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. Author manuscript; available in PMC: 2018 Apr 23.
Published in final edited form as: Cancer Res. 2009 Oct 27;69(22):8775–8783. doi: 10.1158/0008-5472.CAN-09-2312

Figure 2. Phosphorylation of serine 331 is essential for conferring resistance to MMC.

Figure 2

A). FANCD2 (S331A) fails to correct MMC sensitivity in FA-D2 cells. Resistance of FA-D2 deficient cell PD20+vector, PD20 + wild type FlagEGFP-FANCD2, or PD20 + FlagEGFP-D2 S331A cells against MMC were measured by clonogenic survivial assays. Points, means; bars,SE. n=3. B).

Disruption of phosphorylation at serine 331 abolishes inducible monoubiquitylation. FA-D2 deficient PD20 cells corrected with Flag-EGFP tagged wild FANCD2, FANCD2 (K561R) or FANCD2 (S331A) were treated with or without 0.5 μM MMC for 20 hours before analyzed by immunoblotting against total FANCD2.The result shows loss of monoubiquitylated FANCD2 (L-isoform) in the cases of S331A and K561R, compared with wild type FANCD2 (top panel). Immunoblot for Ku70 served as a loading control (bottom panel).

C). Hydroxyurea also induces monoubiquityation in a phosphoserine 331-dependent fashion. FA-D2 mutant cells transduced with either Flag-FANCD2 or Flag-FANCD2 (S331A) were treated with 1 mM hyroxyurea or 0.1 μM MMC for 24 hours. Immunoblotting revealed that hydroxyurea induced monoubiquitylation of FANCD2 only in wild type FANCD2 but not in FANCD2 (S331A).

D). FANCD2 (S331A) localizes to the nucleus. FA-D2 mutant cells expressing Flag-GFP-FANCD2 wild type or S331A mutant were plated and examined by direct fluorescent microscopy. The micrographs show that Flag-EGFP tagged FANCD2 (S331A) has similar nuclear cellular distribution as that of wild type FANCD2.