Cancer Research Meeting Calendar Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 11186, December 1, 2007. doi: 10.1158/0008-5472.CAN-07-2578
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sankaran, S.
Right arrow Articles by Parvin, J. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sankaran, S.
Right arrow Articles by Parvin, J. D.

Molecular Biology, Pathobiology, and Genetics

Aurora-A Kinase Regulates Breast Cancer–Associated Gene 1 Inhibition of Centrosome-Dependent Microtubule Nucleation

Satish Sankaran1, Donna E. Crone2,3, Robert E. Palazzo2,3 and Jeffrey D. Parvin1,4

1 Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; 2 Department of Biology, Rensselaer Polytechnic Institute, Troy, New York; 3 Marine Biological Laboratory, Woods Hole, Massachusetts; and 4 Department of Biomedical Informatics, The Ohio State University Medical Center, Columbus, Ohio

Requests for reprints: Jeff Parvin, Department of Biomedical Informatics, The Ohio State University, 460 West 12th Avenue, 904 Biomedical Research Tower, Columbus, OH 43210. Phone: 614-292-0523; Fax: 614-688-8675; E-mail: Jeffrey.Parvin{at}osumc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Breast cancer–associated gene 1 (BRCA1) regulates the duplication and the function of centrosomes in breast cells. We have previously shown that BRCA1 ubiquitin ligase activity directly inhibits centrosome-dependent microtubule nucleation. However, there is a paradox because centrosome microtubule nucleation potential is highest during mitosis, a phase when BRCA1 is most abundant at the centrosome. In this study, we resolve this conundrum by testing whether centrosomes from cells in M phase are regulated differently by BRCA1 when compared with other phases of the cell cycle. We observed that BRCA1-dependent inhibition of centrosome microtubule nucleation was high in S phase but was significantly lower during M phase. The cell cycle–specific effects of BRCA1 on centrosome-dependent microtubule nucleation were detected in living cells and in cell-free experiments using centrosomes purified from cells at specific stages of the cell cycle. We show that Aurora-A kinase modulates the BRCA1 inhibition of centrosome function by decreasing the E3 ubiquitin ligase activity of BRCA1. In addition, dephosphorylation of BRCA1 by protein phosphatase 1{alpha} enhances the E3 ubiquitin ligase activity of BRCA1. These observations reveal that the inhibition of centrosome microtubule nucleation potential by the BRCA1 E3 ubiquitin ligase is controlled by Aurora-A kinase and protein phosphatase 1{alpha}–mediated phosphoregulation through the different phases of the cell cycle. [Cancer Res 2007;67(23):11186–94]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Centrosomes nucleate microtubules, resulting in the organization of a dynamic polymer matrix that is necessary for numerous cell functions including the maintenance of cell shape and polarity, intracellular transport, and the segregation of chromosomes during cell division (reviewed in ref. 1). At the end of the G1 phase of the cell cycle, the single centrosome in the cell replicates such that by the G2-M transition, the cell contains two centrosomes that form the poles of the bipolar mitotic spindle. The mitotic spindle ensures equal segregation of duplicated chromosomes to the two dividing daughter cells. The control of centrosome duplication is crucial because any aberration in centrosome number could lead to multipolar spindles and subsequent missegregation of chromosomes, resulting in aneuploidy and genomic instability in the daughter cells.

As centrosomes progress through the G2 and M phases of the cell cycle, they undergo a process called maturation, which raises their microtubule nucleation potential to high levels during mitosis (reviewed in ref. 2). As a result of multiple structural changes, mitotic centrosomes increase their microtubule nucleation potential 5-fold during mitosis, a process that is reversed and reset to a relatively low nucleation potential when the daughter cells return to interphase (3). Centrosomes are thus subject to cell cycle–dependent regulatory mechanisms that control their microtubule nucleation potential.

Breast cancer–associated gene 1 (BRCA1), the breast and ovarian cancer–specific tumor suppressor, localizes to the centrosomes (4, 5) and is important in the regulation of centrosome number (6–9). The control of centrosome number is of interest because breast cancer tumor cells accumulate supernumerary centrosomes, which likely contribute to the aneuploidy that is common in lesions (10, 11). Importantly, it has been shown that BRCA1 directly regulates centrosome microtubule nucleation activity (5, 12). BRCA1, a 1,863 amino acid phosphoprotein, exhibits E3 ubiquitin ligase activity as a heterodimer with BRCA1-associated RING domain 1 (BARD1; refs. 13, 14). The ubiquitin ligase activity is critical for the inhibition of centrosome function because the expression in cells of a point mutant of BRCA1 that can no longer function as a ubiquitin ligase causes the dominant-negative phenotype of centrosome amplification and hyperactive centrosomes (12). Many cancer-associated mutations of the BRCA1 gene directly affect the E3 ubiquitin ligase activity because they alter the structure of the RING domain and inactivate this enzymatic activity (15, 16). Thus, the E3 ubiquitin ligase activity is critical to the tumor suppression function of BRCA1. The most prevalent mutations of the BRCA1 gene are frame-shift or nonsense mutations, which result in the loss of the COOH terminus but often leave the RING domain and the E3 ubiquitin ligase activity intact. Although these truncated BRCA1 proteins, in complex with BARD1, are active as ubiquitin ligases in a nonspecific assay, these carboxyl-terminal truncation mutant BRCA1 proteins are defective for the regulation of centrosome function (8, 12). Thus, there is a correlation between a variety of cancer-associated BRCA1 mutations and the BRCA1 ubiquitin ligase–mediated control of centrosome function.

BRCA1 ubiquitinates multiple centrosomal proteins, and one has been identified as {gamma}-tubulin (8). Expression in cells of a mutant {gamma}-tubulin, in which the lysine that is an ubiquitin acceptor site was altered to an arginine, resulted in the same phenotype as those caused by inhibition of BRCA1: centrosome amplification and hyperactivity (5, 8). Because BRCA1 localizes to centrosomes at all phases of the cell cycle (5), it is likely that the BRCA1 ubiquitin ligase activity is directly involved in regulating centrosome function. Although BRCA1 localization to the centrosome was questioned in one study in which the green fluorescent protein fusion of BRCA1 failed to localize to the centrosome (17), a number of other reports have shown endogenous BRCA1 localization to the centrosome (4, 5, 18, 19).

RNA interference (RNAi) of BRCA1 in cells blocked in early S phase by hydroxyurea treatment resulted in markedly enhanced centrosomal microtubule nucleation activity (5). This result suggested that in early S phase cells, centrosomes have relatively low microtubule nucleation potential, and this low level of activity depends on BRCA1 down-regulation of centrosome-dependent microtubule nucleation potential. During mitosis, however, centrosome microtubule nucleation is 5-fold higher (3), even though the BRCA1 concentration at centrosomes is at peak levels (4), bringing confusion on the role of BRCA1 in the regulation of centrosome-dependent microtubule nucleation. To resolve this issue, in the present study, we compare the BRCA1-dependent inhibition of microtubule nucleation potential in centrosomes from early S phase with those from M phase. We assayed centrosomes in tissue culture cells using purified centrosomes in cell-free reactions. We find that centrosomes in mitotic cells are refractory to inhibition by BRCA1 due to Aurora-A kinase (AURKA) activity. In addition, protein phosphatase 1{alpha} (PP1a) potentiates the activity of BRCA1 in regulating centrosome function. These results, along with published findings, suggest that a phosphorylation cascade exists which controls BRCA1 ubiquitin ligase–mediated inhibition of centrosome function in live cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies. Antibodies specific to {gamma}-tubulin and {alpha}-tubulin (Sigma), ubiquitin (raised in rabbit), centrin (a kind gift from Dr. Jeffrey Salisbury, The Mayo Clinic, Rochester, MN) were used at 1:1,000 dilution for both immunoblots and immunofluorescence.

Cell culture and transfection. Hs578T (ATCC cell line HTB-1216) and HeLa S3 cells (ATCC cell line CCL-2.2) were blocked in S phase either by treatment with 20 mmol/L of hydroxyurea for 18 h or by a double thymidine block protocol: incubating with 2 mmol/L of thymidine (Sigma) for 18 h, followed by growth in thymidine-free medium for 9 h and reblocking in 2 mmol/L of thymidine for another 18 h.

Cells were blocked at M phase either by treatment with 50 µmol/L of monastrol (Sigma) for 12 h, or by thymidine + nocodazole block. For thymidine + nocodazole block, cells were grown in 2 mmol/L of thymidine for 18 h, grown in thymidine-free medium for 3 h, followed by growth in 100 ng/mL of nocodazole (Sigma) for 12 h. Tissue culture experiments involving the transient addition of Aurora kinase inhibitor II (EMD Biosciences Inc.) were serum-free when in the presence of the drug.

The microtubule regrowth assays were done 48 h posttransfection with short interfering RNA (siRNA). The BRCA1-specific oligonucleotide was described earlier (BRCA1-a; ref. 5) and the control oligonucleotide was specific for luciferase (5, 8).

Microtubule regrowth assay. The assay was performed as described earlier (5). In the case of mitotic cells, 50 mitotic cells were identified based on the absence of a nuclear membrane and the presence of condensed chromosomes, and their aster morphology scored. The inStat program (Graph Software Inc.) was used to compute statistical significance of the biochemical data. A two-tailed t test compared control with test data series. Statistically significant P values (P < 0.01) are indicated in the figure legends.

Protein purification. The BRCA1/BARD1 protein preparations as well as centrosome isolation were done as described earlier (8). Sea urchin tubulin was cycled thrice and prepared as described (20). Purified recombinant AURKA (Upstate), PP1a (EMD Biosciences), and ubiquitin (Sigma) were purchased.

In vitro ubiquitination and aster formation assays. Ubiquitination reactions were performed as described earlier (5). The ubiquitinated centrosomes were centrifuged onto glass coverslips and allowed to grow asters in the presence of either 0.35 mg/mL of purified tubulin in reassembly buffer [100 mmol/L PIPES (pH 6.9), 1 mmol/L EGTA, 5 mmol/L MgSO4, and 2 mmol/L GTP] or 200 µg of Xenopus extract at 25°C for 12 to 15 min. Xenopus extract was used in experiments described in Fig. 1C and D . Some reactions included phosphatase inhibitor cocktail (50 mmol/L NaF, 10 mmol/L β-glycerophosphate, 10 mmol/L sodium PPi, and 1 mmol/L sodium orthovanadate).


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. BRCA1-mediated inhibition of centrosomal microtubule nucleation is stronger at S phase than during M phase. A, histogram of the percentage of cells with large centrosomal asters in cells blocked either in S or the M phase or asynchronously growing cells. Columns, mean from two independent experiments; bars, SE. Cells were transfected with a control siRNA (blue) or a BRCA1-specific siRNA (red). In the presence of monastrol, the difference between BRCA1 and control was not significantly different. B, centrosomes were purified from asynchronously growing HeLa S3 cells as well as those blocked in S and M phases of the cell cycle. Centrosomes were ubiquitinated in cell-free assays and tested for aster formation using isolated tubulin. Asynchronous centrosomes formed asters only when BRCA1 was omitted from the reaction (a and b), whereas centrosomes from monastrol blocked cells (M phase) formed smaller asters in the presence of BRCA1 (c and d). Bar, 10 µm. C, quantitation of the results from B are shown as a ratio of microtubule content (plus BRCA1: minus BRCA1) for centrosomes purified from asynchronous cells (Asy) or from M phase cells using purified tubulin (P < 0.001). D, centrosomes purified from cells blocked either in S phase or M phase were assayed for microtubule nucleation using crude mitotic Xenopus extracts as a source of tubulin. The mean microtubule content from 20 asters from asynchronous cells or those blocked at S or M phase were quantified. The values were normalized to the paired reaction with no BRCA1. Columns, normalized mean values; bars, SE (P < 0.001).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BRCA1 inhibits centrosome function in early S phase but not M phase. BRCA1 regulates the microtubule nucleation potential of centrosomes in breast cells via its E3 ubiquitin ligase activity (5, 12). Centrosomal microtubule nucleation is responsible for maintaining the cytoskeletal microtubule network in cells during interphase and is involved in spindle formation during mitosis.

We have shown that BRCA1-mediated inhibition of centrosome microtubule nucleation potential was more pronounced in S phase cells than in asynchronously growing cells (5). To further investigate the cell cycle dependence of BRCA1 regulation of centrosome function, we used Hs578T breast cancer cells blocked in S phase by treatment with hydroxyurea or in mitotic phase by treatment with monastrol (21). Using an in vivo microtubule nucleation assay, we compared centrosome microtubule nucleation potential in Hs578T cells with normal BRCA1 to cells with reduced BRCA1 levels. Hs578T cells grown on glass coverslips were transfected with siRNA targeting either BRCA1 or a control gene (luciferase). Forty-eight hours later, these cells were subjected to microtubule regrowth assay. Large asters were identified based on the criterion that they had at least 10 long radiating microtubules (5) as compared with small asters that had almost undetectable {alpha}-tubulin staining. This criterion was useful in quantifying asters from asynchronously growing cells and cells synchronized by blockage with hydroxyurea. Asters in mitotic cells had a smaller diameter and were more densely labeled by the {alpha}-tubulin–specific antibody. The morphologic differences observed between asynchronous and M phase centrosomes are consistent with previous observations of a 5-fold higher microtubule nucleation potential for isolated mitotic centrosomes (22–25) and a change in microtubule dynamics as cells enter M phase (reviewed in ref. 26). In this study, large mitotic asters were defined as those having microtubule arrays emanating from the centrosome with a diameter >2.5 µm (Supplementary Fig. S1). The effect of blocking the cell cycle using monastrol on the percentage of cells with large asters in microtubule regrowth assays from cells transfected with the two siRNAs are shown in the histogram (Fig. 1A). Cultures of asynchronously growing cells with BRCA1 silenced by RNAi had a 2.5-fold higher fraction of cells with large asters. In cells blocked in early S phase using hydroxyurea, RNAi of BRCA1 resulted in a 12-fold higher fraction of cells with large asters. These results are consistent with our prior observations (5) and support the notion that during S phase, BRCA1 restrains centrosome function. In cells blocked with monastrol, inhibition of BRCA1 by RNAi resulted in a minimal change of the fraction of cells that contained large asters.

The modest inhibitory effect of BRCA1 on mitotic centrosomes was further evident when centrosomes purified from cells blocked at mitosis were tested using an in vitro aster formation assay. Centrosomes were isolated from HeLa cells blocked at G2-M by blocking with thymidine followed by release into nocodazole. Although BRCA1 localizes to the centrosome during mitosis, isolated centrosomes contain no detectable BRCA1 protein (data not shown). Centrosomes isolated from these cells were ubiquitinated in vitro in the presence of E1, E2, ubiquitin, and BRCA1/BARD1 (the BRCA1/BARD1 heterodimer is referred to as "BRCA1" in the subsequent sections). The ubiquitinated centrosomes were centrifuged onto glass coverslips, washed, and allowed to nucleate microtubules and form asters in the presence of isolated tubulin. The asters were fixed and immunostained with {alpha}-tubulin–specific (red) and {gamma}-tubulin–specific (green) antibodies to label microtubules and centrosomes, respectively. The asters were observed as red microtubules radiating from a yellow center (the centrosome). Asters formed by centrosomes purified from asynchronous cells were morphologically distinct from those formed from mitotic centrosomes. The patterns of the asters were consistent with what was observed with living cells: interphase or asynchronous centrosomes formed asters that were longer and wispier than the counterparts from mitotic centrosomes (Fig. 1B). This observation indicated that centrosomes from the two different stages of the cell cycle had intrinsic differences in the way they nucleated microtubules. Importantly, upon inclusion of the BRCA1 ubiquitin ligase in the nucleation reaction, microtubule nucleation of asynchronous centrosomes was strongly inhibited (Fig. 1Bb). We did detect acentrosomal microtubules, not associated with {gamma}-tubulin. Quantitation of the microtubule content in these BRCA1-inhibited asters was difficult because there were far fewer detectable asters in those reactions that contained active BRCA1. The value of 10% to 20% microtubule content is a minimum value because that much of an aster must remain in order to be scored. Clearly, BRCA1 strongly inhibited aster formation when using centrosomes purified from asynchronous cells to nucleate tubulin. In contrast, asters formed in vitro from mitotic centrosomes contained many more microtubules that were shorter, perhaps in bundles (Fig. 1Bc). Importantly, inclusion of BRCA1 in the ubiquitination reaction resulted in only a partial decrease of the aster size (Fig. 1Bd). Previously, when using mitotic Xenopus extracts as the source of tubulin, we observed that BRCA1 inhibition of aster formation was strong, but not as complete as observed in Fig. 1Bb (5). The effect of BRCA1 inhibition on aster formation is significantly stronger when using isolated tubulin to assay microtubule nucleation potential, as in these experiments. We suggest that the use of the mitotic Xenopus extracts in earlier experiments might have blunted the inhibitory effect of BRCA1.

Centrosomes isolated from asynchronous, S phase, or M phase cells formed asters in vitro in the absence or presence of 30 nmol/L of BRCA1, and in the presence of mitotic Xenopus extracts. Whereas the microtubule content of asters from asynchronous and S phase centrosomes was reduced 5-fold by BRCA1 ubiquitination activity, mitotic aster function was only inhibited by ~2-fold (Fig. 1C). Even though the source of tubulin in the experiment shown in Fig. 1D was the mitotic extract, there was a differential degree of sensitivity of the cell cycle–specific centrosomes to the inhibition by the BRCA1 ubiquitin ligase, suggesting that some factor is present or a covalent modification(s) occurs within the centrosome that confers either resistance or sensitivity to the inhibition by the BRCA1 ubiquitin ligase.

Resistance of mitotic centrosomes to BRCA1-dependent inhibition is mediated by phosphorylation. BRCA1 is present at centrosomes during all phases of the cell cycle, and levels are particularly high during metaphase when the DNA is condensed, the nuclear membrane dissolves, and most of the nuclear BRCA1 relocates to the centrosomes (4, 5). Because the microtubule nucleation potential of centrosomes is highest during mitosis, it is puzzling that BRCA1 is most abundantly localized to the organelle at this time. Because the ubiquitination activity of the BRCA1 protein, and not just the presence of BRCA1, is important for regulating centrosome function (12), it is possible that the BRCA1 associated with mitotic centrosomes has decreased E3 ubiquitin ligase activity. Alternatively, it is possible that the centrosome-bound substrates of the BRCA1 ubiquitin ligase are resistant to inhibition by ubiquitination. Both possibilities could be affected by protein phosphorylation, and there is a marked increase in centrosome protein phosphorylation during mitosis (reviewed in refs. 27, 28). Furthermore, there is a direct correlation between the increased centrosome protein phosphorylation and the higher microtubule nucleation potential of centrosomes (29). If protein phosphorylation is responsible for either inactivating the E3 ubiquitin ligase activity of BRCA1 or decreasing its accessibility to substrates, removal of the phosphate through the action of a phosphatase should restore the BRCA1-dependent inhibition of mitotic centrosome function. PP1a localizes to the nucleus and centrosomes during interphase, and is localized to the centrosome exclusively during mitosis (30). Importantly, PP1a has been shown to dephosphorylate BRCA1 (31). Based on these reports, we tested the effect of PP1a on in vitro aster formation using purified mitotic centrosomes incubated with isolated tubulin. The inclusion of 1 unit of recombinant PP1a in the assay along with 30 nmol/L of BRCA1 increased the BRCA1 effect, resulting in a 1.6- to 5-fold higher inhibition of mitotic centrosome microtubule nucleation potential (Fig. 2A ). Inclusion of phosphatase inhibitors in the reaction mixture containing BRCA1 and PP1a resulted in a near-complete loss of BRCA1 inhibition of mitotic centrosome function, thus reversing the effect of the phosphatase. These data clearly show that mitotic centrosomes are resistant to inhibition by the BRCA1 ubiquitin ligase due to phosphorylation events mediated by one or more kinases.


Figure 2
View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Phosphorylation attenuates the sensitivity of mitotic centrosomes to BRCA1. A, purified centrosomes from mitotic HeLa S3 cells were ubiquitinated and assayed for aster formation with isolated tubulin in the presence of 1 unit (U) of PP1a and/or phosphatase inhibitor cocktail (PI) as indicated. The microtubule content of 20 asters from each treatment was calculated and normalized to the control reaction without BRCA1; columns, normalized mean values; bars, SE (P < 0.001). Varying concentrations of the Polo-like kinase inhibitor, Wortmannin (B), and Aurora kinase inhibitor II (C) were included in the in vitro aster formation assay with mitotic centrosomes and isolated tubulin. The mean microtubule content for each sample minus BRCA1 ({diamondsuit}) or plus BRCA1 ({blacksquare}) was normalized to its equivalent sample without any inhibitor. C, these normalized values are plotted against the corresponding inhibitor concentration used (P < 0.001).

 
AURKA modulates the BRCA1-dependent inhibition of mitotic centrosome function. Polo-like kinase I (32) and Aurora kinase (reviewed in refs. 33, 34) are two important enzymes that are essential for the maturation of centrosomes before the onset of mitosis. We used kinase -specific inhibitors to test whether either of these two kinases were involved in the protection of mitotic centrosomes from inhibition by BRCA1.

Wortmannin selectively inhibits the phosphoinositide-3-kinases, including mammalian Polo-like kinase 1 at an IC50 of 24 nmol/L (35). Various concentrations of Wortmannin were used in the in vitro aster formation assay using centrosomes purified from mitotic cells and isolated tubulin. The addition of up to 48 nmol/L of Wortmannin had no effect on the level of inhibition of mitotic centrosome activity by BRCA1 (Fig. 2B).

To test if AURKA was involved, various concentrations of aurora kinase inhibitor II, which inhibits AURKA at an IC50 of 300 nmol/L (36), were included in the in vitro assay. If AURKA modulates BRCA1-mediated inhibition of centrosome function, then this specific inhibitor should cause a BRCA1-dependent decrease in microtubule nucleation by mitotic centrosomes. The addition of 120 nmol/L of the inhibitor to the in vitro assay reduced the microtubule content of asters by 2-fold compared with the control sample (Fig. 2C). This result indicates that active AURKA present in mitotic centrosomes blocked the inhibitory activity of BRCA1. By inhibiting AURKA, BRCA1 became effective in reducing the microtubule nucleation potential of mitotic centrosomes. From these observations, we identified AURKA as a modulator of BRCA1 inhibition of mitotic centrosome microtubule nucleation potential. Because the inhibition of AURKA occurred during the course of the in vitro reaction, the AURKA was most likely modifying the recombinant BRCA1 protein added to the reactions. Alternatively, phosphorylated epitopes on the centrosome might be unstable and require continuous AURKA activity to maintain a resistance to the BRCA1 ubiquitin ligase.

AURKA, which localizes to centrosomes in late S phase (34), is known to phosphorylate BRCA1 (37). To test whether AURKA blocks the BRCA1-mediated inhibition of centrosome-dependent microtubule nucleation, we added purified recombinant AURKA to centrosomes purified from asynchronous cells. Most of these asynchronous cells are in the G1 phase of the cell cycle, as determined by flow cytometry (data not shown), and these cells are sensitive to the inhibition of centrosome-dependent microtubule nucleation by BRCA1 (Fig. 1A). We hypothesized that the inclusion of AURKA would confer resistance against BRCA1-mediated inhibition of centrosome microtubule nucleation potential. In support of this idea, inclusion of 1.4 mmol/L of AURKA in the in vitro centrosome microtubule nucleation assay did indeed block the inhibition of centrosome microtubule nucleation by BRCA1 (Fig. 3 ). Quantitation of the microtubule content of asters formed in this assay clearly indicated that the addition of AURKA to asynchronous centrosomes almost completely protected them from the inhibitory effect of BRCA1 (Fig. 3B). These results indicate that either by targeting proteins that are present at the centrosomes at interphase as well as mitosis, or by modifying BRCA1 enzymatic activity, AURKA offsets the inhibitory effect of BRCA1 on centrosome function.


Figure 3
View larger version (7K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. AURKA renders centrosomes from asynchronous cells refractory to BRCA1-mediated inhibition. A, the in vitro aster formation assay was performed with asynchronous centrosomes with or without 1.4 mmol/L of AURKA. BRCA1 inhibited centrosome aster formation (b) but the addition of AURKA (d) negated the inhibitory effect of BRCA1. Bar, 10 µm. B, microtubule content of the asters formed in the assay were quantified and the mean values for each sample were normalized to the sample with no BRCA1. Columns, normalized mean values; bars, SE (P < 0.001).

 
BRCA1 ubiquitin ligase activity is decreased in the presence of mitotic centrosomes. The data presented thus far suggest that mitotic centrosomes are refractory to inhibition by BRCA1 due to decreased BRCA1-dependent ubiquitination of centrosome proteins. To test whether the BRCA1-mediated ubiquitination activity was different in the presence of asynchronous or mitotic centrosomes, these centrosomes were ubiquitinated in vitro in the presence of E1, E2, ubiquitin, and BRCA1 and the ubiquitinated proteins were detected by Western blot analysis using antibody specific to ubiquitin. In each reaction, the same recombinant BRCA1 protein was tested. Importantly, in the presence of centrosomes purified from asynchronous cells, the ubiquitin ligase was more active than when in the presence of mitotic centrosomes. In reactions containing BRCA1 plus asynchronous centrosomes (Fig. 4A, lane 3 ), a high molecular weight smear was more intense when compared with BRCA1 ubiquitination products generated in the presence of mitotic centrosomes (Fig. 4A, lane 2). Centrosome content in these assays was standardized based on the quantitation of centrin content (Fig. 4A, bottom), indicating that the difference in ubiquitinated products was indeed due to the difference in the activity of the ubiquitin ligase. Although there is a direct correlation between the degree of inhibition of centrosome function by BRCA1 and the ubiquitin ligase activity of BRCA1, it was not clear if the phosphorylation of BRCA1 directly affects its ubiquitin ligase activity or whether centrosomal substrates were refractory to ubiquitination by BRCA1.


Figure 4
View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. BRCA1 ubiquitin ligase activity is attenuated by phosphorylation with AURKA. A, mitotic (lane 2) and asynchronous (lane 3) HeLa centrosomes were ubiquitinated in the presence of E1, E2, ubiquitin, and BRCA1. The sample analyzed in lane 1 contained no BRCA1. The reactions were subjected to electrophoresis on a 4% to 12% SDS-PAGE gel, blotted and probed with antiubiquitin antibody. The same blot was probed for centrin as a loading control. B–D, the efficiency of BRCA1-dependent ubiquitin polymerization was analyzed in the presence of PP1a or AURKA. The ubiquitin polymers were analyzed on 4% to 12% gel, blotted and probed with antiubiquitin antibody. B, lane 1 contained no BRCA1, PP1, or PI. Reactions contained 30 nmol/L of BRCA1 without PP1 (lane 2), 30 nmol/L of BRCA1 with 1 unit of PP1 (lane 3), and 30 nmol/L of BRCA1 plus 1 unit of PPI + phosphatase inhibitor cocktail (PI; lane 4). C, titration of ubiquitin polymerization activity of 15 nmol/L of BRCA1 in the presence of 0, 0.05, 0.2, and 1 unit of PP1 (lanes 1–4, respectively). Bottom, short exposure of the antiubiquitin blot (top), to show the extent of monomer ubiquitin utilization in these reactions. D, titration of ubiquitin polymerization activity of 30 nmol/L of BRCA1 in the presence of 0, 0.07, 0.35, 1.4, and 2.8 mmol/L of AURKA (lanes 1–5, respectively).

 
Phosphatase treatment of BRCA1 enhances its E3 ubiquitin ligase activity. The BRCA1 protein used in this study was expressed in insect cells using recombinant baculoviruses that each expressed BRCA1 and BARD1. Although the proteins were recombinant, insect cells could phosphorylate proteins; thus, the BRCA1 preparation likely has baseline phosphorylation. To study the direct effect of phosphorylation/dephosphorylation of BRCA1 on its ubiquitin polymerizing activity, nonspecific ubiquitin polymerization by BRCA1 was analyzed in the presence of PP1a and AURKA. Ubiquitin polymers were synthesized in the presence of E1, E2, and BRCA1, making this analysis independent of added centrosomes.

BRCA1 catalyzes the polymerization of long ubiquitin chains (Fig. 4B, lanes 2–4). Inclusion of 1 unit of PP1a in the BRCA1-mediated ubiquitin polymerization reaction caused a significant increase in the high–molecular weight ubiquitin chains observed as a smear migrating at positions consistent with molecular mass between 40 and 250 kDa (Fig. 4B, lane 3). The inclusion of a phosphatase inhibitor in the reaction containing BRCA1 and PP1a caused a reduction in the yield of ubiquitin chain formation, similar to the amount in the reaction containing only BRCA1 and no PP1a. Titrating different concentrations of PP1a into the ubiquitin polymerization reaction resulted in a dose-dependent increase in the concentration of high–molecular mass ubiquitin chains (Fig. 4C). As little as 0.05 units of PP1a strongly stimulated the BRCA1 ubiquitin ligase activity. These data clearly indicate that PP1a specifically increased BRCA1-mediated ubiquitin chain formation. Because no centrosomal proteins were present in this reaction, we suggest that the recombinant BRCA1 protein, purified from baculovirus-infected insect cells, has a baseline level of phosphorylation that inhibits its E3 ubiquitin ligase activity. PP1a thus raised the activity level of the BRCA1 preparation.

Conversely, we found that phosphorylating BRCA1 directly repressed its ubiquitin polymerization activity in this nonspecific assay. When increasing concentrations of recombinant AURKA were titrated into the polymerization reaction, a modest decrease in the ubiquitin polymerization activity by BRCA1 was observed (Fig. 4D). The low-magnitude effect by AURKA was likely due to the baseline phosphorylation of the purified BRCA1 protein. Based on these results, we suggest that the AURKA kinase activity inhibits BRCA1 ubiquitin ligase activity directly. It is also possible that AURKA renders at least some of the centrosomal substrates refractory to BRCA1 ubiquitination.

Transient inhibition of AURKA in cells blocked in G2-M increases the inhibition of centrosome function by BRCA1. Thus far, the cell cycle stage–specific effects on centrosome function of blocking BRCA1 expression and the cell-free assays for centrosome function have been consistent. The BRCA1 ubiquitin ligase inhibits centrosome function, but this inhibitory effect of BRCA1 is blocked in mitotic centrosomes by active AURKA. We tested whether this correlation is true in cells arrested in mitosis by pretreating cells with monastrol followed by a brief exposure to the AURKA inhibitor. We predicted that the inhibitory effect of BRCA1 on centrosome function would be more pronounced under these conditions because AURKA would be inhibited, but we anticipated that the effect would be small due to the predicted slow decay of phosphorylated epitopes on the BRCA1 protein. To our surprise, the effect of blocking BRCA1 expression was remarkably strong, with severe changes to centrosome structure and significant changes to centrosome function.

Hs578T cells grown on glass coverslips were transfected with either BRCA1-specific or control luciferase siRNA. Thirty-six hours posttransfection, these cells were treated with monastrol for 12 h. Forty-six hours posttransfection, cells were treated with 600 nmol/L of Aurora kinase inhibitor II for 2 h. The cells were then stained for {gamma}-tubulin. Centrosomes in BRCA1 knockdown cells exhibited increased {gamma}-tubulin staining in multiple adjacent foci associated with centrosomes (Fig. 5A and B ). The inhibition of BRCA1 resulted in a 3.5-fold increase in the number of cells with aberrant {gamma}-tubulin accumulation at the centrosomes (Fig. 5C). Although clearly aberrant, these supernumerary aggregations of {gamma}-tubulin do not reflect centrosome amplification because the stain for centrin, which marks the centrioles, was normal (data not shown).


Figure 5
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Inhibition of AURKA in mitotic cells results in aberrant {gamma}-tubulin accumulation at centrosomes. Hs578T cells transfected with the control siRNA (A) or the BRCA1-specific siRNA (B) were blocked at M phase by monastrol treatment. These cells were stained for {gamma}-tubulin (to mark centrosomes) and 4',6-diamidino-2-phenylindole (to visualize the nucleus). Centrosomes (white arrows). Amplified images of the centrosome (insets in A and B). Bar, 10 µm. C, cells with aberrant {gamma}-tubulin staining were scored in the control and the BRCA1 siRNA-treated samples; columns, mean percentages; bars, SE (P < 0.006). D, a histogram of the percentage of mitotic cells with large asters after being transfected with either the control or BRCA1-specific siRNA and with or without AURKA inhibitor (as indicated). Columns, mean from two independent experiments; bars, SE.

 
These cells were also assayed for centrosome function. Mitotic cells were assayed for microtubule nucleation capacity in the presence of normal or decreased BRCA1 when AURKA activity is reduced by the addition of the inhibitor and compared with when AURKA is fully active (Fig. 5D). The aster sizes of the different samples were normalized with respect to that of the control siRNA–treated cells in the absence of AURKA inhibitor. In the absence of AURKA inhibitor, BRCA1 knockdown caused only a modest increase in the percentage of cells with large asters. In contrast, cells with reduced BRCA1 and in the presence of AURKA inhibitor had a 1.5-fold increase in the percentage of cells with large asters as compared with cells with normal BRCA1 content but reduced AURKA activity.

These observed changes to the centrosome due to AURKA and BRCA1 remarkably require only 2 h to occur. These results strongly confirm that the in vitro experiments using purified centrosomes and purified BRCA1, PP1a, and AURKA enzymes apply to the cell in mitosis. Thus, one of the mechanisms by which AURKA positively stimulates microtubule nucleation activity of the mitotic centrosome is by blocking the inhibitory effects of the BRCA1 ubiquitin ligase.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, analyzing centrosome function in breast cancer cells in tissue culture and in cell-free reactions, we found that phosphorylation by AURKA inhibits BRCA1 ubiquitin ligase–mediated control of centrosomal microtubule nucleation. We propose a mechanism by which BRCA1, together with AURKA and PP1a, regulates centrosome-dependent microtubule nucleation potential during the cell cycle (Fig. 6 ). We do not suggest that this is the only mechanism by which AURKA stimulates microtubule nucleation activity of centrosomes, but rather this AURKA-BRCA1 axis significantly contributes to how AURKA stimulates centrosome function.


Figure 6
View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Model for BRCA1-mediated regulation of centrosomal microtubule nucleation through different phases of the cell cycle. A, based on published data and results from this study, the factors that control BRCA1 E3 ubiquitin ligase activity during M phase are shown: (–) inhibition and (+) activation of enzymatic activity. BRCA1 is maintained in a phosphorylated state during M phase by the concerted effects of AURKA kinase activity and the inhibition of PP1a phosphatase. Phosphorylated BRCA1 has reduced E3 ubiquitin ligase activity, resulting in reduced inhibition of centrosomal microtubule nucleation. B, relative enzymatic activity at the centrosome of BRCA1, PP1a, and AURKA through the cell cycle phases is shown schematically. At G1 and S phases, BRCA1 ubiquitin ligase activity is potentiated by high PP1a activity. At late G2-M phase, AURKA activity peaks resulting in the inactivation of both BRCA1 and PP1a with the net effect being enhanced centrosomal microtubule nucleation. At the beginning of the next G1, the BRCA1-mediated inhibition of centrosomal nucleation activity is re-established.

 
We show that BRCA1-mediated inhibition of centrosome microtubule nucleation is stronger during S phase but weak during M phase, even though BRCA1 is present at higher concentrations at the centrosomes at the early phases of mitosis (4, 5). We propose that PP1a, a serine-threonine phosphatase with a cellular distribution similar to BRCA1 (30), dephosphorylates BRCA1, resulting in an increased ubiquitin ligase activity, thus strongly inhibiting centrosome-dependent microtubule nucleation. Furthermore, because AURKA localizes to centrosomes during late S phase (34), our model proposes that AURKA inhibits the enzymatic activity of BRCA1 directly. PP1a is known to inhibit AURKA (38) and we have now shown that it stimulates BRCA1. PP1a is in turn regulated by cdc2, cdc25, and AURKA (39–42). Hence, through these positive and negative regulatory loops, AURKA antagonizes BRCA1 and PP1a-induced inhibition of centrosome function, promoting the increased microtubule nucleation potential of mitotic centrosomes (Fig. 6A). High BRCA1 ubiquitination activity is promoted by high PP1a phosphatase activity, resulting in decreased centrosome microtubule nucleation during G1 and S phases. With the increase in AURKA localization to the centrosomes during G2 and M phases, both BRCA1 and PP1a activities are suppressed, resulting in a dramatic increase in centrosome-dependent microtubule nucleation (Fig. 6B). With the degradation of AURKA at the end of mitosis, both BRCA1 and PP1a activities are restored, re-establishing the BRCA1-mediated inhibition of centrosome microtubule nucleation. BRCA1 is also degraded at the end of mitosis (43), but some BRCA1 remains at the centrosome in G1 (5).

AURKA is an oncogene that is overexpressed in 62% of breast cancers (44). BRCA1 is phosphorylated by AURKA (37) and overexpression of AURKA leads to phenotypes similar to those associated with BRCA1 knockdown, i.e., absence of the G2-M checkpoint (45) and centrosome amplification (46). Therefore, we propose that these two proteins are part of the same regulatory pathway that is responsible for the control of centrosome function, and a delicate balance in the activities of these two proteins is crucial for tumor suppression.


   Acknowledgments
 
Grant support: Komen Foundation Fellowship (S. Sankaran), National Cancer Institute grant CA111480 (J.D. Parvin), and NIH-NIGMS grant GM43264 (R.E. Palazzo).

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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Current address for S. Sankaran: Massachusetts General Hospital Cancer Center, Boston, MA.

Received 7/10/07. Revised 8/28/07. Accepted 9/26/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Doxsey S. Re-evaluating centrosome function. Nat Rev Mol Cell Biol 2001;2:688–98.[CrossRef][Medline]
  2. Palazzo RE, Vogel JM, Schnackenberg BJ, Hull DR, Wu X. Centrosome maturation. Curr Top Dev Biol 2000;49:449–70.[Medline]
  3. Piehl M, Tulu US, Wadsworth P, Cassimeris L. Centrosome maturation: measurement of microtubule nucleation throughout the cell cycle by using GFP-tagged EB1. Proc Natl Acad Sci U S A 2004;101:1584–8.[Abstract/Free Full Text]
  4. Hsu LC, White RL. BRCA1 is associated with the centrosome during mitosis. Proc Natl Acad Sci U S A 1998;95:12983–8.[Abstract/Free Full Text]
  5. Sankaran S, Starita LM, Groen AC, Ko MJ, Parvin JD. Centrosomal microtubule nucleation activity is inhibited by BRCA1-dependent ubiquitination. Mol Cell Biol 2005;25:8656–68.[Abstract/Free Full Text]
  6. Hsu LC, Doan TP, White RL. Identification of a {gamma}-tubulin-binding domain in BRCA1. Cancer Res 2001;61:7713–8.[Abstract/Free Full Text]
  7. Ko MJ, Murata K, Hwang DS, Parvin JD. Inhibition of BRCA1 in breast cell lines causes the centrosome duplication cycle to be disconnected from the cell cycle. Oncogene 2006;25:298–303.[Medline]
  8. Starita LM, Machida Y, Sankaran S, et al. BRCA1-dependent ubiquitination of {{gamma}}-tubulin regulates centrosome number. Mol Cell Biol 2004;24:8457–66.[Abstract/Free Full Text]
  9. Xu X, Weaver Z, Linke SP, et al. Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol Cell 1999;3:389–95.[CrossRef][Medline]
  10. Lingle WL, Barrett SL, Negron VC, et al. Centrosome amplification drives chromosomal instability in breast tumor development. Proc Natl Acad Sci U S A 2002;99:1978–83.[Abstract/Free Full Text]
  11. Pihan GA, Wallace J, Zhou Y, Doxsey SJ. Centrosome abnormalities and chromosome instability occur together in pre-invasive carcinomas. Cancer Res 2003;63:1398–404.[Abstract/Free Full Text]
  12. Sankaran S, Starita LM, Simons AM, Parvin JD. Identification of domains of BRCA1 critical for the ubiquitin-dependent inhibition of centrosome function. Cancer Res 2006;66:4100–7.[Abstract/Free Full Text]
  13. Hashizume R, Fukuda M, Maeda I, et al. The RING heterodimer BRCA1-1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation. J Biol Chem 2001;276:14537–40.[Abstract/Free Full Text]
  14. Xia Y, Pao GM, Chen HW, Verma IM, Hunter T. Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein. J Biol Chem 2003;278:5255–63.[Abstract/Free Full Text]
  15. Morris JR, Pangon L, Boutell C, Katagiri T, Keep NH, Solomon E. Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility. Hum Mol Genet 2006;15:599–606.[Abstract/Free Full Text]
  16. Ruffner H, Joazeiro CA, Hemmati D, Hunter T, Verma IM. Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity. Proc Natl Acad Sci U S A 2001;98:5134–9.[Abstract/Free Full Text]
  17. Hut HM, Rembacz KP, van Waarde MA, et al. Dysfunctional BRCA1 is only indirectly linked to multiple centrosomes. Oncogene 2005;24:7619–23.[CrossRef][Medline]
  18. Joukov V, Groen AC, Prokhorova T, et al. The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly. Cell 2006;127:539–52.[CrossRef][Medline]
  19. Lotti LV, Ottini L, D'Amico C, et al. Subcellular localization of the BRCA1 gene product in mitotic cells. Genes Chromosomes Cancer 2002;35:193–203.[CrossRef][Medline]
  20. Schnackenberg BJ, Hull DR, Balczon RD, Palazzo RE. Reconstitution of microtubule nucleation potential in centrosomes isolated from Spisula solidissima oocytes. J Cell Sci 2000;113:943–53.[Abstract]
  21. Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchison TJ. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 1999;286:971–4.[Abstract/Free Full Text]
  22. Kuriyama R, Borisy GG. Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle. J Cell Biol 1981;91:822–6.[Abstract/Free Full Text]
  23. Snyder JA, McIntosh JR. Initiation and growth of microtubules from mitotic centers in lysed mammalian cells. J Cell Biol 1975;67:744–60.[Abstract/Free Full Text]
  24. Telzer BR, Rosenbaum JL. Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells. J Cell Biol 1979;81:484–97.[Abstract/Free Full Text]
  25. Zhai Y, Kronebusch PJ, Simon PM, Borisy GG. Microtubule dynamics at the G2-M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implications for spindle morphogenesis. J Cell Biol 1996;135:201–14.[Abstract/Free Full Text]
  26. Kline-Smith SL, Walczak CE. Mitotic spindle assembly and chromosome segregation: refocusing on microtubule dynamics. Mol Cell 2004;15:317–27.[CrossRef][Medline]
  27. Mayor T, Meraldi P, Stierhof YD, Nigg EA, Fry AM. Protein kinases in control of the centrosome cycle. FEBS Lett 1999;452:92–5.[CrossRef][Medline]
  28. Sankaran S, Parvin JD. Centrosome function in normal and tumor cells. J Cell Biochem 2006;99:1240–50.[CrossRef][Medline]
  29. Centonze VE, Borisy GG. Nucleation of microtubules from mitotic centrosomes is modulated by a phosphorylated epitope. J Cell Sci 1990;95:405–11.[Abstract/Free Full Text]
  30. Andreassen PR, Lacroix FB, Villa-Moruzzi E, Margolis RL. Differential subcellular localization of protein phosphatase-1{alpha}, {gamma}1, and {delta} isoforms during both interphase and mitosis in mammalian cells. J Cell Biol 1998;141:1207–15.[Abstract/Free Full Text]
  31. Liu Y, Virshup DM, White RL, Hsu LC. Regulation of BRCA1 phosphorylation by interaction with protein phosphatase 1{alpha}. Cancer Res 2002;62:6357–61.[Abstract/Free Full Text]
  32. Tavares AA, Glover DM, Sunkel CE. The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts. EMBO J 1996;15:4873–83.[Medline]
  33. Brittle AL, Ohkura H. Centrosome maturation: Aurora lights the way to the poles. Curr Biol 2005;15:R880–2.[CrossRef][Medline]
  34. Dutertre S, Descamps S, Prigent C. On the role of aurora-A in centrosome function. Oncogene 2002;21:6175–83.[CrossRef][Medline]
  35. Liu Y, Shreder KR, Gai W, Corral S, Ferris DK, Rosenblum JS. Wortmannin, a widely used phosphoinositide 3-kinase inhibitor, also potently inhibits mammalian polo-like kinase. Chem Biol 2005;12:99–107.[CrossRef][Medline]
  36. Heron NM, Anderson M, Blowers DP, et al. SAR and inhibitor complex structure determination of a novel class of potent and specific Aurora kinase inhibitors. Bioorg Med Chem Lett 2006;16:1320–3.[CrossRef][Medline]
  37. Ouchi M, Fujiuchi N, Sasai K, et al. BRCA1 phosphorylation by Aurora-A in the regulation of G2 to M transition. J Biol Chem 2004;279:19643–8.[Abstract/Free Full Text]
  38. Satinover DL, Leach CA, Stukenberg PT, Brautigan DL. Activation of Aurora-A kinase by protein phosphatase inhibitor-2, a bifunctional signaling protein. Proc Natl Acad Sci U S A 2004;101:8625–30.[Abstract/Free Full Text]
  39. Gautier J, Solomon MJ, Booher RN, Bazan JF, Kirschner MW. cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell 1991;67:197–211.[CrossRef][Medline]
  40. Katayama H, Zhou H, Li Q, Tatsuka M, Sen S. Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle. J Biol Chem 2001;276:46219–24.[Abstract/Free Full Text]
  41. Kwon YG, Lee SY, Choi Y, Greengard P, Nairn AC. Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase. Proc Natl Acad Sci U S A 1997;94:2168–73.[Abstract/Free Full Text]
  42. Strausfeld U, Labbe JC, Fesquet D, et al. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature 1991;351:242–5.[CrossRef][Medline]
  43. Choudhury AD, Xu H, Baer R Ubiquitination and proteasomal degradation of the BRCA1 tumor suppressor is regulated during cell cycle progression. J Biol Chem 2004;279:33909–18.[Abstract/Free Full Text]
  44. Miyoshi Y, Iwao K, Egawa C, Noguchi S. Association of centrosomal kinase STK15/BTAK mRNA expression with chromosomal instability in human breast cancers. Int J Cancer 2001;92:370–3.[CrossRef][Medline]
  45. Anand S, Penrhyn-Lowe S, Venkitaraman AR. AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol. Cancer Cell 2003;3:51–62.[CrossRef][Medline]
  46. Meraldi P, Honda R, Nigg EA. Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53–/– cells. EMBO J 2002;21:483–92.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
P. Kalab and R. Heald
The RanGTP gradient - a GPS for the mitotic spindle
J. Cell Sci., May 15, 2008; 121(10): 1577 - 1586.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sankaran, S.
Right arrow Articles by Parvin, J. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sankaran, S.
Right arrow Articles by Parvin, J. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online