Abstract
Fanconi anemia (FA) is clinically characterized by increased occurrence of leukemias and solid tumors, progressive bone marrow failure, and skeletal abnormalities. Altogether 13 genes have been implicated in FA, and their products constitute a common pathway in DNA damage signaling termed FA pathway. The newest member in the FA pathway, FancI, has been identified through a proteomic screen in an effort to identify novel ATM/ATR kinase substrates, or by positional cloning. FancI physically associates with the key factor FancD2, resulting in the D2-I complex formation. Upon DNA damage or S phase stress, FancD2 and FancI are monoubiqutinated in a manner dependent on each other by multi-subunit E3 ubiquitin ligase (called the FA core complex), which comprises of eight FA gene products. We have recently demonstrated that multiple phosphorylation of FancI is critical for FancD2 monobubiquitination following DNA damage, and serves as molecular switch in the FA pathway. Upon monoubiquitintion, FancD2 and FancI are both targeted to chromatin and form colocalizing foci.
To directly test genetic requirements for FancI phosphorylation, we utilized the SDS-PAGE gel containing Phos-tag reagent that selectively retards migation of phosphorylated proteins by binding to phospho-Ser, Thr, or Tyr. We found that the FA core complex and FancD2 are crucial for phosphorylation of FancI. However, the phosphorylation occurs in a manner independent of the ATR activators Rad17-Rad9. Furthermore, in a cell line that expresses the ATR-activation domain of TopBP1 fused with the estrogen receptor (ER-AD, kindly provided by Dr Fernandez-Capetillo), we could not activate FancD2 monoubiquitination by tamoxifen stimulation. Thus these results suggest a unique activation mechanism linking an upstream checkpoint kinase and the FancI phosphorylation that probably involves the FA core complex.
