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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 5. Phosphorylation of serine 331 is independent of the FA core complex and required for FANCD2/BRCA2 interaction.

Figure 5

A) Phospho-serine 331 FANCD2 is expressed in FA core complex mutant cell lines. FA-D2 deficient PD20 cell and its derived cells expressing Flag tagged FANCD2 S331A(PD20-S331A), FANCD2 561R(PD20-K561R) or wild type FANCD2 (PD20-315) , lymphoblast cell lines BD180 (WT), HSC72 (FA-A), HSC536 (FA-C), EUFA143 (FA-G) and EUFA868 (FA-L), fibroblast cell line GM6914 (FA-A) and their respective cDNA complemented derivatives were analyzed by immunoblotting. Expression of phospho-FANCD2 was present in each of the uncomplemented core complex mutant cell lines and wild type cells, except PD20 and PD20-S331A.

B) FANCD1/BRCA2 is co-immunoprecipitated with FANCD2. Cell lysates were immunoprecipitated with antibody to FANCD2 and probed either with an anti-BRCA2 C-terminal antibody (amino acids 3245–3418) to determine FANCD2-BRCA2 interaction (upper panel) or with the same FANCD2 antibody used for immunoprecipitation (lower panel). BRCA2 co-precipitated with FANCD2 in cell lines expressing wild type (PD20-3-15, PD20+FANCD2) and FANCD2 (K561R) proteins. (PD20-3-15 cells are derived from complementing FA-D2 deficient PD20 cells with human chromosome 3p). Co-precipitation was absent in FA-D2 deficient PD20 cells and expression of FANCD2 (S331A) failed to restore this interaction. In contrast, in PD20 cells expressing the phospho-mimetic mutation S331D, interaction between FANCD2 and BRCA2 was restored. The input lane represents 1/50 of the protein extract used for immunoprecipitation in the cell lines indicated.

C) FANCD2 is co-immunoprecipitated with FANCD1/BRCA2. The reciprocal immunoprecipiatation with anti-BRCA2 antibody was performed using the same cells as in (B) and subsequently immunoblotted with either FANCD2 antibody (upper panel) or BRCA2 antibody (lower panel). Blotting with FANCD2 antibody yielded two adjacent FANCD2 protein bands in PD20-3-15, PD20+FANCD2 and PD20+FANCD2 (S331D) cell lines, indicating that both isoforms of FANCD2 interact with BRCA2. No interaction was detected in PD20 cells. Neither isoform was co-precipitated in PD20+FANCD2 (S331A) cells, indicating that prior phosphorylation of FANCD2 at serine 331 is a requirement for interaction with BRCA2 regardless of monoubiquitylation status. BRCA2 is expressed in all cell lines (lower panel). The input lane represents 1/50 of the protein extract used for immunoprecipitation in the cell lines indicated.