Download repp86: A Human Protein Associated in the Progression of Mitosis

Vol. 1, 271 – 279, February 2003
Molecular Cancer Research
repp86: A Human Protein Associated in the Progression
of Mitosis
Hans-Juergen Heidebrecht, 1 Sabine Adam-Klages, 2 Monika Szczepanowski,1 Marc Pollmann,1
Friedrich Buck, 4 Elmar Endl,5 Marie-Luise Kruse, 3 Pierre Rudolph,1 and Reza Parwaresch1
1
Department of Hematopathology and Lymph Node Registry, 2Department of Immunology and 31st Department of
Medicine, University of Kiel, Kiel, Germany; 4Department of Cell Biochemistry and Clinical Neurobiology, University of
Hamburg, Hamburg, Germany; and 5Division of Molecular Immunology, Research Center Borstel, Germany
Abstract
Human repp86 becomes detectable in the nucleoplasm
of cycling cells at the G1-S boundary, condenses at
the centrosomes with the onset of mitosis, during
which it progressively locates to the mitotic spindle
and to the midbody, and vanishes at the completion
of cytokinesis. The repp86 cDNA was cloned and
sequenced. Full-length repp86 and its COOH-terminal
domain cosediment with polymerized microtubules,
linking repp86 to the family of microtubule-associated
proteins. During prophase and metaphase, repp86
interacts on the mitotic spindle with the putative
motor protein Hklp2. Thus, repp86 may function in
targeting Hklp2 to the microtubule minus ends, its
activity being regulated by phosphorylation of
serine/threonine residues. Exogenous overexpression
of repp86 provokes accumulation of cells in G2-M
phase and subsequent polyploidization, suggesting
that excess repp86 may interfere with correct
nuclear division.
Introduction
Accurate segregation and redistribution of daughter chromosomes during mitosis depends on a flawless function of the
mitotic spindle apparatus (1, 2). The latter is composed of a
highly dynamic microtubular skeleton associated with a variety
of proteins including kinesin-like proteins and microtubuleassociated proteins (MAPs) which modulate its structure and
regulate its mobility (3).
Many proteins such as MAPs, protein kinases, phosphatases,
and nuclear matrix-associated proteins become enriched at the
Received 8/16/02; revised 1/6/03; accepted 1/8/03.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: While this manuscript was in review, Gruss et al. (Nat. Cell Biol., 11 ,
871 – 879, 2002) reported that overexpression of repp86/hTPX2 blocks cells in a
prometaphase-like state. These results confirm our data concerning repp86
overexpression.
Grant support: Deutsche Forschungsgemeinschaft (He2837/1-2) and by the
Kinderkrebs-Initiative Buchholz, Holm-Seppensen, Germany.
Requests for reprints: Hans-Juergen Heidebrecht, Department of Hematopathology, University of Kiel, Michaelisstr. 11, D-24105 Kiel, Germany. Phone: 49431-597-3393; Fax: 49-431-597-3428. E-mail: [email protected]
Copyright D 2003 American Association for Cancer Research.
centrosomes when cells enter mitosis (4 – 6). The activity of
these proteins leads to a dramatic reorganization of the
interphase microtubule array. The minus ends of microtubules
(MTs) are focused into two poles, the plus ends being oriented
toward the chromosomes, which enables motor proteins such as
dynein and kinesin-like proteins to exert a diametric traction to
separate chromosome pairs (2, 7, 8).
Herein we describe a novel cell cycle-associated human
protein, repp86 (restrictedly expressed proliferation-associated
protein; formerly termed p100). Discovered as a nuclear antigen
reactive with the monoclonal antibody (mAb) Ki-S2, repp86 is
a protein of about 100 kDa apparent molecular mass encoded
by a gene located on human chromosome band 20q11.2 (9, 10).
Its expression is tightly cell cycle regulated as demonstrated by
fluorescence-activated cell sorting (FACS) analysis, becoming
detectable at the G1-S transit and vanishing at the completion of
cytokinesis. During S and G2 phase, repp86 is diffusely
distributed throughout the cell nucleus, whereas in mitotic cells,
repp86 is strictly associated with the mitotic spindle (9).
Independently of us, the same protein was described in
Xenopus and subsequently in the human system as a protein
implicated in spindle pole organization through interaction with
the motor protein Xklp2 (11, 12). Its function was shown to be
regulated by the GTPase Ran, which mediates its dissociation
from the nuclear import factors importins a and h in the
presence of condensing DNA (12). On the basis of data
obtained from Xenopus models, they named this protein TPX2
or hTPX2. However, considering its distinctive association with
specific cell cycle phases, we believe that ‘‘repp86’’ describes
the characteristics of this protein more adequately. To gain
deeper insights into the biological function of repp86 in human
cells, we transiently overexpressed repp86 and investigated its
interaction with other proteins.
Results
Cloning and Sequencing of repp86
By immunopurification of repp86 from nuclear lysates of
HeLa cells using mAb Ki-S2 and peptide sequencing after
proteolytic digestion of the purified protein, we were able to
identify 20 different peptides. PCR experiments with degenerated oligonucleotides and subsequent screening of a HeLa
cDNA library by primer walking yielded a 3151-bp cDNA
(AF098158), which includes the complete coding frame for
repp86. All previously identified peptides were found in the
deduced amino acid sequence. Translation analysis of our
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272 Human Protein repp86 and Progression of Mitosis
FIGURE 1. Genomic organization of repp86. The repp86 gene consists of 18 exons. Untranslated regions are indicated by light boxes, and coding regions
by gray boxes. The translation start site (ATG ) is located within exon 3. The translation stop codon TAA and polyadenylation signal AATAAA are part of exon
18. Intron and exon lengths are drawn according to the length scale of 1 kb. The two longest introns with 14.7 and 8.8 kb are indicated.
cDNA predicted a 747-amino-acid protein with a theoretical
molecular mass of 85,653 Da. The repp86 protein contains four
potential nuclear localization sites, and one ATP/GTP binding
site motif A (P-loop) that, in many instances, is essential for
enzymatic activity. To further verify the authenticity of the
cDNA, we expressed a fragment of repp86 in a bacterial system
and used the protein product to immunize BALB/c mice. One
mAb was obtained, which stained the same pattern as mAb
Ki-S2 in immunohistochemistry. This antibody also recognized
immunopurified repp86 in Western blot experiments, indicating
that the elicited cDNA actually encodes repp86.
Screening of a genomic data bank with the cDNA sequence
of repp86 led to the identification of the human chromosome
20 clone RP11-243J16. This clone allowed us to generate a
scheme of the genomic organization of the repp86 gene. The
gene is organized into 18 exons with sizes ranging from 98 to
522 bp, and 17 introns with sizes between 372 and 14,755 bp,
the translation initiation site being located in exon 3 (Fig. 1).
repp86 Distribution During the Cell Cycle
Repp86 expression is tightly cell cycle associated. In freshly
prepared peripheral blood lymphocytes (PBLs) of healthy
donors, neither repp86 specific mRNA (data not shown) nor
repp86 protein expression is detectable (Fig. 2). Western blot
analysis of lysates of freshly prepared PBLs and mitogenstimulated PBLs revealed that repp86 expression is upregulated in mitogen-stimulated PBLs. After 1 day, a very
weak staining of repp86 is detectable, whereas after 3 days of
stimulation, repp86 is significantly expressed (Fig. 2). Immunofluorescence microscopy showed that repp86 is diffusely
distributed in interphase nuclei, and slightly condensed along
the nuclear membrane and a branching tubular structure. There
was no apparent association with interphase tubulin or DNA
(Fig. 3B). During prophase and prometaphase, repp86 became
gradually concentrated at the spindle poles and the mitotic
asters. Mitotic MTs were stained by repp86, leaving astral MTs
almost unstained (Fig. 3A). This pattern remained present
through anaphase, at the end of which repp86 was relocated to
the midzone resulting in a strong labeling of the midbody in late
telophase (Fig. 3B). The staining intensity then rapidly
diminished so that repp86 became undetectable with the
completion of cytokinesis.
repp86 Interacts With MTs
The close association of repp86 with the mitotic spindle
suggested an interaction with MTs. To verify this, endogenous
repp86 and two different GST-repp86 fusion proteins were
tested on taxol-stabilized MTs. One of the GST fusion proteins,
GST-repp86(1 – 747), contained the complete coding frame, the
other, the COOH terminus of repp86(601 – 747). By sequence
analysis, the COOH terminus of repp86 was characterized as a
coiled-coil region. Such structures are frequently responsible
for protein-protein interactions.
The different repp86 preparations were incubated with or
without taxol-stabilized MTs for 20 min at room temperature
FIGURE 2. Up-regulation of repp86 expression in lysates of mitogenstimulated PBLs. Equal amounts of freshly prepared and mitogenstimulated PBLs were lysed at the indicated times, separated by
SDS-PAGE and blotted. The blot was stained with the repp86 specific
mAb Ki-S2. Freshly prepared PBLs do not express repp86, whereas
repp86 expression is clearly detectable after 72 h of mitogen stimulation.
Positive control: lysate of L428 cells.
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Molecular Cancer Research
endogenous repp86 and about 75% of GST-repp86(1 – 747) is
bound to MTs. Cosedimentation of about 35% of the GSTrepp86(601 – 747) proved that the coiled-coil region of repp86 is
definitely involved in this interaction.
repp86 Interacts With Hklp2 in Human Cells
Given the high degree of identity between repp86 and TPX2,
we investigated whether the human kinesin-like protein 2
(Hklp2, the human homologue of the TPX2-associated
Xenopus kinesin-like protein, Xklp2) interacts with repp86.
Xklp2 localizes to the spindle poles and is required for
centrosome separation during spindle assembly in Xenopus egg
extracts (13). The human homologue of Xklp2, human kinesinlike protein 2 (Hklp2), was recently identified, but its link to
repp86 remained unknown (14). After immunoprecipitation and
Western blot analysis, one of the coprecipitated proteins could
be identified as Hklp2 by means of immunostaining with a
specific polyclonal antibody. A single band at approximately
160 kDa was observed, which is consistent with the molecular
mass reported for Hklp2 (Fig. 5).
We furthermore performed double immunofluorescence
staining with Ki-S2 and anti-Hklp2 antibody. Hklp2 was
present both in the nucleus and the cytoplasm, mostly as a
finely punctuate pattern with occasional confluence to larger,
irregularly shaped dots. In interphase nuclei of cycling cells, the
two proteins were dispersed throughout the nucleus without
apparent colocalization (data not shown). Conversely, during
mitosis, a strong additive fluorescence as revealed by yellow
coloring became evident on the mitotic spindle, while a portion
of Hklp2 remained in the cytoplasm (Fig. 6, A – C). From
metaphase through telophase, the additive signal gradually
diminished due to the disappearance of Hklp2. These results
show that, like its Xenopus homologue TPX2, repp86
associates with Hklp2 during mitosis, suggesting an analogous
function.
FIGURE 3.
Repp86 distribution in interphase and mitotic cells.
Immunofluorescence staining of a cytospin preparation from L428 cells
with mAb Ki-S2 and an Alexa 488-labeled goat anti-mouse polyclonal
antibody. A. In mitotic cells, it associates with the mitotic spindle and the
spindle poles during metaphase. B. In interphase cells, repp86 is diffusely
distributed throughout the nucleus. At cytokinesis, repp86 is detectable at
the spindle poles and the midbody. Images represent optical slices of 0.5
Am through the center of the cell.
and then centrifuged for 40 min at 100,000 g. The pelleted
proteins were boiled in loading buffer, separated by SDSPAGE, and blotted. After staining with mAb Ki-S2, cosedimentation of endogenous repp86 and GST-repp86(1 – 747) with
MTs became evident (Fig. 4). Because Ki-S2 detects an NH2terminal epitope of repp86, we used a polyclonal anti-GST
antibody to determine cosedimentation of the COOH-terminal
GST-repp86(601 – 747) with MTs. No pelleted endogenous repp86
and GST-repp86 could be detected when MTs were omitted.
Densitometric evaluation revealed that about 45% of the
FIGURE 4. Interaction of endogenous repp86, GST-repp86(1 – 747), and
the COOH-terminal GST-repp86(601 – 747) with MTs. The different repp86
preparations were incubated with or without taxol-stabilized MTs for
20 min at room temperature and then centrifuged for 40 min at 100,000 g.
The pelleted proteins were boiled in loading buffer. Control proteins (a),
MT pelleted proteins (b), and proteins pelleted without MTs (c ) were
separated by SDS-PAGE and blotted. After staining with mAb Ki-S2 and a
polyclonal antibody specific for GST, cosedimentation of endogenous
(endogen. ) repp86, GST-repp86(1 – 747), and the COOH-terminal fusion
protein GST-repp86(601 – 747) with MTs is clearly detectable (b ). No pelleted
endogenous repp86 and GST-repp86 could be detected when MTs were
omitted (c). For densitometric evaluation, control proteins and MT pelleted
proteins were compared.
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274 Human Protein repp86 and Progression of Mitosis
approximately 50% of the cells in each experiment. In addition
to the nuclear signal regularly observed in a fraction of the cells,
those overexpressing repp86 showed abundant accumulation of
repp86 in the cytoplasm, whereas no cytoplasmic labeling was
seen in control cells. By FACS analysis of HEK293 cells
FIGURE 5. Coprecipitation of Hklp2 with repp86. Interaction of repp86
with Hklp2 is demonstrated by coprecipitation experiments. Lysates had
previously been intensively precleared with protein A-Sepharose beads.
Lanes a and c show Western blot experiments with lysates from L428
cells stained with mAb Ki-S2 (a) and polyclonal anti-Hklp2 (c ). In lanes b
and d , the lysate was immunoprecipated with Ki-S2, and the membranes
were immunostained with Ki-S2 (b) and anti-Hklp2 (d). A protein of about
160 kDa that coprecipitated with the repp86 immunocomplex is detected
by the antibody specific for Hklp2. Molecular weight markers are shown on
the right.
repp86 Is a Mitotic Phosphoprotein
Labeling of asynchronously growing L428 cells and
nocodazole-arrested L428 cells with 32P and subsequent
immunoprecipitation of the extracts with mAb Ki-S2 revealed
a much stronger signal in the extracts of arrested cells compared
with the signal obtained from asynchronously growing cells,
showing that repp86 is more abundantly phosphorylated during
mitosis than in interphase cells (9). It remained to be
determined which residues of repp86 are targets of phosphorylation in mitotic (nocodazole-arrested) cells. To this end,
immunoprecipitated repp86 from asynchronous and nocodazole-arrested cells was tested in Western blot experiments either
with PY99, an antibody specific for phosphotyrosine residues,
or MPM2, an antibody specific for phosphorylated serine/
proline (S/P) or threonine/proline (T/P) residues. Immunostaining of repp86 from nocodazole-arrested L428 cells with PY99
revealed a signal, indicating that mitotic repp86 is phosphorylated on one or several tyrosine residues during mitosis (Fig. 7,
A/B). Similarly, we visualized with mAb MPM2 a signal
corresponding to repp86 in extracts from nocodazole-arrested
HeLa cells that was almost undetectable in asynchronously
growing cells (Fig. 7D). This differential phosphorylation
during interphase and mitosis suggests that phosphorylation on
tyrosine, S/P, and T/P residues constitutes an important step in
regulating the function of repp86 at the G2-M transition.
Overexpression of repp86 Leads to Accumulation of
Cells in G2 Phase
To gain further insights into the function of repp86, we
sought to modulate repp86 expression in human cells. Because
repp86 repression using antisense oligonucleotides was repeatedly unsuccessful, we investigated the effect of repp86
overexpression. For this purpose, HEK293 cells were transfected with the eukaryotic vector pRK5 containing repp86,
and with the vector alone. Judging by immunohistochemistry,
exogenous overexpression of repp86 was achieved in
FIGURE 6. Colocalization of repp86 and Hklp2 in mitotic cells. A.
Colocalization of repp86 and Hklp2 is visualized by yellow additive
fluorescence concentrated on the spindle pole and the mitotic spindle in
mitogen-stimulated PBL. B. Spatial arrangement of repp86 (red) and DNA
(blue ). C. Association of Hklp2 (green ) with the mitotic spindle plus
additional dotlike immunoreactivity throughout the cell. Original magnification, 400; scan zoom, 4.
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FIGURE 7. Tyrosine and serine/threonine phosphorylation of repp86
in nocodazole-arrested cells. Repp86 was immunoprecipitated from
unsynchronized and nocodazole-arrested L428 (A/B) or HeLa cells
(C/D). Immunoprecipitates from asynchronously growing cells (A a ; B
a; C c ; D c ) and arrested cells (A b ; B b ; C d ; D d) were stained with
mAbs Ki-S2 (A/C), PY99 (B), and mpm2 (D). The broader signal
visualized with Ki-S2 in extracts from arrested cells is indicative of
additional phosphorylation. Accordingly, signals corresponding to tyrosine
phosphorylation (B b) and S/P or T/P phosphorylation (D d ) are apparent
only in extracts from nocodazole-arrested cells. Molecular weight markers
are shown on the right.
transfected with the full-length repp86 expression vector, we
observed a significant accumulation of repp86-overexpressing
cells in the G2-M phase after 24 and 48 h. With increasing time
after transfection, repp86-overexpressing cells exhibited
marked DNA polyploidy, suggesting that excess repp86 may
inhibit nuclear division (Fig. 8). Interestingly, primary experiments indicate that exogenously overexpressed repp86 is not
significantly phosphorylated (personal observations). Immunofluorescence staining of repp86-overexpressing COS7 cells
with mAb Ki-S2 72 h after transfection revealed that many of
the overexpressing cells are multinucleated and contain
malformed polymorphous nuclei (Fig. 9). Western blot analysis
of lysates of control and repp86-transfected cells demonstrates a
significant overexpression of repp86.
Discussion
Repp86 was identified by immunostaining with the mAb
Ki-S2 as a proliferation-associated nuclear protein that becomes
detectable at the transit from G1 into S phase and vanishes at
the end of mitosis (9). During this period, repp86 undergoes
dramatic changes in its subcellular distribution, being diffusely
nucleoplasmic in interphase to associate with the mitotic
spindle as cells enter M phase. Consistent with these
observations, repp86 is closely associated with mitotic MTs,
but not with interphase tubulin. During cytokinesis, the protein
relocates to the midbody. Western blot analysis revealed that
repp86 is up-regulated in mitogen-stimulated PBLs.
We cloned and sequenced the corresponding cDNA, which
includes the complete coding frame for repp86. A homologous
cDNA was recently reported by two other groups. Using
suppression subtractive hybridization, this sequence was
identified among genes differentially expressed in colorectal
cancer (15). An identical cDNA, C20orf2, was retrieved by
comparing cancerous and noncancerous lung cells by mRNA
differential display (16). This sequence appeared to encode a
novel protein of unknown function. The repp86 protein actually
shares 52% sequence homology with a recently characterized
Xenopus MAP, TPX2 (11).
The changes in the subcellular location of repp86 strongly
suggested an important role in mitosis, the idea being further
supported by the high sequence homology shared between
repp86 and the Xenopus spindle-associated protein TPX2 (11).
TPX2 was shown to be required for microtubule assembly and
spindle pole organization, and for targeting Xklp2, a putative
plus end-directed motor protein, to the minus ends of mitotic
MTs (11). In this process, which involves the dynein-dynactin
complex, TPX2 alone appears to be sufficient to induce
microtubule nucleation and formation of a polar spindle (12),
whereas the function of Xklp2 appears to be redundant.
However, once associated with the spindle apparatus, Xklp2 is
likely to exert a function as a motor element in antiparallel
sliding of the MTs.
Like the Xenopus MAP TPX2, human repp86 cosediments
with polymerized MTs, demonstrating that repp86 belongs to
the large family of MAPs (11). As coiled-coil regions of
proteins are often responsible for protein-protein interactions,
we performed cosedimentation experiments with the bacterially
expressed COOH-terminal GST-repp86(601 – 747) containing the
coiled-coil region (17, 18). As expected, the COOH-terminal
GST-repp86(601 – 747) cosedimented with MTs, indicating that
the coiled-coil motif is involved in the binding to MTs.
To gain further insights into the function of repp86, we
looked for protein interactions. These were assessed by
coprecipitation and verified by double immunofluorescence
staining with subsequent confocal laser scanning microscopy.
We were able to identify an interaction between Hklp2 and
repp86. This result led us to suppose that repp86 is involved in
the segregation of mitotic chromosomes and cell division.
Indeed, at the onset of mitosis, repp86 intimately associates at
the mitotic spindle with the human homologue of the Xenopus
kinesin-like protein Xklp2, Hklp2 (19). The colocalization with
Hklp2 implies that repp86 may function in a similar manner as
its Xenopus homologue TPX2 (11).
This putative function might be established by showing a
failure of chromosomes to attach to the spindle in the absence
of repp86. However, we were unable to knock out repp86
expression by means of antisense oligonucleotides even using
different types of human cells. As an alternative, we
transiently overexpressed repp86 in HEK293 cells. After
24 h, this exogenous overexpression led to an accumulation
of cells in G2-M phase as shown by flow cytometric DNA
analysis. Forty-eight and 72 h later, many of the overexpressing cells became polyploid. Immunofluorescence
staining of COS7 cells overexpressing repp86 demonstrates
that many of these cells are multinucleated with nuclear
malformations.
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276 Human Protein repp86 and Progression of Mitosis
FIGURE 8. Cell cycle kinetic analysis of the effect of repp86 overexpression in HEK293 cells. Cell cycle analysis by FACS of HEK293 vector-transfected
cells (pRK5 ), HEK293 pRK5-repp86-transfected cells (all cells ), and HEK293 pRK5-repp86 (transfected cells ) 24, 48, and 72 h after transfection. Compared
with control cells (29% G2-M), repp86-overexpressing cells accumulate in G2-M phase (54% G2-M) after 24 h. Forty-eight and 72 h after transfection, repp86overexpressing cells become polyploid (71%), indicating a failure of cells to divide completely while DNA replication is sustained.
It is conceivable that an excess of repp86 inhibits the
ultimate step in cell division, while chromosomal DNA
undergoes one or several rounds of replication. The experiments with phosphorylation-specific antibodies, on the other
hand, indicate that phosphorylation of repp86 on both tyrosine
and serine/threonine residues is essential for mitotic cell
division, so that the availability of certain repp86-phosphorylating kinases may be the rate-limiting factor for a correct
function of repp86 during mitosis. Indeed, primary data
(not shown) suggest that the exogenously overexpressed
fraction of repp86 is not or only weakly phosphorylated.
Another likely explanation is the fact that exogenously
overexpressed repp86 was found in high amounts in the
cytoplasm contrary to its physiological location in the nucleus.
Indeed, a possible mechanism of negative and positive
regulation of repp86 may be understood by analogy to the
control of TPX2. This protein is one of the cargoes interacting
with members of a family of soluble receptor proteins termed
importins or exportins, another being NuMA (19), which also
plays a role in spindle organization and interacts with the
dynein-dynactin complex (20, 21). After its transfer into the
nucleus, TPX2 remains bound to importins a and h in a ternary
complex. This association is regulated by the small GTPase
Ran. On DNA condensation, RanGDP is converted by the
DNA-associated nucleotide exchange factor RCC1 to its active
form, RanGTP. The latter binds to importin h, thereby
provoking the dissociation of cargo from importin a, which
redistributes to the cytoplasm. TPX2 then can bind to tubulin
and induce microtubule polymerization, for which it appears to
be both necessary and sufficient (12). This regulatory
mechanism would not only explain the failure of repp86 to
induce untimely microtubule nucleation despite its abundant
presence in interphase nuclei, but it also suggests that an excess
of cytoplasmic repp86 might interfere with the physiological
pathways of repp86 activation.
Nevertheless, there is yet another aspect of repp86 overexpression that deserves consideration. We previously observed
that a high percentage of repp86-expressing cells in human
cancers is indicative of a highly malignant behavior and
associated with cell cycle deregulation (22 – 24). Although we
possess no information concerning the repp86 content of
individual tumor cells in these cases, one may conceive a
disturbance analogous to that produced by overexpression of
the centrosomal kinase STK15/Aurora A, which is encoded by
a gene neighboring the repp86 gene locus on chromosome 20q
(25). Both simple overexpression and gene amplification of
STK15 were shown to result in centrosome amplification and
consequent aneuploidy (25, 26), and they appear to play a role
in cellular immortalization (27, 28). Given that global gains of
chromosome 20q have been observed (29 – 31), the polyploidization of cells following enforced repp86 expression would be
consistent with an effect of excess repp86 comparable to that of
STK15. This idea is further supported by experiments
demonstrating a physical interaction of repp86 with STK15
(32, 33). Lastly, an association between high repp86 expression
and telomerase activity intimates a role for repp86 in cellular
immortalization (34, 35). Experiments designed to clarify these
associations are under way.
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The emerging picture is that repp86 is required for both
the initiation and the progression of mitosis. Besides spindle
pole organization, repp86 is likely to mediate and to regulate
microtubule kinetics by interaction with motor proteins such
as Hklp2. Circumstantial evidence further suggests a function
in cell proliferation that requires further investigation. The
striking reorganization of repp86 at the onset of mitosis
points to an interaction with other spindle pole and spindleassociated proteins. Moreover, the shift in repp86 distribution
during the progression of mitosis is suggestive of multiple
functions that may be modulated by differential phosphorylation. Conceivably, an aberrant expression of repp86 may
play a role in oncogenesis or the promotion of malignancy.
Ongoing functional characterization of repp86 will hopefully
shed more light on the biochemical and biophysical processes
taking place during cell proliferation and mitosis.
Materials and Methods
Immunocytochemistry and Immunofluorescence
Microscopy
For confocal laser scanning microscopy and double
immunofluorescence staining, cytospin preparations from
mitogen-stimulated PBLs and L428 cells were fixed for 10
min in 2% paraformaldehyde/PBS, and for another 10 min in
acetone. mAb Ki-S2 (9) was detected with a goat anti-mouse
IgG polyclonal antibody coupled with Alexa 488 (Molecular
Probes, Eugene, OR). Polyclonal rabbit anti-Hklp2 was kindly
provided by M. Takagi, Department of Cell Biology and
Neuroscience, Osaka University, Japan. After washing with
PBS, the slides were incubated in 2 AM TOTO-3 (Molecular
Probes) in PBS for staining of nucleic acids, washed again in
PBS, and mounted in DABCO (Sigma, Deisenhofen,
Germany) mounting medium. For confocal laser scanning
microscopy, a Zeiss LSM 510 laser scanning microscope
(Carl Zeiss Jena, Jena, Germany) was used. Double-stained
images represent optical slices of 0.5 Am through the center of
individual cells.
FIGURE 9. Immunofluorescence staining of repp86-overexpressing
COS7 cells. A. The DNA was stained with propidium iodide. B. Seventytwo hours after transfection with pRK5-repp86, adherently growing COS7
cells were stained with mAb Ki-S2 (green ). The overlay picture (C) shows
that many of the repp86-overexpressing cells are multinucleated with
malformed polymorphous nuclei. Inset, expression of repp86 in control (a )
and transfected cells (b) is demonstrated by Western blotting. Significant
overexpression is clearly detectable in transfected cells (b).
Coprecipitation and Western Blot Experiments
At least 1 108 L428 cells were used for these experiments.
All steps were performed on ice or at 4jC. After intensive
washing with ice-cold PBS, the cells were lysed with 2% Triton
X-100, 1 mM EDTA, and a protease inhibitor cocktail
(Boehringer, Mannheim, Germany), and the nuclear lysate
was centrifuged at 15,000 g. The supernatant was precleared
with protein A-Sepharose CL-4B (SpA). For the determination
of coprecipitating proteins by Western blot analysis, the SpA
antibody-protein complex was boiled in loading buffer under
reducing conditions. Coprecipitated proteins were separated by
SDS-PAGE and transferred to nitrocellulose membranes overnight. The membranes were allowed to react with a polyclonal
antibody specific for Hklp2 (kindly provided by M. Takagi).
Tyrosine-phosphorylated proteins were detected by mAb PY99
(Santa Cruz Biotechnology, Heidelberg, Germany; Ref. 36);
serine/threonine phosphorylation was detected by mAb MPM2
(Biomol, Hamburg, Germany; Ref. 37). Visualization was
achieved with 4-chloro-1-naphthol after incubation of the
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278 Human Protein repp86 and Progression of Mitosis
membranes with peroxidase-conjugated rabbit anti-mouse IgG
and goat anti-rabbit IgG (Sigma), or by chemiluminescence
staining (Amersham, Freiburg, Germany) according to the
delivered protocol.
Protein Sequencing of the Ki-S2 Antigen
For protein sequencing experiments, a cytoplasmic lysate
prepared from 1 109 L428 cells was used. Immunoprecipitation was performed as described above. The immunoprecipitates were separated by SDS-PAGE and transferred to
polyvinylidene difluoride membranes (Immobilon P, Millipore, Eschborn, Germany). The membranes were stained with
coomassie brilliant blue R250. After destaining with 50%
methanol, the protein band was excised from the membrane
and further processed for sequencing. Because attempts to
sequence the intact proteins from the blot membrane were
unsuccessful, the protein was digested with Lys C and the
proteolytic fragments were separated by narrowpore HPLC
(130 A, Applied Biosystems, Weiterstadt, Germany) on a
reverse-phase column (Vydac C4, 300 A pore size, 5 mm
particle size, 2.1 250 mm) (38). Peptides were eluted with
a linear mobile phase gradient (0 – 80% B in 50 min; solvent
A: water/0.1% TFA; solvent B: 70% acetonitrile/0.09% TFA)
at a flow rate of 200 Al/min. Peptide-containing fractions
detected at 210 nm were collected manually into siliconized
Eppendorf tubes and frozen immediately. Protein sequences
of about 20 peptides were determined by standard Edman
degradation on an automatic sequencer (473 A, Applied
Biosystems).
PCR Experiments and cDNA Cloning
Reverse transcription-PCR experiments were performed
under the following conditions: template: 0.5 Al per reaction;
reaction volume: 25 Al; 50 pM of each primer; magnesium
concentration: 2.5 mM; temperature cycle: 1 min 94jC, 1 min
55jC, 1.5 min 72jC; 35 cycles with 1 min predenaturation at
94jC. Degenerated oligonucleotides were derived from three
of the sequenced peptides (KPVDNTYYK; KSVDFHFRTD;
KNVTQIEP) for PCR experiments with cDNA from L428
cells. Subsequent screening of a HeLa cDNA library by
primer walking yielded a cDNA of 3151 bp. PCR experiments using only one of the degenerated primers were
performed as controls. PCR products were purified by
agarose gel electrophoresis and cloned using the TOPO TA
cloning kit (Invitrogen, Karlsruhe, Germany). DNA sequences were determined by automatic sequencing (model 377,
PE Biosystems, Weitershausen, Germany). In all cases, both
strands were sequenced by means of a primer walking
strategy.
Cloning of repp86 for Eukaryotic Expression
The coding region of the repp86 cDNA was amplified by
PCR (primer: 5V-CTCGGATCCATGTCACAAGTTAAAAGC3V and 5V-CTCGGATCCCAGCTCAC AGCTGAG-3V) and
cloned into the EcoRI site of the eukaryotic expression vector
pRK5 (39). The authenticity of the construct was verified by
DNA sequencing.
Transient Transfection of HEK293 and COS7 Cells
with repp86
HEK293 and COS7 cells were originally obtained from the
American Type Culture Collection and cultivated in DMEM
without HEPES, supplemented with 10% FCS, 2 mM glutamine,
and 50 Ag/ml each of streptomycin and penicillin. For
transfection experiments, 1 106 HEK293 cells were seeded
per 10-cm plate and transiently transfected with 5 Ag of a repp86
expression construct (full-length repp86 cloned into the vector
pRK5) or 5 Ag of vector alone using the calcium phosphate
precipitation method (40). COS7 cells were transfected with
pRK5 or the repp86 construct by electroporation (40).
Cell Cycle Analysis
Transiently transfected HEK293 cells were incubated for the
indicated times to allow expression of repp86, and detached
using 1% Trypsin-PBS/5 mM EDTA. After washing in PBS,
cells were fixed for 30 min at 4jC in 50% ethanol, and
incubated with Ki-S2 for 1 h at 4jC. The cells were then
washed twice in PBS, stained with 1:100 diluted FITC-labeled
goat-anti-mouse antiserum (DAKO, Hamburg, Germany), and
resuspended in 0.1 ml PBS containing 40 Ag/ml RNaseA after
additional washing in cold PBS. The suspension was incubated
for 30 min at room temperature, then the cells were stained with
0.5 ml staining solution (50 Ag/ml propidium iodide in PBS/5
mM EDTA). Cell cycle analysis was performed by flow
cytometry using a FACSCalibur Analyzer (Becton Dickinson,
Heidelberg, Germany). In addition, an aliquot of the transfected
cells was directly fixed in 50% ethanol and stained with
propidium iodide as described above.
Expression of repp86(1 – 747) and Its COOH
Terminus(601 – 747) in Escherichia coli
A repp86 cDNA fragment containing the entire coding
sequence was subcloned from vector pRK5 into the bacterial
expression vector pGEX-6P-1. The COOH-terminal cDNA
fragment of repp86 was amplified by PCR using the repp86pRK5 construct as the template and then cloned into pGEX-5X1 vector (both vectors from Amersham Biosciences, Freiburg,
Germany). In-frame cloning and accuracy of the DNA
sequences were confirmed by automated sequencing. The
constructs were then transformed into E. coli BL21 and singlerecombinant clones were used for subsequent expression
experiments.
Microtubule Binding Assay
Nuclear lysates of L428 cells, GST-repp86(1 – 747) full-length
fusion protein, and GST-repp86(601 – 747) were incubated with
or without a defined amount of taxol-stabilized MTs according
to the delivered protocol (Cytoskeleton Inc., Denver, CO).
After 40 min of centrifugation (100,000 g; RT), the
supernatant was carefully removed. The pelleted proteins were
boiled in loading buffer and separated by SDS-PAGE. After
transfer to nitrocellulose, the membranes were stained as
described above.
The amount of endogenous repp86, GST-repp86(1 – 747), and
GST-repp86(601 – 747) bound to taxol-stabilized MTs was
determined by Western blot analysis using Ki-S2 or an anti-
Downloaded from mcr.aacrjournals.org on August 3, 2017. © 2003 American Association for Cancer Research.
Molecular Cancer Research
GST antibody (Amersham Biosciences) followed by densitometry. For quantification, the measuring software ‘‘quantity one’’
(part of the gel documentation system ‘‘gel doc 2000’’ of
Bio-Rad, München, Germany) was used. The gel background
was set as 0% and the repp86 band of controls as 100%. The
amount of endogenous repp86 and GST fusion proteins bound
to MTs was compared to this standardization by the software.
Acknowledgments
The authors thank K. Andersen, B. Dettmann, and S. Ussat for their excellent
technical assistance and Dr. M. Takagi (Osaka, Japan) for generously providing
the polyclonal anti-Hklp2 antiserum.
References
1. Sharp, D. J., Rogers, G. C., and Scholey, J. M. Microtubule motors in mitosis.
Nature, 407: 41 – 47, 2000.
2. Wittmann, T., Hyman, A., and Desai, A. The spindle: a dynamic assembly of
microtubules and motors. Nat. Cell Biol., 3: 28 – 34, 2001.
3. Desai, A. and Mitchison, T. J. Microtubule polymerization dynamics. Annu.
Rev. Dev. Biol., 13: 83 – 117, 1997.
4. Kalt, A. and Schliwa, M. Molecular components of the centrosome. Trends
Cell Biol., 3: 118 – 128, 1993.
5. Mayor, T., Meraldi, P., Stierhof, Y. D., Nigg, E. A., and Fry, A. M. Protein
kinases in control of the centrosome cycle. FEBS Lett., 452: 92 – 95, 1999.
6. Meraldi, P. and Nigg, E. A. Centrosome cohesion is regulated by a balance of
kinase and phosphatase activities. J. Cell Sci., 114: 3749 – 3757, 2001.
7. Hyman, A. A. and Karsenti, E. Morphogenic properties of microtubules and
mitotic spindle. Cell, 84: 401 – 410, 1996.
8. Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman,
A., and Karsenti, E. Self-organization of microtubules into bipolar spindles
around artificial chromosomes in Xenopus egg extracts. Nature, 382: 420 – 425,
1996.
9. Heidebrecht, H. J., Buck, F., Steinmann, J., Sprenger, R., Wacker, H. H., and
Parwaresch, R. p100: a novel proliferation-associated nuclear protein specifically
restricted to cell cycle phases S, G2 and, M. Blood, 90: 226 – 233, 1997.
10. Zhang, Y., Heidebrecht, H. J., Rott, A., Schlegelberger, B., and Parwaresch,
R. Assignment of human proliferation associated p100 gene (C20orf1 ) to human
chromosome band 20q11.2 by in situ hybridisation. Cytogenet. Cell Genet., 84:
182 – 183, 1999.
11. Wittmann, T., Wilm, M., Karsenti, E., and Vernos, I. TPX2, a novel Xenopus
map involved in spindle pole organization. J. Cell Biol., 149: 1405 – 1418, 2000.
12. Gruss, O. J., Carazo-Salas, R. E., Scatz, C. A., Guarguaglini, G., Knast, J.,
Wilm, M., Le Bot, N., Vernos, I., Karsenti, E., and Mattaij, I. W. Ran induces
spindle assembly by reversing the inhibitory effect of importin on TPX2 activity.
Cell, 104: 83 – 93, 2001.
13. Wittmann, T., Boleti, H., Antony, C., Karsenti, E., and Vernos, I. Localisation
of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a
microtubule-associated protein and dynein. J. Cell Biol., 143: 673 – 685, 1998.
14. Sueishi, M., Tagaki, M., and Yoneda, Y. The forkhead associated domain of
Ki-67 antigen interacts with the novel kinesin like protein Hklp2. J. Biol. Chem.,
275: 28888 – 28892, 2000.
15. Hufton, S. E., Moerkerk, P. T., Brandwijk, R., de Bruine, A. P., Arends, J. W.,
and Hoogenboom, H. R. A profile of differentially expressed genes in primary
colorectal cancer using suppression subtractive hybridisation. FEBS Lett., 46:
77 – 82, 1999.
16. Manda, R., Kohno, T., Matsuno, Y., Takenoshita, S., Kuwano, H., and
Yokota, J. Identification of genes (SPON2 and C20orf2 ) differentially expressed
between cancerous and noncancerous lung cells by mRNA differential display.
Genomics, 61: 5 – 14, 1999.
17. Mack, G. J. and Compton, D. A. Analysis of mitotic microtubule-associated
proteins using mass spectrometry identifies astrin, a spindle-associated protein.
Proc. Natl. Acad. Sci., 98: 14434 – 14439, 2001.
18. Benson, M. A., Newey, S. E., Martin-Rendon, E., Hawkes, R., and Blake,
D. J. Dysbindin, a novel coiled-coil-containing protein that interacts with the
dystrobrevins in muscle and brain. J. Biol. Chem., 276: 24232 – 24241, 2001.
19. Nachury, M. V., Maresca, T. J., Salmon, W. C., Waterman-Storer, C. M.,
Heald, R., and Weis, K. Importin h is a mitotic target of the small GTPase Ran in
spindle assembly. Cell, 104: 95 – 106, 2001.
20. Gaglio, T., Saredi, A., and Compton, D. A. NuMA is required for the
organization of microtubules into aster-like mitotic arrays. J. Cell Biol., 131:
693 – 708, 1995.
21. Merdes, A., Heald, R., Samejima, K., Earnshaw, W. C., and Cleaveland,
D. W. Formation of spindle poles by dynein/dynactin-dependent transport of
NuMA. J. Cell Biol., 149: 851 – 862, 2000.
22. Bonatz, G., Lüttges, J., Hedderich, J., Inform, D., Jonat, W., Rudolph, P., and
Parwaresch, R. Prognostic significance of a novel proliferation marker, antirepp86, for endometrial carcinoma: a multivariate study. Hum. Pathol., 30: 949 –
956, 1999.
23. Rudolph, P., Alm, P., Heidebrecht, H. J., Bolte, H., Rathjen, V., Baldetorp, B.,
Ferno, M., Olsson, H., and Parwaresch, R. Immunologic proliferation marker
Ki-S2 as a prognostic indicator for lymph node-negative breast cancer. J. Natl.
Cancer Inst., 91: 271 – 278, 1999.
24. Rudolph, P., Alm, P., Olsson, H., Heidebrecht, H. J., Fernö, M., Baldetorp,
B., and Parwaresch, R. Concurrent overexpression of p53 and c-erbB-2 correlates
with accelerated cycling and concomitant poor prognosis in lymph node-negative
breast cancer. Hum. Pathol., 32: 311 – 319, 2001.
25. Zhou, H., Kuang, J., Zhong, L., Kuo, W. L., Gray, J. W., Sahin, A., Brinkley,
B. R., and Sen, S. Tumor amplified kinase STK15/BTAK induces centrosome
amplification, aneuploidy and transformation. Nat. Genet., 20: 189 – 193, 1998.
26. Bischoff, J. R. and Plowmann, G. D. The aurora/Ipl1p kinase family:
regulators of chromosome segregation and cytokinesis. Trends Cell Biol., 9:
454 – 459, 1999.
27. Savelieva, E., Belair, C. D., Newton, M. A., DeVries, S., Gray, J. W.,
Waldman, F., and Reznikoff, C. A. 20q gain associates with immortalization:
20q13.2 correlates with genome instability in human papilloma virus 16 E7
transformed human uroepithelial cells. Oncogene, 14: 551 – 560, 1997.
28. Cuthill, S., Agarwal, P., Sarkar, S., Savelieva, E., and Reznikoff, C. A.
Dominant genetic alteration in immortalization: role for 20q gain. Genes
Chromosomes & Cancer, 26: 304 – 311, 1999.
29. Kokkola, A., Monni, O., Puolakkainen, P., Nordling, S., Haapiainen, R.,
Kivilaakso, E., and Knuutila, S. Presence of high-level DNA copy number gains
in gastric carcinoma and severely dysplastic adenomas but not in moderately
dysplastic adenomas. Cancer Genet. Cytogenet., 107: 32 – 36, 1998.
30. De Angelis, P. M., Clausen, O. P., Schjolberg, A., and Stokke, T.
Chromosomal gains and losses in primary colorectal cancer detected by CGH
and their associations with tumour DNA ploidy, genotypes and phenotypes. Br. J.
Cancer, 80: 526 – 535, 1999.
31. Korn, W. M., Yasutake, T., Kuo, W. L., Warren, R. S., Collins, C., Tomita,
M., Gray, J., and Waldman, F. M. Chromosome arm 20q gains and other genomic
alterations in colorectal cancer metastatic to the liver, as analyzed by comparative
genomic hybridization and fluorescence in situ hybridization. Genes Chromosomes & Cancer, 25: 82 – 90, 1999.
32. Kufer, T. A., Sillje, H. H. W., Körner, R., Gruss, O. J., Meraldi, P., and Nigg,
E. A. Human TPX2 is required for targeting Aurora-A kinase to the spindle.
J. Cell Biol., 158: 617 – 623, 2002.
33. Heidebrecht, H. J., Kruse, M. L., Andersen, K., Szczepanowski, M.,
Pollmann, M., and Parwaresch, R. repp86 interacts with the serine/threonine
kinase STK15/Aurora-A. Cell Motil. Cytoskelet., 54: 177 – 178, 2003.
34. Rudolph, P., Schubert, C., Tamm, S., Heidorn, K., Hauschild, A., Michalska,
I., Majewski, S., Krupp, G., Jablonska, S., and Parwaresch, R. Telomerase activity
in melanocytic lesions—a potential marker of tumor biology. Am. J. Pathol., 156:
1425 – 1432, 2000.
35. Bonatz, G., Frahm, S. O., Klapper, W., Helfenstein, A., Heidorn, K., Jonat,
W., Krupp, G., Parwaresch, R., and Rudolph, P. High telomerase activity is
associated with cell cycle deregulation and rapid progression in endometrioid
adenocarcinoma of the uterus. Hum. Pathol., 32: 605 – 614, 2001.
36. Odenbreit, S., Puls, J., Sedlmaier, B., Gerland, E., Fischer, W., and Haas, R.
Translocation of Heliobacter pylori CagA into gastric epithelial cells by type IV
secretion. Science, 287: 1497 – 1500, 2000.
37. Westendorf, J. M., Rao, P. N., and Gerace, L. Cloning of cDNAs for M-phase
phosphoproteins recognized by the MPM2 monoclonal antibody and determination of the phosphorylated epitope. Proc. Natl. Acad. Sci., 91: 714 – 718, 1994.
38. Bauw, G., van den Bulcke, M., van Damme, J., Puype, M., van Montagu, M.,
and Vandekerckhove, J. Protein electroblotting on polybase coated glass-fiber and
polyvinylidene difluoride membranes: an evaluation. J. Prot. Chem., 7: 194 – 196,
1988.
39. Tartaglia, L. A., Ayres, T. M., Wong, G. H. W., and Goeddel, D. V. A novel
domain within the 55 kd TNF receptor signals cell death. Cell, 74: 845 – 853,
1993.
40. Sambrook, J., Frisch, E. F., and Maniatis, T. Molecular Cloning: A
Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory, 1989.
Downloaded from mcr.aacrjournals.org on August 3, 2017. © 2003 American Association for Cancer Research.
279
repp86: A Human Protein Associated in the Progression of
Mitosis 1 1 Deutsche Forschungsgemeinschaft (He2837/1-2)
and by the Kinderkrebs-Initiative Buchholz,
Holm-Seppensen, Germany.
2 2 Note: While this manuscript was in review, Gruss et al.
(Nat. Cell Biol., 11, 871−879, 2002) reported that
overexpression of repp86/hTPX2 blocks cells in a
prometaphase-like state. These results confirm our data
concerning repp86 overexpression.
Hans-Juergen Heidebrecht, Sabine Adam-Klages, Monika Szczepanowski, et al.
Mol Cancer Res 2003;1:271-279.
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