Resistance to mitomycin C requires direct interaction between the Fanconi anemia proteins FANCA and FANCG in the nucleus through an arginine-rich domain.
Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, birth defects, and chromosomal instability. Because FA cells are sensitive to mitomycin C (MMC), FA gene products could be involved in cellular defense mechanisms. The FANCA and FANCG proteins deficient in FA groups A and G interact directly with each other. We have localized the mutual interaction domains of these proteins to amino acids 18-29 of FANCA and to two noncontiguous carboxyl-terminal domains of FANCG encompassing amino acids 400-475 and 585-622. Site-directed mutagenesis of FANCA residues 18-29 revealed a novel arginine-rich interaction domain (RRRAWAELLAG). By alanine mutagenesis, Arg(1), Arg(2), and Leu(8) but not Arg(3), Trp(5), and Glu(7) appeared to be critical for binding to FANCG. Similar immunolocalization for FANCA and FANCG suggested that these proteins interact in vivo. Moreover, targeting of FANCA to the nucleus or the cytoplasm with nuclear localization and nuclear export signals, respectively, showed concordance between the localization patterns of FANCA and FANCG. The complementation function of FANCA was abolished by mutations in its FANCG-binding domain. Conversely, stable expression of FANCA mutants encoding intact FANCG interaction domains induced hypersensitivity to MMC in HeLa cells. These results demonstrate that FANCA-FANCG complexes are required for cellular resistance to MMC. Because the FANCC protein deficient in FA group C works within the cytoplasm, we suggest that FANCC and the FANCA-FANCG complexes suppress MMC cytotoxicity within distinct cellular compartments.
Kruyt
F A
FA
Department of Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Abou-Zahr
F
F
Mok
H
H
Youssoufian
H
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005199:Fanconi Anemia / Q000378:metabolism; D052217:Fanconi Anemia Complementation Group A Protein; D052241:Fanconi Anemia Complementation Group G Protein; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D016685:Mitomycin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
D000596:Amino Acids; D004268:DNA-Binding Proteins; C498471:FANCA protein, human; C499909:FANCG protein, human; D052217:Fanconi Anemia Complementation Group A Protein; D052241:Fanconi Anemia Complementation Group G Protein; D011506:Proteins; D011993:Recombinant Fusion Proteins; D016685:Mitomycin; D001120:Arginine