FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway

Abstract

In response to DNA damage or replication fork stress, the Fanconi anemia (FA) pathway is activated, leading to monoubiquitination of FancD2 and FancI and their co-localization in foci. Here we show that, in the chicken DT40 cell system, multiple alanine-substitution mutations in 6 conserved and clustered S/TQ motifs of FancI largely abrogate monoubiquitination as well as focus formation of both FancI and FancD2, resulting in loss of DNA repair function. Conversely, FancI carrying phospho-mimic mutations on the same 6 residues induces constitutive monoubiquitination and focus formation of FancI and FancD2, and protects against cell killing and chromosome breakage by DNA interstrand crosslinking agents. We propose that the multiple phosphorylation of FancI serves as a molecular switch in activation of the FA pathway. Mutational analysis of putative phosphorylation sites in human FANCI indicates that this switch is evolutionarily conserved.

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

“…31,32 FANCI also possesses numerous S/TQ motifs, known ATM/ATR phosphorylation consensus sites, and mass spectrometry revealed that at least 5 of these sites are phosphorylated in human and mouse FANCI. 15,33,34 The alignment of human and chicken FANCI sequences revealed that 8 S/TQ motifs are highly conserved, with 6 of these motifs clustering proximal to K523, the site of FANCI monoubiquitination. 9,10,33 Importantly, the 3.4A crystal structure of the murine Fancd2-Fanci heterodimer (ID2) was recently solved by the Pavletich laboratory.…”

“…15,33,34 The alignment of human and chicken FANCI sequences revealed that 8 S/TQ motifs are highly conserved, with 6 of these motifs clustering proximal to K523, the site of FANCI monoubiquitination. 9,10,33 Importantly, the 3.4A crystal structure of the murine Fancd2-Fanci heterodimer (ID2) was recently solved by the Pavletich laboratory. While this structure continues to be a major resource for FA researchers, it is important to recognize that structural information remains lacking for several peptide stretches of both proteins.…”

“…9,10 However, somewhat surprisingly, in contrast to FANCD2 K561, mutation of FANCI K523 does not overtly affect its function in ICL repair. 3,9,10,33 In vitro studies have also revealed that mutation of FANCD2 K561 leads to impaired FANCI monoubiquitination, whereas mutation of FANCI K523 has little effect on the monoubiquitination of FANCD2. 45 The E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase for FANCD2 and FANCI monoubiquitination are UBE2T and the RING domain-containing FANCL, respectively.…”

“…74 ID2 dissociation is triggered by ATM/ATR-mediated phosphorylation of a cluster of at least 6 FANCI SQ/TQ motifs, and is followed by the monoubiquitination of FANCD2, see below. 33,74 The NEDD4 E3 ubiquitin ligase can mono-and polyubiquitinate substrates, and the balance between these 2 posttranslational modifications is determined, at least in part, by the nature of the enzyme-substrate interaction. The tryptophan-tryptophan (WW) motif of NEDD4 can interact with relatively high affinity to a substrate PPxY (where x is any amino acid) motif, leading to substrate polyubiquitination.…”

“…These S/TQ sites become phosphorylated in the absence of prior monoubiquitination, indicating that this posttranslational modification acts upstream of monoubiquitination. 33 Importantly, mutation of these S/TQ sites results in abrogation of FANCI and FANCD2 monoubiquitination and nuclear foci formation, 33 indicating that phosphorylation of the FANCI S/TQ cluster functions as a molecular switch to activate the FA-BRCA pathway. However, the underlying molecular mechanism remains unclear.…”