempting to speculate that hNIFK plays a role in rRNA synthesis and
that the silencing of rDNA transcription during mitosis is accomplished
through the mitosis-specific and phosphorylation-dependent
interaction of hNIFK with pKi-67.
The FHA domain has generated considerable interest, not only because of
its nature as a signaling module, which functions through interactions
with phosphorylated target molecules (10), but because of a link with
cancer; genes that are mutated in the Nijmegen breakage syndrome (NBS1)
and the Li-Fraumeni syndrome (CHK2) encode FHA domain-containing
proteins (24-27). Recent progress around the FHA domain, that is the
screening of optimal phosphopeptide sequences recognized by various FHA
domains (28-30) and the determination of the three-dimensional
structures of the Rad53p FHA domains (28-31), has enhanced our
knowledge of the mode of phosphopeptide recognition of FHA domains.
Although the issue of whether this knowledge fits situations that
actually occur in cells requires further testing, the identification of
actual ligands of FHA domains and the mapping of biologically relevant
FHA domain binding sites on these ligands have been accomplished only
in a limited number of cases (5-8). From this viewpoint, our findings
should be of some use as a new example. Further analysis of the
interactions between Ki-FHA and NIFK, along with past studies (10),
might result in a much more general understanding of the mode of
phosphopeptide recognition of FHA domains and thereby provide the basis
for the development of novel classes of therapeutic agents that target FHA-mediated signal transduction pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside for an additional
14 h at 20 °C. Proteins were purified from the bacteria
according to protocols recommended by the manufacturers.
-glycerophosphate, and okadaic acid) were excluded.
Cell extracts were incubated with GST or GST-Ki-FHA-(1-168) coupled to
glutathione-Sepharose beads (GS-4B; Amersham Pharmacia Biotech) for
1 h at 4 °C with occasional agitation, after which the beads
were washed extensively with EB150 containing okadaic acid (0.5 µM). Associated proteins were obtained by boiling the
sample buffer and separating in 10% acrylamide gel. hNIFK was detected
by Western blotting using anti-hNIFK antibodies at 0.5 µg/ml.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Primary structure of NIFK. A,
alignment of amino acid sequences of human and mouse NIFK. Identical
amino acids are marked by asterisks, and the
underline indicates the putative RNA binding domain.
Dashes correspond to gaps introduced to maximize alignment.
B, schematic comparison of human and mouse NIFK. The
putative RNA binding domains are represented by black boxes.
The 22-residue insert, which is found uniquely in mouse NIFK, is
shaded.
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Fig. 2.
The interaction of hNIFK with the FHA domain
of pKi-67 is cell cycle-regulated. A, HeLa cell
extracts prepared from asynchronous (AS),
aphidicolin-arrested (S), or nocodazole-arrested
(M) cultures were analyzed by immunoblotting with antibodies
specific for hNIFK, prior to (lanes 1-3) or after affinity
purification with GST beads (lanes 1'-3') or
GST-Ki-FHA-(1-168) beads (lanes 1"-3"). Lanes
1-3 show 10% of the amount of the input. hNIFK was detected as a
single band (band III) in asynchronous and
aphidicolin-arrested cells. In mitotic cells, hNIFK was detected mainly
as bands I and II. Asterisks indicate background signals due
to a large amount of recombinant GST-Ki-FHA-(1-168). B,
mitosis-specific modification of hNIFK consists of phosphorylation(s).
Cell extracts of mitotic HeLa cells were prepared in the presence
(lane 3) or absence (lanes 2 and 4) of
phosphatase inhibitors (PI), denatured with SDS-containing
sample buffer immediately (lanes 2 and 3) or
after incubation at 30 °C for 30 min (lane 4), and
analyzed by immunoblotting with antibodies specific for hNIFK.
Lane 1 is the cell extracts of aphidicolin-arrested HeLa
cells and represents the position of band III.
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Fig. 3.
Subcellular localization of hNIFK in
relation to pKi-67. A, HeLa cells were extracted with
0.1% Triton X-100 (TX-100) (a-c) or serially
extracted with Triton X-100 (0.1%) and RNase A (50 µg/ml)
(d-f) before fixation with 10% formalin. The cells were
doubly stained for hNIFK (green, b and e) and
pKi-67 (red, c and f). DNA was counterstained
with Hoechst 33342 (a and d). Bar, 10 µm. B, HeLa cells were fixed with 10% formalin and doubly
stained for pKi-67 (red, a and d) and hNIFK
(green, b and e). The right column
(c and f) indicates the merged images of pKi-67
and hNIFK. Arrowheads indicate the regions on mitotic
chromosomes where hNIFK seems to be associated preferentially.
Bar, 10 µm.
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Fig. 4.
Mapping of the FHA domain binding region in
hNIFK. A, identification of the region of hNIFK that
interacts with the FHA domain of pKi-67 (Ki-FHA). Various
hNIFK constructs in pGAD GH were tested for interaction with
Ki-FHA using two-hybrid system in yeast. +, positive interaction
(specific growth in selective media and activation of the
-galactosidase gene);
, lack of interaction (failure of cells to
grow on selective media). B, alignment of the FHA domain
binding region of human NIFK with the corresponding region (amino
acids 248-291) of mouse NIFK. C, identification of the
residues of hNIFK crucial for its binding with Ki-FHA. Various hNIFK
mutants in which each conserved threonine residue was mutated to
alanine were tested for the interaction with the Ki-FHA using
two-hybrid system in yeast. +, positive interaction;
, lack of
interaction.
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Fig. 5.
Interaction of hNIFK with Ki-FHA in a mitotic
environment in a manner dependent on Thr-234 and Thr-238 of hNIFK.
A, pull-down of in vitro translated hNIFK with
GST-Ki-FHA-(1-168). hNIFK was translated in vitro in the
presence of [35S]methionine, incubated with (lanes
2-2") or without (lanes 1-1") the CSF extracts,
pulled down with GST beads (lanes 1' and 2') or
GST-Ki-FHA-(1-168) beads (lanes 1" and 2"), and
analyzed by SDS-PAGE and autoradiography. Lanes 1 and
2 correspond to 24% of the inputs. B, analysis
of the mitotic modification of in vitro translated hNIFK.
Wild-type (lanes 1 and 1'), T234A (lanes
2 and 2'), and T238A (lanes 3 and
3') hNIFK were translated in vitro in the
presence of [35S]methionine, incubated with (lanes
1'-3') or without (lanes 1-3) the CSF extracts, and
analyzed by SDS-PAGE and autoradiography. Note that T234A and T238
hNIFK, but not wild-type hNIFK, are His-tagged. C, pull-down
of hNIFK mutants with GST-Ki-FHA-(1-168). Wild-type (lanes
1-1"), T234A (lanes 2-2"), and T238A (lanes
3-3") hNIFK were translated in vitro in the presence
of [35S]methionine, incubated with the CSF extracts, and
analyzed by SDS-PAGE and autoradiography prior to (lanes
1-3) or after affinity purification with GST beads (lanes
1'-3') or GST-Ki-FHA-(1-168) beads (lanes
1"-3"). Lanes 1-3 correspond to 24% of the
inputs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Takuya Shimamoto (Osaka University, Japan) for providing us with polyadenylated RNA of mouse Ehrlich tumor cells and Drs. Hideo Nishitani and Takeharu Nishimoto (Kyusyu University, Japan) for the hamster Cdc25C clone.
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FOOTNOTES |
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* This work was supported by Grant-in-aid for Scientific Research on Priority Areas (B) 11237202, Grant-in-aid for Scientific Research (B) 12480215, Grant-in-aid for COE Research 12CE2007 from the Japanese Ministry of Education, Science, Sports and Culture, the Mitsubishi Foundation, and the Human Frontier Science Program.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB044971 and AB056870.
¶ To whom correspondence should be addressed. Tel.: 81-6-6879-3210; Fax: 81-6-6879-3219; E-mail: yyoneda@anat3.med.osaka-u.ac.jp.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M102227200
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ABBREVIATIONS |
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The abbreviations used are: FHA, forkhead-associated; ORF, open reading frame; EST, expressed sequence tag; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; mNIFK, mouse NIFK; CSF, cytostatic factor.
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REFERENCES |
---|
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---|
1. | Gerdes, J., Schwab, U., Lemke, H., and Stein, H. (1983) Int. J. Cancer 31, 13-20[Medline] [Order article via Infotrieve] |
2. |
Gerdes, J.,
Lemke, H.,
Baisch, H.,
Wacker, H. H.,
Schwab, U.,
and Stein, H.
(1984)
J. Immunol.
133,
1710-1715 |
3. |
Takagi, M.,
Matsuoka, Y.,
Kurihara, T.,
and Yoneda, Y.
(1999)
J. Cell Sci.
112,
2463-2472 |
4. | Hofmann, K., and Bucher, P. (1995) Trends Biochem. Sci. 20, 347-349[CrossRef][Medline] [Order article via Infotrieve] |
5. | Stone, J. M., Collinge, M. A., Smith, R. D., Horn, M. A., and Walker, J. C. (1994) Science 266, 793-795[Medline] [Order article via Infotrieve] |
6. |
Sun, Z.,
Hsiao, J.,
Fay, D. S.,
and Stern, D. F.
(1998)
Science
281,
272-274 |
7. |
Boudrez, A.,
Beullens, M.,
Groenen, P.,
Van Eynde, A.,
Vulsteke, V.,
Jagiello, I.,
Murray, M.,
Krainer, A. R.,
Stalmans, W.,
and Bollen, M.
(2000)
J. Biol. Chem.
275,
25411-25417 |
8. |
Sueishi, M.,
Takagi, M.,
and Yoneda, Y.
(2000)
J. Biol. Chem.
275,
28888-28892 |
9. | Durocher, D., Henckel, J., Fersht, A. R., and Jackson, S. P. (1999) Mol. Cell 4, 387-394[Medline] [Order article via Infotrieve] |
10. |
Li, J.,
Lee, G.,
Van Doren, S. R.,
and Walker, J. C.
(2000)
J. Cell Sci.
113,
4143-4149 |
11. | Kawai, J., Shinagawa, A., Shibata, K., Yoshino, M., Itoh, M., Ishii, Y., Arakawa, T., Hara, A., Fukunishi, Y., Konno, H., Adachi, J., Fukuda, S., Aizawa, K., Izawa, M., Nishi, K., Kiyosawa, H., Kondo, S., Yamanaka, I., Saito, T., Okazaki, Y., Gojobori, T., Bono, H., Kasukawa, T., Saito, R., Kadota, K., Matsuda, H. A., Ashburner, M., Batalov, S., Casavant, T., Fleischmann, W., Gaasterland, T., Gissi, C., King, B., Kochiwa, H., Kuehl, P., Lewis, S., Matsuo, Y., Nikaido, I., Pesole, G., Quackenbush, J., Schriml, L. M., Staubli, F., Suzuki, R., Tomita, M., Wagner, L., Washio, T., Sakai, K., Okido, T., Furuno, M., Aono, H., Baldarelli, R., Barsh, G., Blake, J., Boffelli, D., Bojunga, N., Carninci, P., de Bonaldo, M. F., Brownstein, M. J., Bult, C., Fletcher, C., Fujita, M., Gariboldi, M., Gustincich, S., Hill, D., Hofmann, M., Hume, D. A., Kamiya, M., Lee, N. H., Lyons, P., Marchionni, L., Mashima, J., Mazzarelli, J., Mombaerts, P., Nordone, P., Ring, B., Ringwald, M., Rodriguez, I., Sakamoto, N., Sasaki, H., Sato, K., Schonbach, C., Seya, T., Shibata, Y., Storch, K. F., Suzuki, H., Toyo-oka, K., Wang, K. H., Weitz, C., Whittaker, C., Wilming, L., Wynshaw-Boris, A., Yoshida, K., Hasegawa, Y., Kawaji, H., Kohtsuki, S., and Hayashizaki, Y. (2001) Nature 409, 685-690[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Carninci, P.,
Shibata, Y.,
Hayatsu, N.,
Sugahara, Y.,
Shibata, K.,
Itoh, M.,
Konno, H.,
Okazaki, Y.,
Muramatsu, M.,
and Hayashizaki, Y.
(2000)
Genome Res.
10,
1617-1630 |
13. | Adachi, Y., and Yanagida, M. (1989) J. Cell Biol. 108, 1195-1207[Abstract] |
14. | Murray, A. (1991) Methods Cell Biol. 36, 581-605[Medline] [Order article via Infotrieve] |
15. | Seki, T., Yamashita, K., Nishitani, H., Takagi, T., Russell, P., and Nishimoto, T. (1992) Mol. Biol. Cell 3, 1373-1388[Abstract] |
16. |
Nishijima, H.,
Nishitani, H.,
Seki, T.,
and Nishimoto, T.
(1997)
J. Cell Biol.
138,
1105-1116 |
17. | Burd, C. G., and Dreyfuss, G. (1994) Science 265, 615-621[Medline] [Order article via Infotrieve] |
18. | Stukenberg, P. T., Lustig, K. D., McGarry, T. J., King, R. W., Kuang, J., and Kirschner, M. W. (1997) Curr. Biol. 7, 338-348[Medline] [Order article via Infotrieve] |
19. |
Winzeler, E. A.,
Shoemaker, D. D.,
Astromoff, A.,
Liang, H.,
Anderson, K.,
Andre, B.,
Bangham, R.,
Benito, R.,
Boeke, J. D.,
Bussey, H.,
Chu, A. M.,
Connelly, C.,
Davis, K.,
Dietrich, F.,
Dow, S. W.,
El Bakkoury, M.,
Foury, F.,
Friend, S. H.,
Gentalen, E.,
Giaever, G.,
Hegemann, J. H.,
Jones, T.,
Laub, M.,
Liao, H.,
Davis, R. W.,
et al..
(1999)
Science
285,
901-906 |
20. | Gonczy, P., Echeverri, G., Oegema, K., Coulson, A., Jones, S. J., Copley, R. R., Duperon, J., Oegema, J., Brehm, M., Cassin, E., Hannak, E., Kirkham, M., Pichler, S., Flohrs, K., Goessen, A., Leidel, S., Alleaume, A. M., Martin, C., Ozlu, N., Bork, P., and Hyman, A. A. (2000) Nature 408, 331-336[CrossRef][Medline] [Order article via Infotrieve] |
21. | Kuhn, A., Vente, A., Doree, M., and Grummt, I. (1998) J. Mol. Biol. 284, 1-5[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Heix, J.,
Vente, A.,
Voit, R.,
Budde, A.,
Michaelidis, T. M.,
and Grummt, I.
(1998)
EMBO J.
17,
7373-7381 |
23. |
Sirri, V.,
Roussel, P.,
and Hernandez-Verdun, D.
(2000)
J. Cell Biol.
148,
259-270 |
24. |
Bell, D. W.,
Varley, J. M.,
Szydlo, T. E.,
Kang, D. H.,
Wahrer, D. C.,
Shannon, K. E.,
Lubratovich, M.,
Verselis, S. J.,
Isselbacher, K. J.,
Fraumeni, J. F.,
Birch, J. M.,
Li, F. P.,
Garber, J. E.,
and Haber, D. A.
(1999)
Science
286,
2528-2531 |
25. | Carney, J. P., Maser, R. S., Olivares, H., Davis, E. M., Le Beau, M., Yates, J. R., Hays, L., Morgan, W. F., and Petrini, J. H. (1998) Cell 93, 477-486[Medline] [Order article via Infotrieve] |
26. | Matsuura, S., Tauchi, H., Nakamura, A., Kondo, N., Sakamoto, S., Endo, S., Smeets, D., Solder, B., Belohradsky, B. H., Der Kaloustian, V. M., Oshimura, M., Isomura, M., Nakamura, Y., and Komatsu, K. (1998) Nat. Genet. 19, 179-181[CrossRef][Medline] [Order article via Infotrieve] |
27. | Varon, R., Vissinga, C., Platzer, M., Cerosaletti, K. M., Chrzanowska, K. H., Saar, K., Beckmann, G., Seemanova, E., Cooper, P. R., Nowak, N. J., Stumm, M., Weemaes, C. M., Gatti, R. A., Wilson, R. K., Digweed, M., Rosenthal, A., Sperling, K., Concannon, P., and Reis, A. (1998) Cell 93, 467-476[Medline] [Order article via Infotrieve] |
28. | Durocher, D., Taylor, I. A., Sarbassova, D., Haire, L. F., Westcott, S. L., Jackson, S. P., Smerdon, S. J., and Yaffe, M. B. (2000) Mol. Cell 6, 1169-1182[Medline] [Order article via Infotrieve] |
29. | Liao, H., Yuan, C., Su, M. I., Yongkiettrakul, S., Qin, D., Li, H., Byeon, I. J., Pei, D., and Tsai, M. D. (2000) J. Mol. Biol. 304, 941-951[CrossRef][Medline] [Order article via Infotrieve] |
30. | Wang, P., Byeon, I. J., Liao, H., Beebe, K. D., Yongkiettrakul, S., Pei, D., and Tsai, M. D. (2000) J. Mol. Biol. 302, 927-940[CrossRef][Medline] [Order article via Infotrieve] |
31. | Liao, H., Byeon, I. J., and Tsai, M. D. (1999) J. Mol. Biol. 294, 1041-1049[CrossRef][Medline] [Order article via Infotrieve] |