Gurtan Allan M, Stuckert Patricia, D'Andrea Alan D
Biological and Biomedical Sciences Program, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2006 Apr 21;281(16):10896-905. doi: 10.1074/jbc.M511411200. Epub 2006 Feb 10.
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.
范可尼贫血（FA）是一种常染色体隐性疾病，其特征为再生障碍性贫血、癌症易感性以及细胞对丝裂霉素C敏感。11个已克隆的范可尼贫血基因产物（FANCA、-B、-C、-E、-F、-G、-L和-M）中的8个形成一个多亚基核复合物（FA核心复合物），该复合物是下游FA蛋白FANCD2单泛素化所必需的。FANCL拥有三个WD40重复序列和一个植物同源结构域（PHD），是FA复合物假定的E3泛素连接酶亚基。在此，我们证明FANCL的WD40重复序列是与FA复合物其他亚基相互作用所必需的。尽管FANCD2单泛素化需要PHD，但该结构域对于这种相互作用并非必需。FANCL的PHD在序列上也与c-CBL的典型RING结构域相似，包括c-CBL与E2结合所需的保守色氨酸。FANCL PHD中该色氨酸的突变显著损害FANCD2在体内的单泛素化以及体外的自身泛素化活性，并部分损害丝裂霉素C抗性的恢复。我们提出一个模型，即FANCL通过其WD40区域结合FA复合物，并通过其PHD募集一个尚未确定的E2来实现FANCD2的单泛素化。
