Characterization of BARD1 targeting and dynamics at the centrosome: the role of CRM1, BRCA1 and the Q564H mutation

BARD1 heterodimerizes with BRCA1, forming an E3 ubiquitin ligase that functions at nuclear foci to repair DNA damage and the centrosome to regulate mitosis. We compared BARD1 recruitment at these structures using fluorescence recovery after photobleaching assays to measure YFP-BARD1 dynamics in live cells. In nuclei at ionizing radiation-induced foci, 20% of the BARD1 pool was immobile and 80% of slow mobility exhibiting a recovery time > 500 s. In contrast, at centrosomes 83% of BARD1 was rapidly mobile with extremely fast turnover (recovery time ~ 20 s). The ~ 25-fold faster exchange of BARD1 at centrosomes correlated with BRCA1-independent recruitment. We mapped key targeting sequences to a combination of the N and C-termini, and showed that mutation of the nuclear export signal reduced centrosome localization by 50%, revealing a role for CRM1. Deletion of the sequence 128-550 increased BARD1 turnover at the centrosome, consistent with a role in transient associations. Conversely, the cancer mutation Q564H reduced turnover by 25%. BARD1 is one of the most highly mobile proteins yet detected at the centrosome, and in contrast to its localization at DNA repair foci, which requires dimerization with BRCA1, targeting of BARD1 to the centrosome occurs prior to heterodimerization and its rapid turnover may provide a mechanism to regulate dimer formation.     

Analysis of centrosome localization of BRCA1 and its activity in suppressing centrosomal aster formation

BRCA1, a product of a familial breast and ovarian cancer susceptibility gene, localizes to centrosomes and physically interacts with γ-tubulin, a key centrosomal protein for microtubule nucleation and anchoring at centrosomes. Here, we performed a rigorous analysis of centrosome localization of BRCA1, and found that BRCA1 is specifically associated with mother centrioles in unduplicated centrosomes, and daughter centrioles acquire BRCA1 prior to initiation of duplication, and thus duplicated centrosomes are both bound by BRCA1. We further found that BRCA1 suppresses centrosomal aster formation. In addition, we identified a new domain of BRCA1 critical for γ-tubulin binding, which confers not only its localization to centrosomes, but also its activity to suppress centrosomal aster formation.     

CEP70 protein interacts with γ-tubulin to localize at the centrosome and is critical for mitotic spindle assembly

Deregulation of the mitotic spindle has been implicated in genomic instability, an important aspect of tumorigenesis and malignant transformation. To ensure the fidelity of chromosome transmission, the mitotic spindle is assembled by exquisite mechanisms and orchestrated by centrosomes in animal cells. Centrosomal proteins especially are thought to act coordinately to ensure accurate spindle formation, but the molecular details remain to be investigated. In this study, we report the molecular characterization and functional analysis of a novel centrosomal protein, Cep70. Our data show that Cep70 localizes to the centrosome throughout the cell cycle and binds to the key centrosomal component, γ-tubulin, through the peptide fragments that contain the coiled-coil domains. Our data further reveal that the centrosomal localization pattern of Cep70 is dependent on its interaction with γ-tubulin. Strikingly, Cep70 plays a significant role in the organization of both preexisting and nascent microtubules in interphase cells. In addition, Cep70 is necessary for the organization and orientation of the bipolar spindle during mitosis. These results thus report for the first time the identification of Cep70 as an important centrosomal protein that interacts with γ-tubulin and underscore its critical role in the regulation of mitotic spindle assembly.     

Characterization of centrosomal association of nucleophosmin/B23 linked to Crm1 activity

Nucleophosmin (NPM)/B23 is a multifunctional protein, involving in a wide variety of basic cellular processes, including ribosome assembly, DNA duplication, nucleocytoplasmic trafficking, and centrosome duplication. It has previously been shown that NPM/B23 localizes to centrosomes, and dissociate from centrosomes upon phosphorylation by Cdk2/cyclin E. However, detail characterization of centrosomal association of NPM/B23 has been hampered by the lack of appropriate antibodies that efficiently detects centrosomally localized NPM/B23, as well as by apparent loss of natural behavior of NPM/B23 when tagged with fluorescent proteins. Here, by the use of newly generated anti-NPM/B23 antibody, we conducted a careful analysis of centrosomal localization of NPM/B23. We found that NPM/B23 localizes between the paired centrioles of unduplicated centrosomes, suggesting the role of NPM/B23 in the centriole pairing. Upon initiation of centrosome duplication, some NPM/B23 proteins remain at mother centrioles of the parental centriole pairs. We further found that inhibition of Crm1 nuclear export receptor results in both accumulation of cyclin E at centrosomes and efficient dissociation of NPM/B23 from centrosomes.     

Differential modulation of BRCA1 and BARD1 nuclear localisation and foci assembly by DNA damage

The BRCA1/BARD1 heterodimer regulates genomic maintenance and contributes to the DNA damage checkpoint response. We previously reported that BRCA1 and BARD1 can shuttle between nucleus and cytoplasm. In this study, we evaluated the localisation patterns of BRCA1 and BARD1 in response to different types of DNA damaging agents and chemotherapeutic drugs. In MCF-7 cells, endogenous BRCA1 increased transiently in the nucleus at 2 h after ionising radiation (IR), whereas BARD1 was unaffected. IR treatment did not induce nuclear export of either protein, in contrast to previous reports. DNA damage by UV radiation, etoposide or camptothecin caused a preferential down-regulation of nuclear BARD1 at 6 h post-treatment. The UV-dependent loss of nuclear BARD1 was blocked by the proteasome inhibitor MG132, but not by leptomycin B, indicating a change in BARD1 nuclear degradation rather than nuclear export. MG132 also blocked the dispersal of BARD1/BRCA1 nuclear foci at 6 h after UV, implicating the proteasome in repair foci disassembly. In the cytoplasm, BRCA1 and BARD1 were detected at centrosomes but their distribution was not altered by DNA damage. BARD1 displayed a stronger mitochondria accumulation than BRCA1, and became phosphorylated at mitochondria in response to DNA damage. The mitotic spindle poisons vincristine and paclitaxel had no effect on BRCA1 or BARD1 subcellular distribution. We conclude that BARD1 phosphorylation, expression and localisation patterns are regulated in the nucleus and at mitochondria in response to different forms of DNA damage, contributing to the role of BRCA1/BARD1 in DNA repair and apoptotic responses.     

BRCA2 mediates centrosome cohesion via an interaction with cytoplasmic dynein

BRCA2 is responsible for familial breast and ovarian cancer and has been linked to DNA repair and centrosome duplication. Here we analyzed the mechanism by which the centrosomal localization signal (CLS) of BRCA2 interacts with cytoplasmic dynein 1 to localize BRCA2 to the centrosome. In vitro pull-down assays demonstrated that BRCA2 directly binds to the cytoplasmic dynein 1 light intermediate chain 2. A dominant-negative HA-CLS-DsRed fusion protein, the depletion of dynein by siRNA, and the inactivation of dynein by EHNA, inhibited the localization of BRCA2 at centrosomes and caused the separation of centrosome pairs during the S-phase. The double depletion of BRCA2 and C-Nap1 caused a larger dispersion of centrosome distances than the silencing of C-Nap1. These results suggest that cytoplasmic dynein 1 binds to BRCA2 through the latter's CLS and BRCA2 mediates the cohesion between centrosomes during the S phase, potentially serving as a cell-cycle checkpoint.     

DIXDC1 co-localizes and interacts with γ-tubulin in HEK293 cells

DIXDC1 is a Dishevelled-Axin (DIX) domain-containing protein involved in neural development and Wnt signaling pathway. Besides the DIX domain, DIXDC1 also contains a coiled-coil domain (MTH domain), which is a common feature of centrosomal proteins. We have demonstrated that exogenously expressed GFP-tag fused DIXDC1 co-localize with γ-tubulin both at interphase and mitotic phase in HEK293 cells. By immunostaining with anti-DIXDC1 and anti-γ-tubulin antibody, endogenous DIXDC1 was also co-localized with γ-tubulin at the centrosomes in HEK293 cells. We confirmed this interaction of DIXDC1 with γ-tubulin by co-immunoprecipitation. The findings suggest that DIXDC1 might play an important role in chromosome segregation and cell cycle regulation.     

The PTEN-Akt pathway impacts the integrity and composition of mitotic centrosomes

Loss of the tumor suppressor PTEN is observed in many human cancers that display increased chromosome instability and aneuploidy. The subcellular fractions of PTEN are associated with different functions that regulate cell growth, invasion and chromosome stability. In this study, we show a novel role for PTEN in regulating mitotic centrosomes. PTEN localization at mitotic centrosomes peaks between prophase and metaphase, paralleling the centrosomal localization of PLK-1 and γ-tubulin and coinciding with the time frame of centrosome maturation. In primary keratinocytes, knockdown of PTEN increased whole-cell levels of γ-tubulin and PLK-1 in an Akt-dependent manner and had little effect on recruitment of either protein to mitotic centrosomes. Conversely, knockdown of PTEN reduced centrosomal levels of pericentrin in an Akt-independent manner. Inhibition of Akt activation with MK2206 reduced the whole-cell and centrosome levels of PLK-1 and γ-tubulin and also prevented the recruitment of PTEN to mitotic centrosomes. This reduction in centrosome-associated proteins upon inhibition of Akt activity may contribute to the increase in defects in centrosome number and separation observed in metaphase cells. Concomitant PTEN knockdown and Akt inhibition reduced the frequency of metaphase cells with centrosome defects when compared with MK2206 treatment alone, indicating that both PTEN and pAkt are required to properly regulate centrosome composition during mitosis. The findings presented in this study demonstrate a novel role for PTEN and Akt in controlling centrosome composition and integrity during mitosis and provide insight into how PTEN functions as a multifaceted tumor suppressor.     

BRCA1-dependent ubiquitination of gamma-tubulin regulates centrosome number

Proper centrosome duplication and spindle formation are crucial for prevention of chromosomal instability, and BRCA1 plays a role in this process. In this study, transient inhibition of BRCA1 function in cell lines derived from mammary tissue caused rapid amplification and fragmentation of centrosomes. Cell lines tested that were derived from nonmammary tissues did not amplify the centrosome number in this transient assay. We tested whether BRCA1 and its binding partner, BARD1, ubiquitinate centrosome proteins. Results showed that centrosome components, including gamma-tubulin, are ubiquitinated by BRCA1/BARD1 in vitro. The in vitro ubiquitination of gamma-tubulin was specific, and function of the carboxy terminus was necessary for this reaction; truncated BRCA1 did not ubiquitinate gamma-tubulin. BRCA1/BARD1 ubiquitinated lysines 48 and 344 of gamma-tubulin in vitro, and expression in cells of gamma-tubulin K48R caused a marked amplification of centrosomes. This result supports the notion that the modification of these lysines in living cells is critical in the maintenance of centrosome number. One of the key problems in understanding the biology of BRCA1 has been the identification of a specific target of BRCA1/BARD1 ubiquitination and its effect on mammary cell biology. The results of this study identify a ubiquitination target and suggest a biological impact important in the etiology of breast cancer.     

Cohesin protein SMC1 is a centrosomal protein

Structural maintenance of chromosome protein 1 (SMC1) is well known for its roles in sister chromatid cohesion and DNA repair. In this study, we report a novel centrosomal localization of SMC1 within the cytoplasm in a variety of mammalian cell lines. We showed that SMC1 localized to centrosomes throughout the cell cycle in a microtubule-independent manner. Biochemically, SMC1 was cofractionated with the centrosomal protein γ-tubulin in centrosomal preparation. Immunohistochemistry and immunoelectron microscopy performed on mouse tissue sections revealed that SMC1 antibody strongly labeled the base of cilia in ciliated epithelia, where basal bodies were located. Furthermore, we showed that SMC1 was associated with both centrioles of a centrosome at G0/G1 stage of the cell cycle. These results demonstrate that SMC1 is a centrosomal protein, suggesting possible involvement of SMC1 in centrosome/basal body-related functions, such as organization of dynamic arrays of microtubules and ciliary formation.     

Differential regulation of centrosome integrity by DNA damage response proteins

MDC1 and BRIT1 have been shown to function as key regulators in response to DNA damage. However, their roles in centrosomal regulation haven’t been elucidated. In this study, we demonstrated the novel functions of these two molecules in regulating centrosome duplication and mitosis. We found that MDC1 and BRIT1 were integral components of the centrosome that colocalize with γ-tubulin. Depletion of either protein led to centrosome amplification. However, the mechanisms that allow them to maintain centrosome integrity are different. MDC1-depleted cells exhibited centrosome overduplication, leading to multipolar mitosis, chromosome missegregation, and aneuploidy, whereas BRIT1 depletion led to misaligned spindles and/or lagging chromosomes with defective spindle checkpoint activation that resulted in defective cytokinesis and polyploidy. We further illustrated that both MDC1 and BRIT1 were negative regulators of Aurora A and Plk1, two centrosomal kinases involved in centrosome maturation and spindle assembly. Moreover, the levels of MDC1 and BRIT1 inversely correlated with centrosome amplification, defective mitosis, and cancer metastasis in human breast cancer. Together, MDC1 and BRIT1 may function as tumor-suppressor genes, at least in part by orchestrating proper centrosome duplication and mitotic spindle assembly.     

PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis

The microtubule-organizing activity of the centrosome oscillates during the cell cycle, reaching its highest level at mitosis. At the onset of mitosis, the centrosome undergoes maturation, which is characterized by a drastic expansion of the pericentriolar matrix (PCM) and a robust increase in microtubule-organizing activity. It is known that PLK1 is critical for the initiation of centrosome maturation. In this paper, we report that pericentrin (PCNT), a PCM protein, was specifically phosphorylated by PLK1 during mitosis. Phosphoresistant point mutants of PCNT did not recruit centrosomal proteins, such as CEP192, GCP-WD (γ-complex protein with WD repeats), γ-tubulin, Aurora A, and PLK1, into the centrosome during mitosis. However, centrosomal recruitment of CEP215 depended on PCNT irrespective of its phosphorylation status. Furthermore, ectopic expression of PLK1-PCNT fusion proteins induced the centrosomal accumulation of CEP192, GCP-WD, and γ-tubulin even in interphase cells, mimicking centrosome maturation. Based on these results, we propose that PLK1-mediated phosphorylation of PCNT initiates centrosome maturation by organizing the spindle pole-specific PCM lattice.     

Dynamic association of a tumor amplified kinase,Aurora-A,with the centrosome and mitotic spindle

Aurora-A kinase, also known as STK15/BTAK kinase, is a member of a serine/threonine kinase superfamily that includes the prototypic yeast Ipl1 and Drosophila aurora kinases as well as other mammalian and non-mammalian aurora kinases involved in the regulation of centrosomes and chromosome segregation. The Aurora-A gene is amplified and overexpressed in a wide variety of human tumors. Aurora-A is centrosome-associated during interphase, and binds the poles and half-spindle during mitosis; its over-expression has been associated with centrosome amplification and multipolar spindles. GFP-Aurora-A was used to mark centrosomes and spindles, and monitor their movements in living cells. Centrosome pairs labeled with GFP-Aurora-A are motile throughout interphase undergoing oscillations and tumbling motions requiring intact microtubules and ATP. Fluorescence recovery after photobleaching (FRAP) was used to examine the relative molecular mobility of GFP-Aurora-A, and GFP-labeled alpha-tubulin, gamma-tubulin, and NuMA. GFP-Aurora-A rapidly exchanges in and out of the centrosome and mitotic spindle (t(1/2) approximately 3 sec); in contrast, both tubulins are relatively immobile indicative of a structural role. GFP-NuMA mobility was intermediate in both interphase nuclei and at the mitotic spindle (t(1/2) approximately 23-30 sec). Deletion mapping identifies a central domain of Aurora-A as essential for its centrosomal localization that is augmented by both the amino and the carboxyl terminal ends of the protein. Interestingly, amino or carboxy terminal deletion mutants that maintained centrosomal targeting exhibited significantly slower molecular exchange. Collectively, these studies contrast the relative cellular dynamics of Aurora-A with other cytoskeletal proteins that share its micro-domains, and identify essential regions required for targeting and dynamics.     

Structure meets function--centrosomes, genome maintenance and the DNA damage response

Centrosomes are cytoplasmic organelles playing a fundamental role in organizing both the interphase cytoskeleton and the bipolar mitotic spindle. In addition, the centrosome has recently come into focus as part of the network that integrates cell cycle arrest and repair signals in response to genotoxic stress--the DNA damage response. One important mediator of this response, the checkpoint kinase Chk1, has been shown to negatively regulate the G(2)/M transition via its centrosomal localization. Moreover, there is growing evidence that a centrosome inactivation checkpoint exists, which utilizes DNA damage-induced centrosome fragmentation or amplification to provoke a "mitotic catastrophe" and eliminate damaged cells. Candidate regulators of this centrosomal checkpoint include the checkpoint kinase Chk2 and its upstream regulators ATM and ATR. In addition, a growing number of other proteins have been implicated in centrosomal regulation of the DNA damage response, e.g. the tumor suppressor p53, the breast cancer susceptibility gene product BRCA1 and mitotic regulators such as Aurora A, Nek2 and the Polo-like kinases Plk1 and Plk3. However, many missing links and discrepancies between different model systems remain.     

Nek9 Phosphorylation of NEDD1/GCP-WD Contributes to Plk1 Control of γ-Tubulin Recruitment to the Mitotic Centrosome

The accumulation of γ-tubulin at the centrosomes during maturation is a key mechanism that ensures the formation of two dense microtubule (MT) asters in cells entering mitosis, defining spindle pole positioning and ensuring the faithful outcome of cell division ([1] and references herein; [2]). Centrosomal γ-tubulin recruitment depends on the adaptor protein NEDD1/GCP-WD [3, 4] and is controlled by the kinase Plk1 [5-8]. Surprisingly, and although Plk1 binds and phosphorylates NEDD1 at multiple sites [9, 10], the mechanism by which this kinase promotes the centrosomal recruitment of γ-tubulin has remained elusive. Using Xenopus egg extracts and mammalian cells, we now show that it involves Nek9, a NIMA-family kinase required for normal mitotic progression and spindle organization [11,?12]. Nek9 phosphorylates NEDD1 on Ser377 driving its recruitment and thereby that of γ-tubulin to the centrosome in mitotic cells. This role of Nek9 requires its activation by Plk1-dependent phosphorylation [13] but is independent from the downstream related kinases Nek6 and Nek7 [14]. Our data contribute to understand the mechanism by which Plk1 promotes the recruitment of γ-tubulin to the centrosome in dividing cells and position Nek9 as a key regulator of centrosome maturation.     

A functional cooperativity between Aurora A kinase and LIM kinase1

Aurora kinase A (Aur-A), a mitotic kinase, regulates initiation of mitosis through centrosome separation and proper assembly of bipolar spindles. LIM kinase 1 (LIMK1), a modulator of actin and microtubule dynamics, is involved in the mitotic process through inactivating phosphorylation of cofilin. Phosphorylated LIMK1 is recruited to the centrosomes during early prophase, where it colocalizes with γ-tubulin. Here, we report a novel functional cooperativity between Aur-A and LIMK1 through mutual phosphorylation. LIMK1 is recruited to the centrosomes during early prophase and then to the spindle poles, where it colocalizes with Aur-A. Aur-A physically associates with LIMK1 and activates it through phosphorylation, which is important for its centrosomal and spindle pole localization. Aur-A also acts as a substrate of LIMK1, and the function of LIMK1 is important for its specific localization and regulation of spindle morphology. Taken together, the novel molecular interaction between these two kinases and their regulatory roles on one another’s function may provide new insight on the role of Aur-A in manipulation of actin and microtubular structures during spindle formation.     

Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.