ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function

Abstract

Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage-specific event that is downstream of ATR and is functionally important in the FA pathway.

Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations

“…Studies on residual function of the resulting mutant proteins often lead to the discovery of functionally important protein domains. However, apart from NLS and phosphorylation residues, 41,42 no additional domains have been so far described for FANCA. The results of this study show that all missense mutations analyzed, independently of their position within the gene, lead to common functional consequences: a mutant protein is expressed at rather normal levels relative to gene dosage, but the FA/BRCA pathway remains inactive because of the impaired nuclear transport of the mutant protein.…”

The Fanconi Anemia Proteins FANCD2 and FANCJ Interact and Regulate Each Other's Chromatin Localization

“…Supernatants were saved, and equal amounts of extract were used for each immunoprecipitation, with 1 g of antibody (FANCD2 H-300, or normal rabbit IgG; Santa Cruz, Santa Cruz, CA) or 2 l of Brip1/FANCJ rabbit polyclonal antiserum (Novus Biologicals, Littleton, CO). Immunoprecipitations were then performed as previously described (30,31).…”

“…EUFA0030 TERT fibroblasts (kindly provided by H. Joenje) and EUFA0030 TERT FANCJ corrected fibroblasts were cultured in RPMI (Invitrogen) with 20% fetal bovine serum. FA-D2 mutant cells PD20, PD20 Flag-FANCD2 and FA-A mutant cells GM6914 and GM6914 Flag-FANCA were cultured in DMEM containing 15% fetal bovine serum and penicillin/streptomycin (30,31).…”

Defects in homologous recombination repair behind the human diseases: FA and HBOC

“…The FA core complex includes FA proteins FANCA, B, C, E, F, G, J, L, M and FA-associated proteins, such as FAAP24, FAAP20 and FAAP100 , Bogliolo & Surrallés 2015, Ceccaldi et al 2016. In response to an ICL and/or a stalled replication fork, the FA core complex is somehow activated downstream of the checkpoint kinase ATR-ATRIP through multiple phosphorylation of FANCI (Ishiai et al 2008), FANCM (Singh et al 2013) or FANCA (Collins et al 2009) and monoubiquitinates FANCD2 at lysine 561, which is a critical activating event in the FA pathway (Garcia-Higuera et al 2001, Matsushita et al 2005. Recent studies indicate that ubiquitinlike with PHD and RING finger domain 1 (UHRF1) protein functions as an ICL recognition factor and may participate in these steps (Liang et al 2015.…”