Identification of multiple nuclear export sequences in Fanconi anemia group A protein that contribute to CRM1-dependent nuclear export.

Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
Human Molecular Genetics (Impact Factor: 6.68). 06/2005; 14(10):1271-81. DOI: 10.1093/hmg/ddi138
Source: PubMed

ABSTRACT The Fanconi anemia (FA) pathway plays an important role in maintaining genomic stability, and defects in this pathway cause cancer susceptibility. The FA proteins have been found to function primarily in a nuclear complex, although a cytoplasmic localization and function for several FA proteins has also been reported. In this study, we investigated the possibility that FANCA, FANCC and FANCG are subjected to active export out of the nucleus. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, the accumulation of epitope-tagged FANCA in the nucleus increased, whereas FANCC was affected to a lesser extent and FANCG showed no response. CRM1-mediated export of FANCA was further confirmed using CRM1 cotransfection, which led to a dramatic relocalization of FANCA to the cytoplasm. Five functional leucine-rich nuclear export sequences (NESs) distributed throughout the FANCA sequence were identified and characterized using an in vivo export assay. Simultaneous inactivation of three of these NESs resulted in a discrete but reproducible increase of FANCA nuclear accumulation. However, these NES mutations did not affect the ability of FANCA to complement the mitomycin C or cisplatin sensitivity of FA-A lymphoblasts. Surprisingly, mutations in the other two NESs resulted in an almost complete relocation of the protein to cytoplasm, suggesting that these motifs overlap with domains that are crucial for nuclear import. Taken together, these findings indicate that FANCA can be actively exported out of the nucleus by CRM1, revealing a new mechanism to regulate the function of the FA protein complex.

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    ABSTRACT: Fanconi anemia (FA) proteins are known to play roles in the cellular response to DNA interstrand cross-linking lesions; however, several reports have suggested that FA proteins play additional roles. To elucidate novel functions of FA proteins, we used yeast two-hybrid screening to identify binding partners of the Fanconi anemia complementation group A (FANCA) protein. The candidate proteins included never-in-mitosis-gene A (NIMA)-related kinase 2 (Nek2), which functions in the maintenance of centrosome integrity. The interaction of FANCA and Nek2 was confirmed in human embryonic kidney (HEK) 293T cells. Furthermore, FANCA interacted with γ-tubulin and localized to centrosomes, most notably during the mitotic phase, confirming that FANCA is a centrosomal protein. Knockdown of FANCA increased the frequency of centrosomal abnormalities and enhanced the sensitivity of U2OS osteosarcoma cells to nocodazole, a microtubule-interfering agent. In vitro kinase assays indicated that Nek2 can phosphorylate FANCA at threonine-351 (T351), and analysis with a phospho-specific antibody confirmed that this phosphorylation occurred in response to nocodazole treatment. Furthermore, U2OS cells overexpressing the phosphorylation-defective T351A FANCA mutant showed numerical centrosomal abnormalities, aberrant mitotic arrest, and enhanced nocodazole sensitivity, implying that the Nek2-mediated T351 phosphorylation of FANCA is important for the maintenance of centrosomal integrity. Taken together, this study revealed that FANCA localizes to centrosomes and is required for the maintenance of centrosome integrity, possibly through its phosphorylation at T351 by Nek2.
    The international journal of biochemistry & cell biology 06/2013; DOI:10.1016/j.biocel.2013.06.012 · 4.89 Impact Factor
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    ABSTRACT: At the cellular level, defects in Fanconi anemia (FA) genes manifest themselves as hypersensitivity to DNA damaging agents which can be assessed by increased chromosome breakage and cell cycle changes. As long-term manifestations of cellular genetic instability, FA patients are at high risk for the development of a rather narrow spectrum of malignancies. Neoplasia occurs at a much younger age than in non-FA patients, and chiefly includes acute myeloid leukemia (AML) and squamous cell carcinomas (SCC). Given the role of FA genes in the maintenance of genetic stability, one would expect that FA genes are frequently altered in various kinds of tumors arising in non-FA patients. Much to our surprise and with the possible exception of pancreatic carcinoma, there appears to be no convincing evidence for a frequent association between either germline or somatic alterations in FA genes and malignancies arising in non-FA patients. Another notable exception is the FANCF gene which features prominently among the many genes that are silenced in cancer via promoter CpG island hypermethylation. However, the map location of FANCF adjacent to the 11p hotspot region of hypermethylation raises concerns about a strictly causal relationship between FANCF inactivation and tumorigenesis. Altogether, the available evidence suggests that tumor cells just like normal cells benefit from intact FA genes, possibly by keeping their replication machinery intact and by optimizing their defense against the adverse effects of reactive oxygen species.

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