BRCA1 Interaction of Centrosomal Protein Nlp Is Required for Successful Mitotic Progression

Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability.The successful mitosis requires the assembly of a strictly bipolar mitotic apparatus that will ensure that chromosomes equally distribute to the daughter cells. This process is controlled by the centrosomes that are required for spindle formation and function (1). Abnormalities of centrosome have been demonstrated to cause chromosomal missegregation and generation of aneuploidy, consequently leading to cell malignant transformation and tumorigenesis (2, 3). The machinery that controls centrosome stability involves multiple important cellular proteins, including p53 (4), BRCA1 (5), Gadd45 (6, 7), p21 (8), and Cdk2/cyclin E (9). The precise coordination among those regulators maintains centrosome duplication and stability. Prior to mitosis, centrosomes undergo maturation (10), which is characterized by centrosome enlargement, recruitment of γ-tubulin, and an increased microtubule nucleation activity (11, 12). Centrosome maturation is regulated by several mitotic kinases (13), such as Plk1 (Polo-like kinase 1) (14), Aurora-A (15), and Nek2, a member of NIMA (never in mitosis gene A)-related kinase (16). Recently, a Plk1-regulated ninein-like protein, termed Nlp, has been characterized as an important molecule involved in centrosome maturation (17). Nlp interacts with γ-tubulin ring complex and stimulates microtubule nucleation in the interphase. Upon the G₂/M transition, Nlp is subjected to phosphorylation by Plk1 and Nek2 (17, 18) and departs from the centrosome. It is thus suggested that the delicate association of Nlp with the centrosome is required for proper centrosome maturation and spindle assembly (17).BRCA1, a breast cancer susceptibility gene that accounts for more than 70% of hereditary breast cancer cases, is a critical regulator in the control of cell cycle progression (19, 20). BRCA1 interacts with multiple important cellular proteins, including RAD51 (21), BRCA2 (22), p53 (23), c-Myc (24), and p300 proteins (25). It is speculated that the BRCA1 protein may exert its control over cellular functions by acting as a platform for these proteins to converge and interact and may, therefore, create interactive modes for regulating their respective functions. BRCA1 is linked to the control of centrosome stability (26). Mouse embryonic fibroblasts (MEFs)³ carrying targeted deletion of exon 11 of the Brca1 gene exhibit centrosome amplification and abnormalities of spindle formation (5). BRCA1 may regulate centrosome duplication, probably through its interacting proteins such as p53 (23), BRCA2 (27), Cdk2 (28), and γ-tubulin (29–31), or its downstream genes such as p21 (32) and Gadd45a (33, 34). Most recently, BRCA1 was reported to be required for mitotic spindle assembly through its interaction with three spindle pole proteins, TPX2, NuMA, nuclear mitotic apparatus protein; and XRHAMM, Xenopus homolog to human RHAXX (35). These findings strongly suggest that BRCA1 is involved in the mitotic machinery. However, the importance of BRCA1 in the control of mitotic progression still remains to be further defined.In this report, we demonstrate that BRCA1 physically interacts and colocalizes with Nlp. Nlp centrosomal localization and its protein stability are likely dependent on normal cellular BRCA1 function. Suppression of Nlp using the siRNA approach disturbs the process of chromosomal segregation and results in aberrant spindle formation, failure of chromosomal segregation, and aneuploidy.