FIG. 3.

Phosphorylation of T691 and S717 is required for optimal FANCD2 monoubiquitination and downstream functional activity. (A) FA-D2 fibroblasts were stably transduced with the cDNAs encoding the indicated wild-type (wt) or mutant FANCD2 proteins. Cells were treated as indicated, and whole-cell extracts were immunoblotted with anti-FANCD2 antiserum. The autoradiograph was scanned by densitometry, and the ratio of FANCD2-L (monoubiquitinated FANCD2 isoform) to FANCD2-S (unubiquitinated isoform) was calculated (L/S). (B) Cells were examined by the MMC cytotoxicity assay. The results are shown for vector alone (closed triangles), FANCD2 (closed squares), FANCD2(T691A,S717A) (closed circles), FANCD2(T691A) (open triangles), and FANCD2(S717A) (open circles).