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Cell Growth & Differentiation Vol. 10, 769-775, November 1999
© 1999 American Association for Cancer Research


Articles

BUBR1 Phosphorylation Is Regulated during Mitotic Checkpoint Activation1

Wenqing Li, Zhengdao Lan, Huiyun Wu, Shechao Wu, Juliana Meadows, Jie Chen, Veronica Zhu and Wei Dai2

Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Abstract

Eukaryotic cells have evolved a mechanism that delays the progression of mitosis until condensed chromosomes are properly positioned on the mitotic spindle. To understand the molecular basis of such monitoring mechanism in human cells, we have been studying genes that regulate the mitotic checkpoint. Our early studies have led to the cloning of a full-length cDNA encoding MAD3-like protein (also termed BUBR1/MAD3/SSK1). Dot blot analyses show that BUBR1 mRNA is expressed in tissues with a high mitotic index but not in differentiated tissues. Western blot analyses show that in asynchronous cells, BUBR1 protein primarily exhibits a molecular mass of 120 kDa, and its expression is detected in most cell lines examined. In addition, BUBR1 is present during various stages of the cell cycle. As cells enter later S and G2, BUBR1 levels are increased significantly. Nocodazole-arrested mitotic cells obtained by mechanical shake-off contain BUBR1 antigen with a slower mobility on denaturing SDS gels. Phosphatase treatment restores the slowly migrating band to the interphase state, indicating that the slow mobility of the BUBR1 antigen is attributable to phosphorylation. Furthermore, purified recombinant His6-BUBR1 is capable of autophosphorylation. Our studies indicate that BUBR1 phosphorylation status is regulated during spindle disruption. Considering its strong homology to BUB1 protein kinase, BUBR1 may also play an important role in mitotic checkpoint control by phosphorylation of a critical cellular component(s) of the mitotic checkpoint pathway.

Introduction

Eukaryotic cells have evolved mechanisms, commonly referred to as checkpoints, that monitor their readiness to enter the next stage of the cell cycle (1 , 2) . Extensive research in the past has identified at least two major checkpoints (G2-M and mitotic checkpoints) that control the onset of mitosis and mitotic progression. The mitotic checkpoint is a conserved function in eukaryotic cells that delays the onset of anaphase until the microtubules are properly connected to all chromosomes. Genetic analyses have identified at least seven distinct yeast genes (MPS1, BUB1, BUB2, BUB3, MAD1, MAD2, and MAD3) that are important in regulating the mitotic checkpoint (3, 4, 5) . Recently, several research groups have identified and characterized mammalian counterparts of the mitotic checkpoint components (6, 7, 8, 9) . The human MAD1 protein is hyperphosphorylated during S, G2, and mitosis, and it undergoes dramatic subcellular translocalization during mitosis (6) . It localizes to the centrosome during metaphase and to the spindle midzone and the midbody subsequently (6) . The loss of human MAD1 function, because of sequestration by T-cell leukemia viral product Tax, appears to be responsible for the transformation of T cells by the tumor virus (6) . The human MAD2 protein, structurally and functionally conserved (7) , is localized at the kinetochore after chromosome condensation but not after metaphase (7) . In addition, MAD2 associates with APC3 (or cyclosome; Ref. 10 ). Purified MAD2 is capable of arresting cycling Xenopus egg extracts at metaphase and blocking cyclin B degradation by preventing its ubiquitination (10) . Microinjection of a MAD2 antibody into Ptk cells in mitosis induces premature anaphase (11) . Both murine and human BUB1 genes have been cloned and characterized recently (8 , 9 , 12) . Murine and human BUB1 proteins also localize to the kinetochore during early mitosis and mitotic checkpoint activation (8 , 12) . The human BUB1 gene has been implicated recently in the development of certain colorectal cancers (9 , 13) .

We and others have cloned a MAD3-like gene (alternatively termed BUBR1/MAD3/SSK1). BUBR1 encodes a protein of 120 kDa, the amino acid sequences of which resemble both MAD3 and BUB1 of the budding yeast. Structural abnormalities have been detected in the BUBR1 gene isolated from colorectal cancer samples (9) , suggesting that it may also play a tumor-suppressor role. Recently, it has been shown that BUBR1 is localized to kinetochore from prophase to mid-anaphase and may have multiple functions during mitosis (14) . In the present study, we report that BUBR1 is an active protein kinase capable of autophosphorylation and that its phosphorylation is regulated during mitotic checkpoint activation.

Results

Our previously cloned MAD3-like cDNA (GenBank accession number AF068760) was identical to BUBR1 (9) , MAD3 (15) , and SSK1 (16) . To gain insights into the biological function of the putative mitotic checkpoint kinase, we first examined the pattern of BUBR1 mRNA expression in various human tissues. A survey of 50 primary human tissue blots obtained from Clontech, Inc. for BUBR1 expression (Fig. 1A)Citation showed that thymus (Fig. 1ACitation , blot E5), bone marrow (Fig. 1ACitation , blot E8), and various fetal tissues (Fig. 1ACitation , blots G1–G7) expressed a moderate-to-high level of BUBR1 transcripts. Testes contained the most abundant level of BUBR1 transcripts (Fig. 1ACitation , blot D1). On the other hand, differentiated tissues, such as various brain tissues (Fig. 1ACitation , blots A1–A8), heart (Fig. 1ACitation , blot C1), and muscle (Fig. 1ACitation , blot C3) expressed little or no detectable level of BUBR1 transcripts. The multiple tissue dot-blot was also probed with a control gene (ß-actin), confirming that an approximately equal amount of RNA from various tissues was loaded onto the blot (Fig. 1B)Citation . The summarized results of BUBR1 mRNA expression, normalized by ß-actin expression, are shown in Table 1Citation .



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Fig. 1. Analyses of BUBR1 mRNA expression in primary human tissues. A dot blot containing an approximately equal amount of total RNA isolated from 50 primary human tissues was probed for BUBR1 (A) and ß-actin (B) expression. Specific signals were quantified by densitometric scanning. BUBR1 mRNA expression levels, normalized by ß-actin expression levels, and the sample identifications are shown in Table 1Citation .

 

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Table 1 Summary of BUBR1 expression in 50 primary human tissues

 
To study the biological role of BUBR1, we raised antipeptide antisera against BUBR1 protein using a synthetic peptide corresponding to residues 421–438 of the protein (8 , 15) as an immunogen. Western blot analyses showed (Fig. 2A)Citation that the antiserum from rabbit no. 35 (Lanes 3 and 4), but not the preimmune serum (Lanes 1 and 2), recognized an antigen {approx}120 kDa in both HEL (Lane 3) and Dami cell lysates (Lane 4). The antigen was barely detectable when the antiserum was preabsorbed with the BUBR1 peptide (Fig. 2ACitation , Lanes 5 and 6), indicating that the antiserum recognized BUBR1 antigen. Immunoprecipitation followed by Western blotting confirmed the specificity of the anti-BUBR1 antiserum (Fig. 2B)Citation . Compared with the cell lysates (Fig. 2BCitation , Lane 7), the anti-BUBR1 antiserum (Lanes 4–6), but not the preimmune serum (Lanes 1–3), immunoprecipitated BUBR1 antigen. To ascertain that the anti-BUBR1 antibody did not have a cross-reactivity with BUB1 antigen, interphase or mitotic HeLa cell lysates were immunoprecipitated with the anti-BUBR1 antibody. The immunoprecipitates were analyzed by Western blotting using the BUB1 antibody. Fig. 3ACitation shows that the BUBR1 antibody did not bring down BUB1 antigen (Lanes 3 and 4). Reciprocal experiments were also performed. Fig. 3BCitation shows that BUB1 immunoprecipitates did not contain the BUBR1 antigen either. These combined studies strongly suggest that the anti-BUBR1 antibody does not show a cross-reactivity with BUB1, although we should point out that the anti-BUB1 antibody was not efficient in immunoprecipitation. Further studies by surveying various cell lines via Western blotting revealed that BUBR1 antigen was present, albeit at different levels, in many cell lines that were examined (Fig. 4ACitation , Lanes 2–8). BUBR1 was barely detectable in HIMeg-1 (a myeloid progenitor; Fig. 4ACitation , Lane 1) and PC-3 (prostatic carcinoma; Lane 9) cell lines.



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Fig. 2. Characterization of anti-BUBR1 antibody. A, three sets of HEL (odd lanes) and Dami (even lanes) cell lysates were analyzed by SDS-PAGE, followed by immunoblotting using the preimmune serum (Lanes 1 and 2), the anti-BUBR1 antiserum (Lanes 3 and 4), or the anti-BUBR1 antiserum preabsorbed with the BUBR1 peptide (Lanes 5 and 6). B, asynchronous HeLa cell lysates were immunoprecipitated with the anti-BUBR1 antiserum (Lanes 4–6) or with the preimmune serum (Lanes 1–3). Immunoprecipitates were analyzed by SDS-PAGE, followed by Western blotting using the anti-BUBR1 antiserum. Lane 7, HeLa cell lysates.

 


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Fig. 3. BUBR1 antibody does not recognize BUB1. A, BUBR1 immunoprecipitated from interphase (Lane 3) or Noc-arrested mitotic (Lane 4) HeLa cells were analyzed by Western blotting using the anti-BUB1 antibody. Interphase (Lane 1) and mitotic cell (Lane 2) lysates were used as controls. B, interphase (Lanes 3 and 5) or mitotic (Lanes 4 and 6) HeLa cell lysates were immunoprecipitated with the anti-BUB1 (Lanes 3 and 4) or the anti-BUBR1 antibody (Lanes 5 and 6). The immunoprecipitates (IP) were analyzed by Western blotting for BUBR1. Lanes 1 and 2 were the interphase and mitotic cell lysates, respectively.

 


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Fig. 4. BUBR1 protein levels in various cell lines. A, equal amounts of lysates from various cell lines were analyzed for BUBR1 expression via Western blotting. B, a part of the stained blot was shown as a loading control.

 
To study the potential role of BUBR1 in mitotic checkpoint activation, we analyzed BUBR1 in several cell lines treated with a microtubule-disrupting agent Noc via Western blotting. Fig. 5ACitation shows that BUBR1 was detected in asynchronous GMOO637D (Lane 1), A549 (Lane 3), and HeLa (Lane 5) cells primarily as a single band of {approx}120 kDa. However, upon overnight Noc treatment, a new BUBR1 antigen with a slow mobility was induced in these cell lines (Lanes 2, 4, and 6). We noticed that in mitotic shake-off cells, BUBR1 existed primarily as the high molecular weight form (Fig. 5ACitation , Lane 8), whereas the interphase cells contain only the low molecular weight form (Fig. 5ACitation , Lane 7). Many mitotic checkpoint proteins are phosphorylated during mitotic checkpoint activation. To determine whether the mobility shift of BUBR1 was attributable to phosphorylation, we immunoprecipitated BUBR1 from Noc-treated HeLa cell lysates using the anti-BUBR1 antibody. The immunoprecipitates were treated with {lambda}-phosphatase (PPase) in the presence or absence of okadaic acid, a phosphatase inhibitor. Fig. 5BCitation shows that the slow mobility form of BUBR1 in both mitotic shake-off cell lysates (Lane 5) and asynchronous cell lysates (Lane 6) was converted to the fast mobility one after phosphatase treatment. In the presence of okadaic acid, the phosphatase failed to convert the mobility of BUBR1 (Lane 4), indicating that Noc treatment induces BUBR1 phosphorylation.



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Fig. 5. Phosphorylation of BUBR1 upon mitotic checkpoint activation. A, GMOO637D, A549, or HeLa cells were cultured in the presence or absence of Noc for 16 h. Total cell lysates were analyzed for BUBR1 expression by Western blotting. Interphase HeLa cell lysates (Lane 7) and mitotic cell lysates obtained by mechanical shake-off (Lane 8) were analyzed for BUBR1 expression via Western blotting. B, HeLa cells were treated with Noc for 16 h. The entire population of treated cells (Lanes 2, 3, 4, and 6), as well as the mitotic shake-off cells (Lanes 1 and 5), were collected and lysed in the lysis buffer. HeLa cell lysates were immunoprecipitated with the anti-BUBR1 antibody (Lanes 4–6). BUBR1 immunoprecipitates (IP) treated with {lambda}-phosphatase in the presence (Lane 4) or absence of okadaic acid (OA; Lanes 5 and 6), as well as the cell lysates, were analyzed by SDS-PAGE, followed by Western blotting using the anti-BUBR1 antibody.

 
Proteins such as p55CDC involved in mediating mitotic checkpoint activation fluctuate during the cell cycle (17) . In addition, there exists a potential destruction box (RSSLAELKS) in BUBR1 but not in BUB1 (18) . To examine whether BUBR1 is also regulated at the protein level during the cell cycle, equal amounts of A549 cell lysates from various stages of the cell cycle were analyzed for BUBR1 via Western blotting. A549 was used for synchronization because we have previously used the cell line for cell cycle study (19) . Fig. 6ACitation shows that BUBR1 was present in G1 and that its level increased (at least doubled based on densitometric scanning) during later S and G2. No phosphorylated BUBR1 was detected during G1, G1-S, S, and G2 (Fig. 5ACitation , Lanes 2–4). In contrast, M-phase cell lysates contained the phosphorylated form of BUBR1 (Fig. 6ACitation , Lane 5). Cell cycle status was confirmed by flow cytometric analyses of propidium iodide-stained cells (data not shown).



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Fig. 6. BUBR1 protein levels during the cell cycle. A, A549 cells were synchronized at various stages of the cell cycle as described in "Materials and Methods." Equal amounts of cell lysates were analyzed by Western blotting for BUBR1. B, a part of the stained blot was shown as a loading control. Asyn, asynchronous.

 
BUBR1 does not have an obvious ATP-binding consensus sequence (9 , 15) . To determine whether BUBR1 is an active protein kinase, we expressed BUBR1 as a six histidine-tagged fusion protein using the baculoviral expression system. The expressed His6-BUBR1 was analyzed by SDS-PAGE, followed by Western blotting. Fig. 7ACitation shows that His6-BUBR1 antigen was present in sf-9 cells infected with BUBR1 recombinant baculoviruses (Lanes 2) but not in the cells infected with wild-type baculoviruses (Lanes 1). Infection of sf-9 cells with a higher titer (10x higher in Lane 3 than that in Lane 2) of the recombinant baculoviruses resulted in a significant increase of the amount of BUBR1 antigens (Lane 3). The recombinant BUBR1 expressed in sf-9 cells (Fig. 7BCitation , Lane 1) was purified to near homogeneity (Lanes 2 and 3) via affinity chromatography. The purified protein was immobilized onto Ni-NTA resins and assayed for autophosphorylation activity in a kinase assay as described in "Materials and Methods." Fig. 7CCitation shows that His6-BUBR1 (Lane 2, 10 µg of protein), but not the vehicle resins (Lane 1), is capable of autophosphorylation. We noticed that the autophosphorylation did not shift BUBR1 to the slowly migrating one.



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Fig. 7. His6-BUBR1 is capable of autophosphorylation. A, equal amounts of protein lysates from sf-9 cells infected with His6-BUBR1 recombinant baculoviruses (Lanes 2 and 3) or with the wild-type baculoviruses (Lane 1) were analyzed for BUBR1 via Western blotting. Ten times more His6-BUBR1 baculoviruses were used for infection of cells shown in Lane 3 than those shown in Lane 2. B, total sf-9 cell lysates infected with His6-BUBR1 baculoviruses (Lane 1) and the purified His6-BUBR1 proteins (Lanes 2 and 3) were analyzed by SDS-PAGE, followed by Coomassie blue staining. C, purified His6-BUBR1 was immobilized onto Ni-NTA resins. The BUBR1 resins (Lane 2), as well as control resins alone (Lanes 1), were analyzed for autokinase activities.

 
Discussion

Several groups have recently cloned BUBR1 (9) , MAD3 (15) , and SSK1 (16) , which are identical to our cloned gene MAD3L (AF068760). Although the putative protein kinase has been implicated in the mitotic checkpoint control (9 , 15) , little was known regarding its regulation and mode of action during the mitotic checkpoint activation. In the present study, we have shown that high levels of BUBR1 mRNA expression are correlated with a high mitotic index. It is known that activation of yeast BUB1 and MAD3 halts mitotic progression when chromosomes fail to align correctly during mitosis because of disruption of microtubules (4) . It is reasonable to speculate that BUBR1 protein also functions as a mitosis-safeguard protein to ensue the order of progression of mitotic events. Therefore, it is conceivable that the BUBR1 gene expression may not be required for quiescent or differentiated cells. Indeed, this notion is supported by our observations that fully differentiated primary tissues such as muscle, heart, and brain express no detectable levels of BUBR1 transcripts (Fig. 1Citation ; Table 1Citation ). Indeed, recent studies by Chan et al. (20) have convincingly shown that BUBR1 is essential for the mitotic checkpoint activation and for normal mitosis.

BUBR1 proteins were present, albeit at different levels, in most cell lines examined (Fig. 4A)Citation . Dami and HEL express the most abundant BUBR1. It is interesting to note that these two cell lines are either megakaryocytic or with megakaryocyte-differentiation potentials (21) , and that a significant fraction of cells are polyploid.4 This implies that high levels of BUBR1 may play a role in terminal differentiation of megakaryocytes by negatively affecting mitotic progression. A recent study showed that BUBR1 and BUB1 sequentially assembled onto kinetochores during prophase, and more BUBR1 and BUB1 proteins are associated with kinetochores of unaligned chromosomes (22) .

We have shown that phosphorylation appears to be a primary mechanism regulating BUBR1 activity during mitotic checkpoint activation. The dramatic mobility shift observed with BUBR1, a protein of 120 kDa, after mitotic checkpoint activation suggests that the protein is phosphorylated on many sites. Our in vitro kinase assays (Fig. 7C)Citation demonstrated that BUBR1 is capable of autophosphorylation, although its autokinase activity was rather low as compared with the immunocomplex kinase assay (data not shown). Autophosphorylated BUBR1 did not result in the mobility shift, suggesting that some other kinase activities are required for the conversion of BUBR1 to the hyperphosphorylated form. Yeast BUB1 protein appears to lie at the beginning of the mitotic checkpoint signaling pathway (1 , 23) , and its kinase activity is activated during the mitotic checkpoint activation (24) . In addition, p38, a mitogen-activated protein kinase family member implicated in mitotic checkpoint control, is rapidly activated by Noc (25) . Therefore, it would be interesting to examine whether BUB1 or p38 is the putative activity that phosphorylates BUBR1 during the mitotic checkpoint activation.

BUBR1 appears to be also regulated, to a certain extend, at protein levels (Fig. 6)Citation . Although present in G1 BUBR1 levels are significantly increased as cell progress into later S and G2 stages of the cell cycle. Recently, it has been reported that a murine BUBR1 counterpart contains a RSSLAELKS motif similar to the destruction box (18) , commonly found in many proteins that are regulated in a cell cycle-dependent manner. This motif is also present in human BUBR1 (amino acids 225–233), supporting the notion that BUBR1 protein levels may fluctuate during the cell cycle. No phosphorylated form of BUBR1 is detected until cells are in mitosis. This is consistent with the function of mitotic checkpoint genes, i.e., to monitor and control metaphase-anaphase transition.

Although the biochemical function of yeast MAD3 is unknown, it has been shown recently that Saccharomyces cerevisiae MAD3, as well as MAD1 and MAD2, associate with CDC20, a protein required for exiting from mitosis (26) . The interaction between MAD3 and CDC20 appears to be cell cycle dependent, peaking during mitosis (26) . It has been suggested that CDC20 is a target of the yeast mitotic checkpoint (26) . Human p55CDC, a CDC20 homologue in mammals, is a phosphoprotein, and the phosphorylation peaks at mitosis (21 , 27) . In addition, it has been shown that p55CDC connects MAD2 to APC (10) . Thus, it would be interesting to examine whether BUBR1 might relay a mitotic checkpoint signal to APC via p55CDC.

Extensive research in the past decade or so has shown that many genes involved in cell cycle regulation or checkpoint controls also play an essential role in the suppression of tumor growth. For example, p53 regulates G1-S and G2-M checkpoint, and the ATM gene product regulates DNA damage checkpoint. Structural abnormalities that result in functional defects of p53 or ATM are closely correlated with the neoplastic transformation (28 , 29) . The mitotic checkpoint regulates the initiation and segregation of chromosomes. A failure in this checkpoint can lead to the loss or gain of genetic materials in daughter cells, which is thought to contribute to the genomic instability associated with cancer development and progression (13) . We and others have mapped the BUBR1 gene to chromosome 15q14–154 or 14q13–15 (9) , a locus shown to exhibit loss of heterozygosity in several types of cancers (30 , 31) . It has been proposed that chromosomal instability in most colorectal cancers, as well as in many other cancers, may be a direct consequence of mutational inactivation of mitotic checkpoint genes such as BUB1 or BUBR1 (9) . The absence of BUBR1 antigen in PC3 (Fig. 4ACitation , Lane 9) may be attributable to abnormalities in the BUBR1 gene because defects in this gene have been reported in colorectal cancer samples (9) . Obviously, further studies will be necessary to fully understand the role of BUBR1 in normal as well as in abnormal cell growth.

Materials and Methods

Materials.
All culture media (RPMI 1640, MEM, Iscove’s modified Dulbecco’s medium, DMEM, and McCoy’s) and antibiotics (penicillin-streptomycin) were purchased from Life Technologies, Inc. (Grand Island, NY). A multiple tissue master dot blot was purchased from Clontech, Inc. (Palo Alto, CA). The pT7Blue plasmid was obtained from Novagen, Inc. (Madison, WI). [{alpha}-32P]dCTP (800 Ci/mmol) was from DuPont NEN (Wilmington, DE). Fetal bovine serum was purchased from Hyclone Laboratories (Logan, UT). Polyclonal anti-BUBR1 antisera were raised in rabbits via Research Genetics (Atlanta, GA). {lambda} phosphatase was from New England Biolab (Beverly, MA). A baculoviral expression system was purchased from PharMingen (San Diego, CA).

Cell Lines and Treatments.
Cell lines were grown in various media supplemented with 10% FBS and antibiotics (100 µg/ml penicillin and 50 µg/ml streptomycin). Cell lines included in the present study were Dami (megakaryoblastic leukemia), HEL (erythroleukemia), HIMeg (myeloid progenitor), SAM-1 (megakaryocytic), LNCaP (prostatic carcinoma), DU145 (prostatic carcinoma), HeLa (cervical carcinoma), GM00637D (fibroblast), Molt4 (T-lymphocytic), A549 (transformed lung fibroblast), and PC3 (prostatic carcinoma).

A549 and HeLa cells were used for synchronization studies. G1 phase A549 cells were obtained by culture in methionine-free DMEM containing 10% FBS for 48 h. A549 cells synchronized at the G1-S boundary were achieved by sequential culture in medium containing aphidicolin (1 µg/ml) for 14 h, normal medium for 14 h, and finally, medium containing thymidine (2 mM) for 14 h. A549 cells arrested at late S and G2 were obtained through washing cells arrested at the G1-S boundary, as described above, with PBS and reculturing in fresh DMEM with 10% FBS for 6–7 h. To obtain mitotic prometaphase cells, A549 cells were treated with Noc (0.4 µg/ml) for 16 h. HeLa cells treated with Noc for 16 h were used for collection of mitotic shake-off cells. Interphase HeLa cells were the adherent fraction of HeLa cell population. The cell cycle status of the treated cells was confirmed by flow cytometric analyses of propidium iodide-stained cells.

To activate mitotic checkpoint, A549, GM00637D, or HeLa cells were treated with Noc (0.4 µg/ml) for 16 h (unless otherwise specified). At the end of the treatment, cells were lysed in a lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM EDTA, 1 mM Na4VO3, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml soy bean trypsin inhibitor, 10 mM NaF, and 300 nM okadaic acid]. Cell lysates were stored at -70°C for subsequent analyses.

Dot Blotting.
A multiple-tissue dot blot purchased from Clontech was hybridized overnight with a 32P-labeled BUBR1 or ß-actin cDNA probe according to the supplier’s protocol. The blot was washed as described (12) and autoradiographed. Specific signals detected on autoradiographs were quantified using an image scanner (Imager Densitometer GS-700; Bio-Rad, Richmond, CA).

Western Blotting.
Equal amounts of cell lysates were analyzed by SDS-PAGE, followed by Western blotting using an anti-BUBR1 antibody. The anti-BUBR1 antiserum was raised in rabbit against BUBR1 peptide (residues 421–438). The protein blots were first probed with the BUBR1 (1:500 dilution) antibody and then with a goat-antirabbit antibody conjugated with horseradish peroxidase as described (19) . The specific signals on the blots were detected with the enhanced chemiluminescence method.

Expression and Purification of Recombinant BUBR1.
Full-length recombinant BUBR1 was expressed using the baculoviral expression system (PharMingen), following the manufacturer’s protocol. Briefly, a cDNA fragment containing the entire open reading frame of human BUBR1 was subcloned into pVL-1393 transfer vector. To facilitate purification of recombinant BUBR1 protein, a short sequence coding for six histidine residues was inserted in-frame immediately after the ATG codon of BUBR1 cDNA. Baculoviral expression vector BaculGold DNA and the transfer plasmid pVL-1393-BUBR1 were then cotransfected into insect sf-9 cells. Insect sf-9 cells expressing recombinant BUBR1 were harvested and lysed for purification of His6-BUBR1 using Ni-NTA resins (Qiagen, Chatsworth, CA).

Immunoprecipitation and BUBR1 Protein Kinase Assays.
Equal amounts of cell lysates were supplemented with an anti-BUBR1 antiserum (1:500 dilution) or with the preimmune serum and incubated at room temperature for 2 h or at 4°C overnight. Protein A/G agarose beads (25 µl) were then added to each immunoprecipitation mixture, and the incubation continued at room temperature for 1 h or at 4°C for 2 h. Immunoprecipitates were collected, washed three times with the lysis buffer, and analyzed by SDS-PAGE, followed by Western blotting. For kinase assays, His6-BUBR1 was purified using Ni-NTA resins. The purified His6-BUBR1 was resuspended in a kinase buffer [10 mM HEPES (pH 7.4), 10 µM MnCl2, and 5 mM MgCl2]. Kinase assays were initiated by the addition of [{gamma}-32P]ATP (5 µCi). The kinase mixtures were incubated at 37°C for 30 min and then analyzed by SDS-PAGE, followed by autoradiography.

Acknowledgments

We thank Andrew Hoyt for valuable suggestions and helpful discussions. We also thank Bin Ouyang and Huiqi Pan for technical assistance.

Footnotes

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.

1 This work was supported by USPHS Grant CA74299. Back

2 To whom requests for reprints should be addressed, at Division of Hematology-Oncology, University of Cincinnati College of Medicine, K-Pavilion, ML-508, 231 Bethesda Avenue, Cincinnati, OH 45267. Phone: (513) 584-4445; Fax: (513) 584-6703; E-mail: wei.dai{at}uc.edu Back

3 The abbreviations used are: APC, anaphase-promoting complex; Noc, nocodazole; FBS, fetal bovine serum; Ni-NTA, Ni-nitrilotriacetic acid. Back

4 Unpublished data. Back

Received for publication 3/19/99. Revision received 10/ 6/99. Accepted for publication 10/ 7/99.

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