Phosphorylation of FANCD2 on Two Novel Sites Is Required for Mitomycin C Resistance

Abstract

The Fanconi anemia (FA) pathway is a DNA damage-activated signaling pathway which regulates cellular resistance to DNA cross-linking agents. Cloned FA genes and proteins cooperate in this pathway, and monoubiquitination of FANCD2 is a critical downstream event. The cell cycle checkpoint kinase ATR is required for the efficient monoubiquitination of FANCD2, while another checkpoint kinase, ATM, directly phosphorylates FANCD2 and controls the ionizing radiation (IR)-inducible intra-S-phase checkpoint. In the present study, we identify two novel DNA damage-inducible phosphorylation sites on FANCD2, threonine 691 and serine 717. ATR phosphorylates FANCD2 on these two sites, thereby promoting FANCD2 monoubiquitination and enhancing cellular resistance to DNA cross-linking agents. Phosphorylation of the sites is required for establishment of the intra-S-phase checkpoint response. IR-inducible phosphorylation of threonine 691 and serine 717 is also dependent on ATM and is more strongly impaired when both ATM and ATR are knocked down. Threonine 691 is phosphorylated during normal S-phase progression in an ATM-dependent manner. These findings further support the functional connection of ATM/ATR kinases and FANCD2 in the DNA damage response and support a role for the FA pathway in the coordination of the S phase of the cell cycle.Fanconi anemia (FA) is an autosomal recessive, or X-linked, cancer susceptibility disorder characterized by cellular hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC) (11, 12). The 11 cloned FA genes (A, B, C, D1, D2, E, F, G, L, M, and J) cooperate in a novel DNA damage response pathway, the FA pathway (6). Eight of the FA proteins (A, B, C, E, F, G, L, and M) are subunits of a nuclear E3 ubiquitin ligase required for monoubiquitination of the downstream FANCD2 protein on lysine 561, which is a critical step for the function of the FA pathway (8). The FANCL subunit is the putative catalytic E3 ligase subunit of the complex (19). Monoubiquitinated D2 interacts with FANCD1/BRCA2 (36). The recently cloned FANCJ protein is a helicase (3,15,16,18) which may work in concert with FANCD2-Ub and BRCA2 or may function independently of the FA pathway. Disruption of any of the proteins in the pathway leads to MMC hypersensitivity and chromosome instability.Increasing evidence supports a role for the FA pathway during normal S-phase progression in mammalian cells. First, the FA complex is associated with chromatin during S phase and interacts with the helicase BLM, suggesting that the complex plays a "sensor" role at the advancing replication fork (21, 22) Second, the newly cloned FANCM subunit of the FA complex appears to promote its chromatin association during S phase (20,26). Third, the FA complex activates FANCD2 monoubiquitination during S phase, leading to the association of monoubiquitinated FANCD2 with BRCA1 and RAD51 in replication foci (34). Fourth, the FA pathway is required for normal homologous recombination repair, a mode of DNA repair executed in S phase (28). Finally, t...

Interplay between ATM and ATR in the regulation of common fragile site stability

“…Recently it was shown that downregulation of the Chk1 kinase has a major effect on fragile site stability , hence the effect of ATM on the level of Chk1 phosphorylation is probably part of the mechanism by which ATM regulates fragile site expression. It is worth noting that in addition to Chk1, other shared targets of ATR and ATM (BRCA1, SMC1 and FANCD2) (Gatei et al, 2001;Kim et al, 2002;Ho et al, 2006) were also shown to regulate fragile site stability (reviewed by Arlt et al, 2006). Further studies are required to investigate the regulation of these proteins by ATR and ATM, under conditions that induce fragile site expression.…”

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

“…98,104 The D'Andrea laboratory established that ATR phosphorylates FANCD2 on T691 and S717 in vitro and in vivo. 98,99 A FANCD2-T691A/S717A double mutant fails to undergo efficient DNA damage-inducible monoubiquitination and fails to correct the MMC hypersensitivity of FA-D2 patient cells. 99 How phosphorylation of these 2 sites promotes FANCD2 monoubiquitination remains unclear.…”

“…96 An important role for the ATR kinase in the regulation of FANCD2 has also been established. 98,99 While ATM primarily responds to DNA DSBs, ATR is a major regulator of the DNA replication checkpoint, primarily responding to agents that disrupt DNA replication, e.g., hydroxyurea, an inhibitor of deoxyribonucleotide reductase, aphidicolin (APH), a processive DNA polymerase inhibitor, and UV irradiation, which promotes the formation of cyclobutane pyrimidine dimers. 100,101 Mutations in ATR underlie Seckel syndrome (SCKL1) a rare autosomal recessive disorder characterized by severe growth retardation, short stature, microcephaly, mental retardation, and increased risk for acute myeloid leukemia (AML), myelodysplasia (MDS), and aplastic anemia, similar to FA.…”

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

“…Both ATR and ATM have been linked to the FA pathway; FANCD2 is phosphorylated by ATR and ATM at different sites in response to replication stress and ionizing irradiation, respectively (2-4, 7). Moreover, phosphorylation by ATR, but not ATM, is required for efficient DNA damage-induced monoubiquitination of FANCD2 in human cells (3,7). We wanted to test whether the observed DNA-stimulated xFANCM-PPP formation in egg extracts is dependent on xATR and/or xATM.…”

“…ATR-dependent phosphorylation of FANCD2 is triggered by various types of DNA damage, including replication stress, and is required for the interstrand cross-link-induced intra-S phase checkpoint response (2,3). Moreover, phosphorylation by ATR is required for efficient FANCD2 monoubiquitination in response to DNA damage, suggesting that the FA pathway might participate in ATR-dependent coordination of the S phase of the cell cycle (3,7).…”