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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 Downloaded from mcr.aacrjournals.org on August 3, 2017. © 2003 American Association for Cancer Research. 271 272 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. Downloaded from mcr.aacrjournals.org on August 3, 2017. © 2003 American Association for Cancer Research. 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. Downloaded from mcr.aacrjournals.org on August 3, 2017. © 2003 American Association for Cancer Research. 273 274 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. Downloaded from mcr.aacrjournals.org on August 3, 2017. © 2003 American Association for Cancer Research. Molecular Cancer Research 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. Downloaded from mcr.aacrjournals.org on August 3, 2017. © 2003 American Association for Cancer Research. 275 276 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. Downloaded from mcr.aacrjournals.org on August 3, 2017. © 2003 American Association for Cancer Research. Molecular Cancer Research 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 Downloaded from mcr.aacrjournals.org on August 3, 2017. © 2003 American Association for Cancer Research. 277 278 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. 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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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