Disparate Requirements for the Phosphorylation of Distinct Sites on the Fanconi Anemia Group I Protein

Fanconi Anemia (FA) is a blood disorder characterized by bone marrow failure, predisposition to hematologic malignancy and sensitivity to interstrand crosslinking agents. Patients with FA carry inherited mutations in any one of at least 16 known Fanconi Anemia Group (FANC) proteins that coordinate to function in a DNA repair pathway (the FA pathway). The activation of this pathway centers on two of these, Fanconi Anemia Group D2 protein (FANCD2) and Fanconi Anemia Group I protein (FANCI), which must undergo both phosphorylation and ubiquitination in order for the pathway to function properly. The latter is catalyzed by the FA core complex ubiquitin ligase, which is composed of 8 other FANC proteins.

Previous studies suggest that, in response to DNA damage, FANCI is phosphorylated at multiple sites within its evolutionarily conserved SQ cluster domain (SCD). This process is essential for activation of the canonical FA pathway. Failure of FANCI to phosphorylate inhibits FANCD2 ubiquitination, FANCD2 foci formation and cellular resistance to interstrand crosslinkers. However, while FANCI phosphorylation is important for the FA pathway to function, little is known about how this phosphorylation is regulated. Studies on the regulation of FANCI phosphorylation have largely been limited to chicken DT40 cells. Furthermore, the detection of FANCI phosphorylation has been restricted to an electrophoretic mobility-based method, which provides little information on the biology of specific phosphorylation sites. The objective of our work is to better understand the precise regulation of FANCI SCD phosphorylation, in human cells, at sites that have been established to be functionally significant.

By performing mass spectrometry on immunoprecipitated human FANCI protein, we established that the human FANCI SCD is indeed phosphorylated on at least two sites. Each of these sites have been found, through mutagenesis studies, to be involved in FA pathway activation. These two sites have also been implicated, through structural studies, in promoting a stable interaction between FANCI and FANCD2. Using this information, we designed immunogenic phospho-peptides to generate antibodies that specifically detect the phosphorylation of each of these two sites. We used these FANCI phospho-antibodies, together with genetically manipulated human cell culture systems, to study factors that modulate FANCI phosphorylation in the context of the human FA pathway.

We first established that these antibodies can be used for both immunoblot and immunofluorescence applications. With immunoblot analysis of cells treated with mitomycin C, we made the interesting observation that the phosphorylation of one of the FANCI sites occurred predominantly in the non-ubiquitinated form of the protein, while the other site was phosphorylated predominantly in the ubiquitinated form. This suggested that the phosphorylation of two distinct FANCI sites occurs at different steps of FA pathway activation. By performing siRNA depletion and biochemical experiments in cultured human cells, we found that the phosphorylation of both sites is at least partially dependent on the Ataxia Telangiectasia and Rad 3 related (ATR) kinase. Surprisingly, we found that only one of these sites could be phosphorylated without prior FANCI/D2 ubiquitination. Phosphorylation of the other site was dependent on both FANCI/D2 ubiquitination and the FA core complex. Therefore, contrary to previous models, we found that both ubiquitination-dependent and -independent phosphorylation sites exist within the FANCI SCD. Different FANCI phosphorylation sites that contribute to FA pathway activation therefore have disparate requirements for their phosphorylation.

Until now, studies on the regulation of FANCI phosphorylation have been limited by the lack of available phospho-specific FANCI antibodies. By developing antibodies that can specifically detect the phosphorylation of distinct sites within the functionally important SCD of FANCI, we have established new and critical reagents that provide additional insight into how the human FA pathway is activated. Our results suggest a novel model of FA pathway activation that involves a dynamic interplay between FANCI phosphorylation and FANCI/D2 ubiquitination, and reveal that activation of the FA pathway by FANCI phosphorylation is more complex a process than previously thought.