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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
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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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    • "Comparable mutations of leucine residues to alanines have frequently been used for the inactivation of NES sites (Ferrer et al., 2005; Jensik et al., 2004; Wen et al., 1995). NES sites are frequently and preferentially located within α-helical structures, and the most C-terminal leucine tends to be exposed and available for interactions (La Cour et al., 2004). "
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    • "In some instances the loss of FANCA, FANCC, FANCE or FANCF results in the formation of smaller sub-complexes, which are incompetent at monoubiquitinating FANCD2 [9] [10] [11]. Furthermore, there is some evidence indicating that the nuclear import of some FA proteins (FANCA and FANCC) is regulated [12] [13]. Since the assembly of the complete core complex requires the availability of all the constituents in the nucleus, it is conceivable that the pathway may be regulated at the point of assembly (Fig. 2B). "
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