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Association of Human SCFSKP2Subunit p19SKP1with Interphase Centrosomes and Mitotic Spindle Poles
Experimental Cell Research 247, 554 –562 (1999) Article ID excr.1999.4386, available online at http://www.idealibrary.com on
RAPID COMMUNICATION Association of Human SCF SKP2 Subunit p19 SKP1 with Interphase Centrosomes and Mitotic Spindle Poles Matthias Gstaiger, Alain Marti, and Wilhelm Krek 1 Friedrich Miescher Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
In Saccharomyces cerevisiae, the initiation of DNA replication and mitotic progression requires SKP1p function. SKP1p is an essential subunit of a newly identified class of E3 ubiquitin protein ligases, the SCF complexes, that catalyze ubiquitin-mediated proteolysis of key cell-cycle-regulatory proteins at distinct times in the cell cycle. SKP1p is also required for proper kinetochore assembly. Little is known about the corresponding human homolog, p19 SKP1 , except that it is expressed throughout the cell cycle and that it too is a component of an S-phase-regulating SCF–E3 ligase complex. Here we show by immunofluorescence microscopy that p19 SKP1 localizes to the centrosomes. Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis. These findings suggest that p19 SKP1 is a novel component of the centrosome and the mitotic spindle, which, in turn, implies a physiological role of this protein in the regulation of one or more aspects of the centrosome cycle. © 1999 Academic Press Key Words: centrosomes; cell cycle; proteolysis; p19 SKP1; F-box proteins.
INTRODUCTION
Progression through the cell cycle is marked by a series of irreversible transitions that separate discrete tasks necessary for cell duplication. The chromosomes must be faithfully replicated once during S phase and identical copies distributed equally to two daughter cells during M phase [1]. Likewise, the centrosomes,
1 To whom correspondence and reprint requests should be addressed. Fax: 1141 61 697 3976. E-mail: [email protected].
0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
which control assembly of microtubules, must duplicate once during each cell cycle, separate, and migrate to the opposite ends of the nucleus and ultimately assemble into a functional spindle to ensure proper segregation of the chromosomes [2]. These complex processes must be coordinated in space and time. Ample evidence suggests that the regulation of protein function through phosphorylation is fundamental in controlling the above-noted processes and that cyclin– cyclin-dependent protein kinase (CDK) complexes play a central role in this regard [3]. In their simplest active form, these consist of a catalytic subunit (the CDK) and a positive regulatory subunit known as cyclin. Diverse families of CDKs and cyclins yield numerous cell-cycle-regulatory enzymes, each with a unique function in cell cycle control [4]. Recent studies emphasize the importance of another class of serine/threonine protein kinases, the polo-like kinases, in the regulation of mitotic progression and centrosome function [5–10]. Moreover, an increasing number of centrosome-associated protein kinases are being identified, including Nek2 [11] and STK15 [12], that appear to regulate essential aspects of centrosome and spindle dynamics. Selective proteolysis of regulatory proteins by the ubiquitin/proteasome pathway has an equally pervasive role in regulating cell cycle progression as protein phosphorylation [13]. Ubiquitin-mediated protein degradation is required for the onset of DNA replication and multiple processes in mitosis [13]. The conjugation of a polyubiquitin chain on substrates requires three activities—an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin protein ligase. Polyubiquitinated substrates are then captured and degraded by the 26S proteasome [14]. E3 ligases play a particularly influential role in ubiquiti-
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nation reactions because they mediate a critical step in substrate recognition. A newly described class of E3 ligases, referred to as SCF complexes (for SKP1–Cullin–F-box protein complexes) utilize F-box proteins as substrate-specific receptors to recruit various substrates into the complex for subsequent ubiquitination [15, 16]. For example, in Saccharomyces cerevisiae, SCF CDC4 and SCF GRR1 catalyze the ubiquitination of the CDK inhibitor SIC1p and the G1 cyclin CLN2p, respectively [17–19]. The full elaboration of the SCF ubiquitination pathway came with the identification of SKP1p in S. cerevisiae [20, 21]. Specifically, SKP1p serves in multiple SCF ubiquitination pathways by binding to the F-box domain of a variety of F-box proteins [20]. Since SKP1p also has the capacity to bind CDC53p (a member of the Cullin family) it is believed that it plays a critical role in the assembly of multiple SCF–E3 ligase complexes [22]. Indeed, genetic analysis reveals that certain SKP1p alleles do not support degradation of key cell cycle regulators, resulting in an arrest of the cell cycle at G1/S or G2/M [20, 21]. A non-SCF function of SKP1p is suggested by the observation that SKP1p plays an essential role in kinetochore assembly [21, 23, 24]. In fact, it appears that SKP1p facilitates an essential modification of the kinetochore subunit p58 Ctf13 by an as yet unidentified kinase [24]. Collectively, these findings suggest that SKP1p participates as a key component of pathways that control S phase entry and chromosomal inheritance in budding yeast. The human homolog of SKP1p, p19 SKP1, was originally identified in conjunction with its binding partner, the F-box protein p45 SKP2, as part of a cyclin A–CDK2 S phase kinase complex [25]. Another protein in this complex is CUL-1, a member of the Cullin family [26 – 29]. The p19 SKP1–CUL-1–p45 SKP2 F-box protein assemblage resembles in its molecular composition SCF CDC4 and thus may be a human representative of SCF–E3 ligases, termed SCF SKP2 [26 –29]. SCF SKP2 accumulates during S/G2 progression, and one of its potential ubiquitination targets appears to be transcription factor E2F-1, which is activated in late G1 [30]. It is believed that SCF SKP2-mediated ubiquitination of E2F-1 contributes to the cessation of E2F-1 activity in S/G2 [30]. Another F-box protein with which p19 SKP1 interacts is cyclin F [20, 31]. Thus, much like its yeast homolog, p19 SKP1 can interact with distinct F-box proteins. The steady-state levels of p19 SKP1 are constant throughout the cell cycle [26], implying that p19 SKP1 might perform multiple functions related to cell growth and division. Here we report that p19 SKP1 is associated with centrosomes during interphase and during all stages of mitosis. These results suggest that p19 SKP1 is a novel component of the centrosome.
MATERIALS AND METHODS Tissue culture and transient transfections. The human osteosarcoma cell line U2-OS was grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (GIBCO) in a 10% CO 2 incubator. For transient expression of recombinant proteins U2-OS cells were transfected with the expression plasmid pcDNA3p19 SKP1 [26] by the calcium phosphate coprecipitation method as described [26] and harvested ;24 h after removal of the precipitate. Taxol and Nocodazole were purchased from Calbiochem and used at concentrations of 5 mM and 6 mg/ml, respectively. Antibody production and immunofluorescence microscopy. For generation of full-length p19 SKP1 antibodies rabbits were injected with gluathione S-transferase (GST)–p19 SKP1 fusion protein purified from bacteria. Antibodies were affinity-purified from rabbit serum by first incubating them with a GST affinity column followed by a GST–p19 SKP1 column. Both columns were prepared by covalently linking the relevant GST fusion proteins to glutathione–Sepharose using dimethylpimelimidate [32]. For indirect immunofluorescence, cells were grown on coverslips for at least 24 h and fixed with paraformaldehyde or methanol essentially as described [9]. Primary antibody incubation was performed at 37°C for 2 h. All antibodies were diluted in phosphate-buffered saline (PBS). For indirect immunofluorescence experiments, affinity-purified anti-p19 SKP1 antibodies were used at 10 –20 mg/ml. As markers for centrosomal localization, anti-g-tubulin antibodies were used from mouse ascites (Sigma) and diluted 1:500 in PBS. b-Tubulin stainings were performed with tissue culture supernatants of monoclonal antibody TU27b directed against human b-tubulin [33]. The antibody was diluted 1:5 in PBS. Following three washes with PBS, affinity-purified goat anti-mouse or goat anti-rabbit IgGs coupled to FITC and Texas red, respectively (Amersham, diluted 1:100), as secondary antibodies were applied together with 4969-diamidino-2-phenylindole (DAPI, 20 mg/ml) for 1 h at room temperature. Cells were washed again 33 in PBS, mounted in Moviol (Gibco), and viewed with a Zeiss fluorescence microscope using a 633 oil immersion objective. Images were taken with a MicroMax cooled CCD camera (Princeton Instruments, Trenton, NJ) and were further processed using MetaMorph imaging software (Universal Imaging Corp., West Chester, PA). Assembly of the panels was performed using Adobe Photoshop. Westerrn blotting and cell fractionation. For preparation of cellular extracts, U2-OS cells were lysed in TNN buffer [250 mM NaCl, Tris–HCl (pH 7.5), 5 mM EDTA (pH 8.0), 50 mM NaF, 0.2 mM NaVO 3, 1 mM PMSF, 10 mg/ml aprotinin, and 1 mM DTT] and analyzed by Western blotting as described previously [26]. In cellular fractionation experiments about 5 3 10 6 cells were harvested, washed once with PBS, resuspended in 400 ml hypertonic buffer [10 mM Mops (pH 7.5), 10 mM KCl, 2 mM MgCl 2, 0.1 mM EDTA (pH 8.0), 1 mM PMSF, 10 mg/ml aprotinin, and 5 mM DTT], and homogenized with 10 strokes in a Dounce homogenizer using a B pestle. After centrifugation for 10 min at 6000 rpm in an Eppendorf centrifuge at 4°C the supernatant was collected (cytoplasmic fraction). The pellet was washed four times with 500 ml hypertonic buffer containing 0.1% NP-40 and lysed in TNN buffer (nuclear fraction). Protein amounts of total, cytoplasmic, and nuclear fractions were estimated by Coomassie blue staining of SDS gels and equal amounts of proteins were analyzed by Western blotting using indicated antibodies. For detection of proteins by Western blotting, antibodies were diluted in 5% nonfat dry milk in TBST [10 mM Tris (pH 8.0), 33 mM NaCl, 0.1% Tween 20] and used at the following concentrations: affinity-purified anti-p19 SKP1 antibody, 2 mg/ml; rabbit anti-human cyclin A serum, 1:2000 dilution; and mouse monoclonal anti-b,-tubulin antibody Tu27b tissue culture supernatant, 1:10 dilution.
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FIG. 1. Specificity of anti-p19 SKP1 antibodies and localization of p19 SKP1 in human cells. (A) Protein extracts were prepared from untransfected U2-OS cells (lane 1) or U2-OS cells transfected with 30 mg of pcDNA3-p19 SKP1 (lane 2) and analyzed by Western blotting with affinity-purified p19 SKP1 antibodies. Note that the p19 SKP1 antibody used in this study recognizes only a single protein in whole cell extracts by Western blotting. Molecular weight markers are represented in kDa on the right side of the panel. The position of p19 SKP1 is marked by an arrow. (B) U2-OS cells were fixed with paraformaldehyde and analyzed by indirect immunofluorescence after incubation with affinitypurified p19 SKP1 antibody (a) or with anti-p19 SKP1 antibody preincubated with GST–p19 SKP1 (c). b and d show the corresponding DNA stains. Note that the spot-like signal present in a disappeared when anti-p19 SKP1 antibody was incubated with GST–p19 SKP1 antigen, demonstrating specificity of the observed p19 SKP1 localization.
RESULTS
To facilitate the subcellular location of p19 SKP1 by indirect immunofluorescence microscopy, an antibody
against p19 SKP1 was generated in rabbits using fulllength human p19 SKP1 linked to GST as immunogen. As shown by Western blotting, the affinity-purified antip19 SKP1 antibody recognized a single band with an ap-
FIG. 2. Colocalization of p19 SKP1 with g-tubulin at the centrosomes. (A) U2-OS cells were triple stained with affinity-purified p19 SKP1 antibody (a), a mononclonal g-tubulin antibody as a centrosomal marker (b), and DAPI (c). Regions consistent with corresponding centrosomes are marked by arrows and a magnification is shown in the insets of a and b. (B) p19 SKP1 localization at the centrosomes is dependent on microtubule dynamics. U2-OS cells grown on coverslips were either left untreated (a, c, e, g) or incubated with either 5 mM Taxol for 4 h (b, f) or 6 mg/ml Nocodazole for 6 h (d, h), fixed with paraformaldehyde, and double-stained with anti-p19 SKP1 (a– d) and anti-b-tubulin antibody Tu27b (e– h). Note that the detachment of microtubules from the centrosomes induced by Taxol did not effect the centrosomal localization of p19 SKP1 (indicated by arrowheads), while Nocodazole treatment resulted in p19 SKP1 localization to foci.
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parent migration of ;23 kDa in whole-cell lysates of human U2-OS cells (Fig. 1A, lane 1). The abundance of this protein detected by immunoblotting with antip19 SKP1 was significantly increased in U2-OS cells transfected with a mammalian expression plasmid encoding untagged p19 SKP1 (Fig. 1A, lane 2). These results suggest that the anti-p19 SKP1 antibody used in this study is specific and recognizes bona fide p19 SKP1. Immunofluorescence staining of exponentially growing human U2-OS osteosarcoma cells with affinity-purified anti-p19 SKP1 antibody revealed that p19 SKP1 is diffusely distributed in the nucleus (Fig. 1B, a). In addition, most cells evidenced a strong anti-p19 SKP1 -labeled spot (or spots) adjacent to the nucleus (Fig. 1B, a), suggestive of the centrosome. This pattern of p19 SKP1 localization was seen in different cell lines and was independent of fixation method as observed following procedures based on either aldehyde or organic solvent (data not shown). Importantly, anti-p19 SKP1 staining of the nuclear compartment and the spots adjacent to the nucleus was blocked by preincubation with the GST–p19 SKP1 fusion protein against which anti-p19 SKP1 was raised (Fig. 1B, c). To establish whether p19 SKP1 is indeed a novel component of the centrosome, double immunofluorescent labeling of U2-OS cells was performed using the anti-p19 SKP1 antibody and a monoclonal antibody (mAb) specific for the centrosomal marker g -tubulin. In addition, DAPI staining was used to locate the nucleus. As shown in Fig. 2A, the anti-p19 SKP1 antibody revealed positive staining of all centrosomes detected by g-tubulin antibody (Fig. 2A, compare a and b and the corresponding insets). This result suggests that centrosomes are loci of endogenous p19 SKP1 . In some cells, p19 SKP1 staining extended to the centrosomal periphery (see Fig. 2A, a, and Fig. 2B, a). Whether this staining represents amorphous pericentriolar material is not known at present. To assess whether the association of p19 SKP1 with centrosomes was dependent on an intact microtubule network, immunostaining on cells was performed under conditions that perturb the microtubule network. U2-OS cells were treated with Taxol and examined for the distribution of p19 SKP1 and b-tubulin. As expected, Taxol administration to these cells resulted in bundling of microtubules and loss of centrosome-nucleated microtubules as shown by b-tubulin antibody staining (Fig. 2B, compare e and f). However, p19 SKP1 remained tightly associated with centrosomes (Fig. 2B, compare a and b). Treatment of U2-OS cells with Nocodazole disrupted the microtubule network as evidenced by b-tubulin antibody staining (Fig. 2B, compare g and h). Under these
conditions, however, p19 SKP1 appeared to localize to specific foci throughout the cell (Fig. 2B, compare c and d). Thus, the state of the intracellular localization of p19 SKP1 is sensitive to the integrity of the microtubule network and p19 SKP1 displays a dynamic behavior that can be initiated by changes in microtubule structure. Previously we have reported that p19 SKP1 is expressed throughout the cell cycle [26]. To determine whether p19 SKP1 localizes to the centrosome also during all stages of mitosis, double immunofluorescent labeling of exponentially growing cells was performed using the anti-p19 SKP1 antibody and g-tubulin mAb. Mitotic figures were selected on the basis of condensation state of DNA as determined by DAPI staining. Figure 3 displays a series of micrographs of U2-OS cells at various stages of mitosis stained with anti-p19 SKP1 antibody (Figs. 3 a, 3d, 3g, and 3j) or mouse g-tubulin mAb (Figs. 3b, 3e, 3h, and 3k). Both antibodies stain the centrosomes during prophase (Figs. 3a and 3b) and localize to the mitotic spindle poles during metaphase (Figs. 3d and 3e). During anaphase, the fairly strong staining of the spindle poles seen with p19 SKP1 antibodies during metaphase was reduced (compare Figs. 3g and 3d), while the intensity of the g-tubulin antibody staining of spindle poles remained unchanged (compare Figs. 3h and 3e). Whether this phenomenon is a direct consequence of a redistribution of p19 SKP1 during mitotic progression or whether it is a reflection of epitope masking is not known at present. To corroborate the above localization data with independent biochemical evidence, nuclei were separated from cytoplasm based on a protocol that employs Dounce homogenization and centrifugation. Partitioning of p19 SKP1 was examined by immunoblotting. As controls, the distributions of a cytoplasmic marker and established nuclear marker were determined in parallel. Figure 4 summarizes the results of these experiments. Efficient separation of nuclear (N) and cytoplasmic (C) fractions is illustrated by the almost complete segregation of the two marker proteins, i.e., nuclear cyclin A (Fig. 4, top) and cytoplasmic b-tubulin (Figure 4, middle). As shown in Fig. 4 (bottom), p19 SKP1 was present in both the cytoplasmic and nuclear fraction. However, a significant enrichment of p19 SKP1 in the cytoplasmic fraction was observed. Since many nuclear proteins are known to leak to the cytoplasm during cell homogenization, we attribute at least part of the cytoplasmic fraction of p19 SKP1 to a redistribution of p19 SKP1 . Taken together, these results support and extend the immunocytochemical data shown above.
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DISCUSSION
We have obtained evidence that in normal dividing cells the SCF SKP2 –E3 ligase subunit p19 SKP1 is located on centrosomes during interphase and on spindle poles during all stages of mitosis. This evidence includes the results of immunofluorescence microscopy using highly specific, affinity-purified, anti-p19 SKP1 antibody raised against full-length p19 SKP1 protein and colocalization studies with the centrosomal marker protein g-tubulin. Since the centrosome controls assembly of microtubules, a process that plays a central role in organizing cell structure and orchestrating formation of the bipolar spindle during mitosis, our results suggest a function of p19 SKP1 related to centrosome dynamics and chromosome segregation. Genetic and biochemical evidence in S. cerevisiae points to a requirement for SKP1p function at the G1/S and G2/M transition of the cell cycle [20, 21, 24]. SKP1p supports entry into S phase by assembling together with the F-box protein CDC4p and CDC53p (a Cullin) into a functional SCF–E3 ligase complex that triggers elimination of the CDK inhibitor SIC1p, thereby allowing induction of S phase [18, 19, 34]. Its human homolog, p19 SKP1 , might perform an analogous role in the induction of S phase in mammalian cells, since it too associates with an F-box protein, p45 SKP2 , CUL-1, and the S-phase kinase cyclin A-CDK2 [26 –29]. These data strongly argue that a key aspect of p19 SKP1 /SKP1p function is linked to ubiquitin-mediated proteolysis of cell-cycleregulatory proteins. Interestingly, existing evidence suggests that ubiquitin modification catalyzed by SCF E3 ligases requires prior phosphorylation of the substrate destined for degradation [16]. Thus, SCF–E3 ligases and protein kinases play an interactive role in promoting the timely and selective degradation of cellular proteins. How do these observations reflect on the findings reported here? One hypothesis that remains to be tested is that p19 SKP1 contributes to the regulation of certain aspects of centrosome duplication/separation and spindle dynamics as part of an SCF–E3 ligase complex. In this regard, it has been reported that C-NAP1, a centrosomal coiled-coil protein with a potential function in centriole– centriole cohesion, is a candidate substrate of the centrosomal kinase Nek2 [35]. It was hypothesized that phosphorylation of C-NAP1 by Nek2 could lead to the degradation or disassociation of C-Nap1 from the centrosome, which in turn would result in the weakening of centriole– centriole cohesion [35]. One might imagine a scenario in which a p19 SKP1 -linked, centrosomally located SCF–E3 ligase targets proteins such asC-NAP1 for degradation,
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thereby contributing to orderly G2/M cell cycle progression. A similar role might be envisioned for the spindle pole-associated p19 SKP1 . In this regard, existing evidence suggests an association of CDC16 and CDC27, established components of the anaphasepromoting complex, a major cell-cycle-regulated E3 ligase complex [36, 37], with the mitotic spindle [38]. Thus, orderly mitotic progression might rely on the collaborative action of distinct ubiquitination pathways. On the other hand, a non-SCF function for p19 SKP1 at the centrosome cannot be excluded. However, it will be possible to investigate this issue only when the relevant components interacting with p19 SKP1 on the centrosomes have been identified. Clearly, the observation reported here provides a basis to initiate a search for centrosome-associated SCF–E3 ligase complexes. In S. cerevisiae, SKP1p plays an important role in the assembly of a functional kinetochore [21, 23, 24]. However, costaining with anti-p19 SKP1 antibodies and a serum from CREST scleroderma patients which recognizes a kinetochore antigen [39] revealed that these two antigens do not colocalize (M. Gstaiger and W. Krek, unpublished). Although we used a p19 SKP1 antibody that has been raised against the full-length gene product, we cannot formally exclude the possibility that this antibody fails to recognize kinetochore-bound p19 SKP1 . Recent evidence suggests that budding yeast SKP1p might participate in the kinetochore assembly pathway by recruiting a kinase for activation of the kinetochore component p58 Ctf13 [24]. Thus, SKP1p might only transiently associate with the kinetochore. Hence, failure to detect p19 SKP1 on kinetochores does not exclude a function of the human protein in this pathway. Biochemical cell fractionation experiments revealed that p19 SKP1 is distributed in the cytoplasm and the nucleus. Taken at face value, it appears that a significant fraction of p19 SKP1 resides in the cytoplasm. The results of immunofluorescence staining localize p19 SKP1 predominantly in the nucleus and a fraction of it in the centrosomes. However, a diffuse distribution of p19 SKP1 in the cytoplasm may simply escape detection by immunofluorescence microscopy. Given the large number of proteins that contain an F-box domain [20], it is conceivable to hypothesize that at least some will participate together with p19 SKP1 in pathways that regulate cytoplasmic activities. Elucidation of the pathways that regulate centrosome function is of critical importance for a better understanding of the process that contributes to accurate chromosome segregation. Abnormalities in the coordination of the chromosome and centrosome
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FIG. 3. Distribution of p19 SKP1 during mitosis in U2-OS cells. Cells were fixed by paraformaldehyde and triple stained with affinitypurified anti-p19 SKP1 antibody (a, d, g, j), monoclonal g-tubulin antibody (b, e, h, k), and DAPI (c, f, i, l). Cells representative for the following mitotic stages are displayed: prophase, metaphase, anaphase, and telophase. Arrowheads indicate positions of the spindle poles.
cycle can result in forms of genetic instability that characterize precancerous and cancerous cells [40]. Identification of p19 SKP1 , a major participant in eukaryotic cell-cycle-regulated ubiquitin-mediated pro-
teolysis, as a component of the centrosome and the mitotic spindle poles provides a new avenue to investigate the role and function of this organelle in the cell cycle.
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FIG. 4. Distribution of p19 SKP1 after biochemical cell fractionation. Total cell lysates (T) and cytoplasmic (C) and nuclear fractions (N) were prepared from U2-OS cells as described under Materials and Methods and equalized for protein content. Individual samples were analyzed by SDS–PAGE followed by Western blotting with anti-cyclin A serum (top), anti-b-tubulin antibody Tu27b (middle), and affinity-purified anti-p19 SKP1 antibody (bottom).
We are indebted to the members of the Matus lab, in particular to Dr. S. Ka¨ch, for advice in immunofluorescence microscopy and the gift of tubulin antibodies. W.K. is a START-fellow and is supported by the Swiss National Science Foundation. M.G. is supported by a grant from the Swiss Cancer League, and A.M. is supported by the Friedrich-Miescher Institut.
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RAPID COMMUNICATION uitin ligase complex (SCF) with SKP1 and an F-box protein. Proc. Natl. Acad. Sci. USA 95, 7451–7456. Michel, J. J., and Xiong, Y. (1998). Human CUL-1, but not other cullin family members, selectively interacts with SKP1 to form a complex with SKP2 and cyclin A. Cell Growth Differ. 9, 435– 449. Yu, Z. K., Gervais, J. L. M., and Zhang, H. (1998). Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins. Proc. Natl. Acad. Sci. USA 95, 11324 –11329. Marti, A., Wirbelauer, C., Scheffner, M., and Krek, W. (1999). Interaction between SCF SKP2 ubiquitin protein ligase and E2F-1 underlies regulation of E2F-1 degradation. Nat. Cell Biol., in press. Bai, C., Richman, R., and Elledge, S. J. (1994). Human cyclin F. EMBO J. 13, 6087– 6098. Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Caceres, A., Binder, L. I., Payne, M. R., Bender, P., Rebhun, L., and Steward, O. (1984). Differential subcellular localization of tubulin and the microtubule- associated protein MAP2 in brain tissue as revealed by immunocytochemistry with monoclonal hybridoma antibodies. J. Neurosci. 4, 394 – 410. Verma, R., Feldman, R. M., and Deshaies, R. J. (1997). SIC1 is ubiquitinated in vitro by a pathway that requires CDC4,
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RAPID COMMUNICATION Association of Human SCF SKP2 Subunit p19 SKP1 with Interphase Centrosomes and Mitotic Spindle Poles Matthias Gstaiger, Alain Marti, and Wilhelm Krek 1 Friedrich Miescher Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
In Saccharomyces cerevisiae, the initiation of DNA replication and mitotic progression requires SKP1p function. SKP1p is an essential subunit of a newly identified class of E3 ubiquitin protein ligases, the SCF complexes, that catalyze ubiquitin-mediated proteolysis of key cell-cycle-regulatory proteins at distinct times in the cell cycle. SKP1p is also required for proper kinetochore assembly. Little is known about the corresponding human homolog, p19 SKP1 , except that it is expressed throughout the cell cycle and that it too is a component of an S-phase-regulating SCF–E3 ligase complex. Here we show by immunofluorescence microscopy that p19 SKP1 localizes to the centrosomes. Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis. These findings suggest that p19 SKP1 is a novel component of the centrosome and the mitotic spindle, which, in turn, implies a physiological role of this protein in the regulation of one or more aspects of the centrosome cycle. © 1999 Academic Press Key Words: centrosomes; cell cycle; proteolysis; p19 SKP1; F-box proteins.
INTRODUCTION
Progression through the cell cycle is marked by a series of irreversible transitions that separate discrete tasks necessary for cell duplication. The chromosomes must be faithfully replicated once during S phase and identical copies distributed equally to two daughter cells during M phase [1]. Likewise, the centrosomes,
1 To whom correspondence and reprint requests should be addressed. Fax: 1141 61 697 3976. E-mail: [email protected].
0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
which control assembly of microtubules, must duplicate once during each cell cycle, separate, and migrate to the opposite ends of the nucleus and ultimately assemble into a functional spindle to ensure proper segregation of the chromosomes [2]. These complex processes must be coordinated in space and time. Ample evidence suggests that the regulation of protein function through phosphorylation is fundamental in controlling the above-noted processes and that cyclin– cyclin-dependent protein kinase (CDK) complexes play a central role in this regard [3]. In their simplest active form, these consist of a catalytic subunit (the CDK) and a positive regulatory subunit known as cyclin. Diverse families of CDKs and cyclins yield numerous cell-cycle-regulatory enzymes, each with a unique function in cell cycle control [4]. Recent studies emphasize the importance of another class of serine/threonine protein kinases, the polo-like kinases, in the regulation of mitotic progression and centrosome function [5–10]. Moreover, an increasing number of centrosome-associated protein kinases are being identified, including Nek2 [11] and STK15 [12], that appear to regulate essential aspects of centrosome and spindle dynamics. Selective proteolysis of regulatory proteins by the ubiquitin/proteasome pathway has an equally pervasive role in regulating cell cycle progression as protein phosphorylation [13]. Ubiquitin-mediated protein degradation is required for the onset of DNA replication and multiple processes in mitosis [13]. The conjugation of a polyubiquitin chain on substrates requires three activities—an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin protein ligase. Polyubiquitinated substrates are then captured and degraded by the 26S proteasome [14]. E3 ligases play a particularly influential role in ubiquiti-
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nation reactions because they mediate a critical step in substrate recognition. A newly described class of E3 ligases, referred to as SCF complexes (for SKP1–Cullin–F-box protein complexes) utilize F-box proteins as substrate-specific receptors to recruit various substrates into the complex for subsequent ubiquitination [15, 16]. For example, in Saccharomyces cerevisiae, SCF CDC4 and SCF GRR1 catalyze the ubiquitination of the CDK inhibitor SIC1p and the G1 cyclin CLN2p, respectively [17–19]. The full elaboration of the SCF ubiquitination pathway came with the identification of SKP1p in S. cerevisiae [20, 21]. Specifically, SKP1p serves in multiple SCF ubiquitination pathways by binding to the F-box domain of a variety of F-box proteins [20]. Since SKP1p also has the capacity to bind CDC53p (a member of the Cullin family) it is believed that it plays a critical role in the assembly of multiple SCF–E3 ligase complexes [22]. Indeed, genetic analysis reveals that certain SKP1p alleles do not support degradation of key cell cycle regulators, resulting in an arrest of the cell cycle at G1/S or G2/M [20, 21]. A non-SCF function of SKP1p is suggested by the observation that SKP1p plays an essential role in kinetochore assembly [21, 23, 24]. In fact, it appears that SKP1p facilitates an essential modification of the kinetochore subunit p58 Ctf13 by an as yet unidentified kinase [24]. Collectively, these findings suggest that SKP1p participates as a key component of pathways that control S phase entry and chromosomal inheritance in budding yeast. The human homolog of SKP1p, p19 SKP1, was originally identified in conjunction with its binding partner, the F-box protein p45 SKP2, as part of a cyclin A–CDK2 S phase kinase complex [25]. Another protein in this complex is CUL-1, a member of the Cullin family [26 – 29]. The p19 SKP1–CUL-1–p45 SKP2 F-box protein assemblage resembles in its molecular composition SCF CDC4 and thus may be a human representative of SCF–E3 ligases, termed SCF SKP2 [26 –29]. SCF SKP2 accumulates during S/G2 progression, and one of its potential ubiquitination targets appears to be transcription factor E2F-1, which is activated in late G1 [30]. It is believed that SCF SKP2-mediated ubiquitination of E2F-1 contributes to the cessation of E2F-1 activity in S/G2 [30]. Another F-box protein with which p19 SKP1 interacts is cyclin F [20, 31]. Thus, much like its yeast homolog, p19 SKP1 can interact with distinct F-box proteins. The steady-state levels of p19 SKP1 are constant throughout the cell cycle [26], implying that p19 SKP1 might perform multiple functions related to cell growth and division. Here we report that p19 SKP1 is associated with centrosomes during interphase and during all stages of mitosis. These results suggest that p19 SKP1 is a novel component of the centrosome.
MATERIALS AND METHODS Tissue culture and transient transfections. The human osteosarcoma cell line U2-OS was grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (GIBCO) in a 10% CO 2 incubator. For transient expression of recombinant proteins U2-OS cells were transfected with the expression plasmid pcDNA3p19 SKP1 [26] by the calcium phosphate coprecipitation method as described [26] and harvested ;24 h after removal of the precipitate. Taxol and Nocodazole were purchased from Calbiochem and used at concentrations of 5 mM and 6 mg/ml, respectively. Antibody production and immunofluorescence microscopy. For generation of full-length p19 SKP1 antibodies rabbits were injected with gluathione S-transferase (GST)–p19 SKP1 fusion protein purified from bacteria. Antibodies were affinity-purified from rabbit serum by first incubating them with a GST affinity column followed by a GST–p19 SKP1 column. Both columns were prepared by covalently linking the relevant GST fusion proteins to glutathione–Sepharose using dimethylpimelimidate [32]. For indirect immunofluorescence, cells were grown on coverslips for at least 24 h and fixed with paraformaldehyde or methanol essentially as described [9]. Primary antibody incubation was performed at 37°C for 2 h. All antibodies were diluted in phosphate-buffered saline (PBS). For indirect immunofluorescence experiments, affinity-purified anti-p19 SKP1 antibodies were used at 10 –20 mg/ml. As markers for centrosomal localization, anti-g-tubulin antibodies were used from mouse ascites (Sigma) and diluted 1:500 in PBS. b-Tubulin stainings were performed with tissue culture supernatants of monoclonal antibody TU27b directed against human b-tubulin [33]. The antibody was diluted 1:5 in PBS. Following three washes with PBS, affinity-purified goat anti-mouse or goat anti-rabbit IgGs coupled to FITC and Texas red, respectively (Amersham, diluted 1:100), as secondary antibodies were applied together with 4969-diamidino-2-phenylindole (DAPI, 20 mg/ml) for 1 h at room temperature. Cells were washed again 33 in PBS, mounted in Moviol (Gibco), and viewed with a Zeiss fluorescence microscope using a 633 oil immersion objective. Images were taken with a MicroMax cooled CCD camera (Princeton Instruments, Trenton, NJ) and were further processed using MetaMorph imaging software (Universal Imaging Corp., West Chester, PA). Assembly of the panels was performed using Adobe Photoshop. Westerrn blotting and cell fractionation. For preparation of cellular extracts, U2-OS cells were lysed in TNN buffer [250 mM NaCl, Tris–HCl (pH 7.5), 5 mM EDTA (pH 8.0), 50 mM NaF, 0.2 mM NaVO 3, 1 mM PMSF, 10 mg/ml aprotinin, and 1 mM DTT] and analyzed by Western blotting as described previously [26]. In cellular fractionation experiments about 5 3 10 6 cells were harvested, washed once with PBS, resuspended in 400 ml hypertonic buffer [10 mM Mops (pH 7.5), 10 mM KCl, 2 mM MgCl 2, 0.1 mM EDTA (pH 8.0), 1 mM PMSF, 10 mg/ml aprotinin, and 5 mM DTT], and homogenized with 10 strokes in a Dounce homogenizer using a B pestle. After centrifugation for 10 min at 6000 rpm in an Eppendorf centrifuge at 4°C the supernatant was collected (cytoplasmic fraction). The pellet was washed four times with 500 ml hypertonic buffer containing 0.1% NP-40 and lysed in TNN buffer (nuclear fraction). Protein amounts of total, cytoplasmic, and nuclear fractions were estimated by Coomassie blue staining of SDS gels and equal amounts of proteins were analyzed by Western blotting using indicated antibodies. For detection of proteins by Western blotting, antibodies were diluted in 5% nonfat dry milk in TBST [10 mM Tris (pH 8.0), 33 mM NaCl, 0.1% Tween 20] and used at the following concentrations: affinity-purified anti-p19 SKP1 antibody, 2 mg/ml; rabbit anti-human cyclin A serum, 1:2000 dilution; and mouse monoclonal anti-b,-tubulin antibody Tu27b tissue culture supernatant, 1:10 dilution.
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FIG. 1. Specificity of anti-p19 SKP1 antibodies and localization of p19 SKP1 in human cells. (A) Protein extracts were prepared from untransfected U2-OS cells (lane 1) or U2-OS cells transfected with 30 mg of pcDNA3-p19 SKP1 (lane 2) and analyzed by Western blotting with affinity-purified p19 SKP1 antibodies. Note that the p19 SKP1 antibody used in this study recognizes only a single protein in whole cell extracts by Western blotting. Molecular weight markers are represented in kDa on the right side of the panel. The position of p19 SKP1 is marked by an arrow. (B) U2-OS cells were fixed with paraformaldehyde and analyzed by indirect immunofluorescence after incubation with affinitypurified p19 SKP1 antibody (a) or with anti-p19 SKP1 antibody preincubated with GST–p19 SKP1 (c). b and d show the corresponding DNA stains. Note that the spot-like signal present in a disappeared when anti-p19 SKP1 antibody was incubated with GST–p19 SKP1 antigen, demonstrating specificity of the observed p19 SKP1 localization.
RESULTS
To facilitate the subcellular location of p19 SKP1 by indirect immunofluorescence microscopy, an antibody
against p19 SKP1 was generated in rabbits using fulllength human p19 SKP1 linked to GST as immunogen. As shown by Western blotting, the affinity-purified antip19 SKP1 antibody recognized a single band with an ap-
FIG. 2. Colocalization of p19 SKP1 with g-tubulin at the centrosomes. (A) U2-OS cells were triple stained with affinity-purified p19 SKP1 antibody (a), a mononclonal g-tubulin antibody as a centrosomal marker (b), and DAPI (c). Regions consistent with corresponding centrosomes are marked by arrows and a magnification is shown in the insets of a and b. (B) p19 SKP1 localization at the centrosomes is dependent on microtubule dynamics. U2-OS cells grown on coverslips were either left untreated (a, c, e, g) or incubated with either 5 mM Taxol for 4 h (b, f) or 6 mg/ml Nocodazole for 6 h (d, h), fixed with paraformaldehyde, and double-stained with anti-p19 SKP1 (a– d) and anti-b-tubulin antibody Tu27b (e– h). Note that the detachment of microtubules from the centrosomes induced by Taxol did not effect the centrosomal localization of p19 SKP1 (indicated by arrowheads), while Nocodazole treatment resulted in p19 SKP1 localization to foci.
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parent migration of ;23 kDa in whole-cell lysates of human U2-OS cells (Fig. 1A, lane 1). The abundance of this protein detected by immunoblotting with antip19 SKP1 was significantly increased in U2-OS cells transfected with a mammalian expression plasmid encoding untagged p19 SKP1 (Fig. 1A, lane 2). These results suggest that the anti-p19 SKP1 antibody used in this study is specific and recognizes bona fide p19 SKP1. Immunofluorescence staining of exponentially growing human U2-OS osteosarcoma cells with affinity-purified anti-p19 SKP1 antibody revealed that p19 SKP1 is diffusely distributed in the nucleus (Fig. 1B, a). In addition, most cells evidenced a strong anti-p19 SKP1 -labeled spot (or spots) adjacent to the nucleus (Fig. 1B, a), suggestive of the centrosome. This pattern of p19 SKP1 localization was seen in different cell lines and was independent of fixation method as observed following procedures based on either aldehyde or organic solvent (data not shown). Importantly, anti-p19 SKP1 staining of the nuclear compartment and the spots adjacent to the nucleus was blocked by preincubation with the GST–p19 SKP1 fusion protein against which anti-p19 SKP1 was raised (Fig. 1B, c). To establish whether p19 SKP1 is indeed a novel component of the centrosome, double immunofluorescent labeling of U2-OS cells was performed using the anti-p19 SKP1 antibody and a monoclonal antibody (mAb) specific for the centrosomal marker g -tubulin. In addition, DAPI staining was used to locate the nucleus. As shown in Fig. 2A, the anti-p19 SKP1 antibody revealed positive staining of all centrosomes detected by g-tubulin antibody (Fig. 2A, compare a and b and the corresponding insets). This result suggests that centrosomes are loci of endogenous p19 SKP1 . In some cells, p19 SKP1 staining extended to the centrosomal periphery (see Fig. 2A, a, and Fig. 2B, a). Whether this staining represents amorphous pericentriolar material is not known at present. To assess whether the association of p19 SKP1 with centrosomes was dependent on an intact microtubule network, immunostaining on cells was performed under conditions that perturb the microtubule network. U2-OS cells were treated with Taxol and examined for the distribution of p19 SKP1 and b-tubulin. As expected, Taxol administration to these cells resulted in bundling of microtubules and loss of centrosome-nucleated microtubules as shown by b-tubulin antibody staining (Fig. 2B, compare e and f). However, p19 SKP1 remained tightly associated with centrosomes (Fig. 2B, compare a and b). Treatment of U2-OS cells with Nocodazole disrupted the microtubule network as evidenced by b-tubulin antibody staining (Fig. 2B, compare g and h). Under these
conditions, however, p19 SKP1 appeared to localize to specific foci throughout the cell (Fig. 2B, compare c and d). Thus, the state of the intracellular localization of p19 SKP1 is sensitive to the integrity of the microtubule network and p19 SKP1 displays a dynamic behavior that can be initiated by changes in microtubule structure. Previously we have reported that p19 SKP1 is expressed throughout the cell cycle [26]. To determine whether p19 SKP1 localizes to the centrosome also during all stages of mitosis, double immunofluorescent labeling of exponentially growing cells was performed using the anti-p19 SKP1 antibody and g-tubulin mAb. Mitotic figures were selected on the basis of condensation state of DNA as determined by DAPI staining. Figure 3 displays a series of micrographs of U2-OS cells at various stages of mitosis stained with anti-p19 SKP1 antibody (Figs. 3 a, 3d, 3g, and 3j) or mouse g-tubulin mAb (Figs. 3b, 3e, 3h, and 3k). Both antibodies stain the centrosomes during prophase (Figs. 3a and 3b) and localize to the mitotic spindle poles during metaphase (Figs. 3d and 3e). During anaphase, the fairly strong staining of the spindle poles seen with p19 SKP1 antibodies during metaphase was reduced (compare Figs. 3g and 3d), while the intensity of the g-tubulin antibody staining of spindle poles remained unchanged (compare Figs. 3h and 3e). Whether this phenomenon is a direct consequence of a redistribution of p19 SKP1 during mitotic progression or whether it is a reflection of epitope masking is not known at present. To corroborate the above localization data with independent biochemical evidence, nuclei were separated from cytoplasm based on a protocol that employs Dounce homogenization and centrifugation. Partitioning of p19 SKP1 was examined by immunoblotting. As controls, the distributions of a cytoplasmic marker and established nuclear marker were determined in parallel. Figure 4 summarizes the results of these experiments. Efficient separation of nuclear (N) and cytoplasmic (C) fractions is illustrated by the almost complete segregation of the two marker proteins, i.e., nuclear cyclin A (Fig. 4, top) and cytoplasmic b-tubulin (Figure 4, middle). As shown in Fig. 4 (bottom), p19 SKP1 was present in both the cytoplasmic and nuclear fraction. However, a significant enrichment of p19 SKP1 in the cytoplasmic fraction was observed. Since many nuclear proteins are known to leak to the cytoplasm during cell homogenization, we attribute at least part of the cytoplasmic fraction of p19 SKP1 to a redistribution of p19 SKP1 . Taken together, these results support and extend the immunocytochemical data shown above.
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DISCUSSION
We have obtained evidence that in normal dividing cells the SCF SKP2 –E3 ligase subunit p19 SKP1 is located on centrosomes during interphase and on spindle poles during all stages of mitosis. This evidence includes the results of immunofluorescence microscopy using highly specific, affinity-purified, anti-p19 SKP1 antibody raised against full-length p19 SKP1 protein and colocalization studies with the centrosomal marker protein g-tubulin. Since the centrosome controls assembly of microtubules, a process that plays a central role in organizing cell structure and orchestrating formation of the bipolar spindle during mitosis, our results suggest a function of p19 SKP1 related to centrosome dynamics and chromosome segregation. Genetic and biochemical evidence in S. cerevisiae points to a requirement for SKP1p function at the G1/S and G2/M transition of the cell cycle [20, 21, 24]. SKP1p supports entry into S phase by assembling together with the F-box protein CDC4p and CDC53p (a Cullin) into a functional SCF–E3 ligase complex that triggers elimination of the CDK inhibitor SIC1p, thereby allowing induction of S phase [18, 19, 34]. Its human homolog, p19 SKP1 , might perform an analogous role in the induction of S phase in mammalian cells, since it too associates with an F-box protein, p45 SKP2 , CUL-1, and the S-phase kinase cyclin A-CDK2 [26 –29]. These data strongly argue that a key aspect of p19 SKP1 /SKP1p function is linked to ubiquitin-mediated proteolysis of cell-cycleregulatory proteins. Interestingly, existing evidence suggests that ubiquitin modification catalyzed by SCF E3 ligases requires prior phosphorylation of the substrate destined for degradation [16]. Thus, SCF–E3 ligases and protein kinases play an interactive role in promoting the timely and selective degradation of cellular proteins. How do these observations reflect on the findings reported here? One hypothesis that remains to be tested is that p19 SKP1 contributes to the regulation of certain aspects of centrosome duplication/separation and spindle dynamics as part of an SCF–E3 ligase complex. In this regard, it has been reported that C-NAP1, a centrosomal coiled-coil protein with a potential function in centriole– centriole cohesion, is a candidate substrate of the centrosomal kinase Nek2 [35]. It was hypothesized that phosphorylation of C-NAP1 by Nek2 could lead to the degradation or disassociation of C-Nap1 from the centrosome, which in turn would result in the weakening of centriole– centriole cohesion [35]. One might imagine a scenario in which a p19 SKP1 -linked, centrosomally located SCF–E3 ligase targets proteins such asC-NAP1 for degradation,
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thereby contributing to orderly G2/M cell cycle progression. A similar role might be envisioned for the spindle pole-associated p19 SKP1 . In this regard, existing evidence suggests an association of CDC16 and CDC27, established components of the anaphasepromoting complex, a major cell-cycle-regulated E3 ligase complex [36, 37], with the mitotic spindle [38]. Thus, orderly mitotic progression might rely on the collaborative action of distinct ubiquitination pathways. On the other hand, a non-SCF function for p19 SKP1 at the centrosome cannot be excluded. However, it will be possible to investigate this issue only when the relevant components interacting with p19 SKP1 on the centrosomes have been identified. Clearly, the observation reported here provides a basis to initiate a search for centrosome-associated SCF–E3 ligase complexes. In S. cerevisiae, SKP1p plays an important role in the assembly of a functional kinetochore [21, 23, 24]. However, costaining with anti-p19 SKP1 antibodies and a serum from CREST scleroderma patients which recognizes a kinetochore antigen [39] revealed that these two antigens do not colocalize (M. Gstaiger and W. Krek, unpublished). Although we used a p19 SKP1 antibody that has been raised against the full-length gene product, we cannot formally exclude the possibility that this antibody fails to recognize kinetochore-bound p19 SKP1 . Recent evidence suggests that budding yeast SKP1p might participate in the kinetochore assembly pathway by recruiting a kinase for activation of the kinetochore component p58 Ctf13 [24]. Thus, SKP1p might only transiently associate with the kinetochore. Hence, failure to detect p19 SKP1 on kinetochores does not exclude a function of the human protein in this pathway. Biochemical cell fractionation experiments revealed that p19 SKP1 is distributed in the cytoplasm and the nucleus. Taken at face value, it appears that a significant fraction of p19 SKP1 resides in the cytoplasm. The results of immunofluorescence staining localize p19 SKP1 predominantly in the nucleus and a fraction of it in the centrosomes. However, a diffuse distribution of p19 SKP1 in the cytoplasm may simply escape detection by immunofluorescence microscopy. Given the large number of proteins that contain an F-box domain [20], it is conceivable to hypothesize that at least some will participate together with p19 SKP1 in pathways that regulate cytoplasmic activities. Elucidation of the pathways that regulate centrosome function is of critical importance for a better understanding of the process that contributes to accurate chromosome segregation. Abnormalities in the coordination of the chromosome and centrosome
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FIG. 3. Distribution of p19 SKP1 during mitosis in U2-OS cells. Cells were fixed by paraformaldehyde and triple stained with affinitypurified anti-p19 SKP1 antibody (a, d, g, j), monoclonal g-tubulin antibody (b, e, h, k), and DAPI (c, f, i, l). Cells representative for the following mitotic stages are displayed: prophase, metaphase, anaphase, and telophase. Arrowheads indicate positions of the spindle poles.
cycle can result in forms of genetic instability that characterize precancerous and cancerous cells [40]. Identification of p19 SKP1 , a major participant in eukaryotic cell-cycle-regulated ubiquitin-mediated pro-
teolysis, as a component of the centrosome and the mitotic spindle poles provides a new avenue to investigate the role and function of this organelle in the cell cycle.
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FIG. 4. Distribution of p19 SKP1 after biochemical cell fractionation. Total cell lysates (T) and cytoplasmic (C) and nuclear fractions (N) were prepared from U2-OS cells as described under Materials and Methods and equalized for protein content. Individual samples were analyzed by SDS–PAGE followed by Western blotting with anti-cyclin A serum (top), anti-b-tubulin antibody Tu27b (middle), and affinity-purified anti-p19 SKP1 antibody (bottom).
We are indebted to the members of the Matus lab, in particular to Dr. S. Ka¨ch, for advice in immunofluorescence microscopy and the gift of tubulin antibodies. W.K. is a START-fellow and is supported by the Swiss National Science Foundation. M.G. is supported by a grant from the Swiss Cancer League, and A.M. is supported by the Friedrich-Miescher Institut.
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Descombes, P., and Nigg, E. A. (1998). The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts. EMBO J. 17, 1328 –1335.
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Fry, A. M., Meraldi, P., and Nigg, E. A. (1998). A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators. EMBO J. 17, 470 – 481.
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Zhou, H., Kuang, J., Zhong, L., Kuo, W. L., Gray, J. W., Sahin, A., Brinkley, B. R., and Sen, S. (1998). Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat. Genet. 20, 189 –193.
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Patton, E. E., Willems, A. R., and Tyers, M. (1998). Combinatorial control in ubiquitin-dependent proteolysis: Don’t Skp the F-box hypothesis. Trends Genet. 14, 236 –243.
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