The WD40 repeats of FANCL are required for Fanconi anemia core complex assembly.
Fanconi anemia (FA) is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. Eight of the 11 cloned Fanconi anemia gene products (FANCA, -B, -C, -E, -F, -G, -L, and -M) form a multisubunit nuclear complex (FA core complex) required for monoubiquitination of a downstream FA protein, FANCD2. FANCL, which possesses three WD40 repeats and a plant homeodomain (PHD), is the putative E3 ubiquitin ligase subunit of the FA complex. Here, we demonstrate that the WD40 repeats of FANCL are required for interaction with other subunits of the FA complex. The PHD is dispensable for this interaction, although it is required for FANCD2 mono-ubiquitination. The PHD of FANCL also shares sequence similarity to the canonical RING finger of c-CBL, including a conserved tryptophan required for E2 binding by c-CBL. Mutation of this tryptophan in the FANCL PHD significantly impairs in vivo mono-ubiquitination of FANCD2 and in vitro auto-ubiquitination activity, and partially impairs restoration of mitomycin C resistance. We propose a model in which FANCL, via its WD40 region, binds the FA complex and, via its PHD, recruits an as-yet-unidentified E2 for mono-ubiquitination of FANCD2.
Biological and Biomedical Sciences Program, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Allan M
AM
Gurtan
Patricia
P
Stuckert
Alan D
AD
D'Andrea
D000477:Alkylating Agents / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002869:Chromosome Aberrations; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005199:Fanconi Anemia / Q000378:metabolism*; D052240:Fanconi Anemia Complementation Group L Protein / Q000737:chemistry*; D005816:Genetic Complementation Test; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008214:Lymphocytes / Q000378:metabolism; D016685:Mitomycin / Q000378:metabolism / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014364:Tryptophan / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry
D000477:Alkylating Agents; D025801:Ubiquitin; D016685:Mitomycin; D014364:Tryptophan; D004247:DNA; D052240:Fanconi Anemia Complementation Group L Protein; D050721:Proto-Oncogene Proteins c-cbl; D005982:Glutathione Transferase