Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway

Abstract

Fanconi anemia (FA) 1 is a genetic disease of cancer susceptibility marked by congenital defects, bone marrow failure, and myeloid leukemia (1-4). To date at least 11 complementation groups have been defined (5-7). Eight genes have been cloned (8 -17). However, the gene products resemble no known proteins and have few identifiable functional protein motifs. One exception is the recently cloned FANCL gene, which contains the ubiquitin ligase motif. In addition, the FANCD1 gene has been identified as BRCA2, one of the familial breast cancer genes.Cells derived from patients with the FA exhibit characteristic hypersensitivity caused by DNA cross-linking agents and generalized decreased survival (18 -22). However, no defined biochemical mechanism for this hypersensitivity has been elucidated, although studies have implicated cytokine dysregulation, excessive, oxidative damage, defects in DNA repair, and lack of cell cycle control (23-27). Patient and cellular phenotypes across all the complementation groups are similar, suggesting an inter-relatedness or cooperativity between the FA proteins.This cooperativity has been borne out by work we have done in showing binding of FANCA and FANCC in a protein complex in both nucleus and cytoplasm (28 -30). Recent work has found the FANCE, FANCF, FANCG, and FANCL proteins in the complex as well (31-34). A large complex is suggested by our recent work (35), and binding does not occur in any of the complementation groups except the FA-D1, D2, I, and J groups (7).One clue to FA function lies in the study of the FANCA protein, which contains a classic bipartite nuclear localization signal and is phosphorylated. Generally, FANCA nuclear localization, phosphorylation, and binding to FANCC are abolished in all complementation groups except the FA-D1 and D2 groups (28 -30, 33). This suggests that a nuclear event is critical to the normal function of the FA proteins, and the aberrant proteins in the FA-D groups may have a role downstream of the FA complex in the nucleus. However, some have found FANCA point mutants that are expressed, translocated to the nucleus, and are phosphorylated to some extent. Some of these mutants are of intermediate MMC sensitivity (36).Over the years, little information has been found that addresses the regulation of the FA proteins. mRNA or protein levels change little in response to DNA damage or the cell cycle. Our recent work has revealed that at least a subset of the FA proteins resides in the nucleus bound to chromatin, where increased protein binding occurs in response to DNA damage (37). One of the non-core complex FA proteins, FANCD2, becomes monoubiquitinated in response to DNA damage (38).In addition, we have shown during the cell cycle that the FA proteins detach from chromatin during mitosis, and FANCG becomes phosphorylated while remaining part of the complex (37). One group has demonstrated that FANCG has a isoform seen in asynchronous cells that is phosphatase sensitive (39).In this paper we report the identification of a phosphopeptide from ...

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

“…In support of this is evidence that FANCD2 and FANCI are mono-ubiquitylated and FANCG is phosphorylated both at S phase and after DNA damage. 16,17,20 The core complex was also known to localize to chromatin under both conditions. 49 This is the first report of an event in the pathway to occur specifically after DNA damage.…”

“…This partial correction of phenotype by a phosphorylation mutant is consistent with previous reports for several other FA proteins. 20 20,22 For FANCD2, these phosphorylations promote mono-ubiquitylation, 24 whereas phosphorylation of Ser7 of FANCG promotes formation of a BRCA2-D2-G-XRCC3 protein complex. 35 The reason for the partial complementation by S1449A FANCA is not yet clear, but it may be postulated that phosphorylation is necessary only for responding to increased cellular stress on the induction of DNA damage, rather than in normal cell activities such as maintaining replication during S phase in the presence of endogenous lesions or low levels of induced DNA damage.…”

“…15,17,19 FANCA, FANCG, FANCM, FANCE, FANCD2, and FANCI are phosphoproteins, underscoring the importance of phosphorylation to the FA core complex and FA pathway. 12,17,[20][21][22][23][24][25] Importantly, identified posttranslational modifications of FA proteins, including mono-ubiquitylation and phosphorylation, FANCD2 foci formation, and translocation of the core complex into the nucleus all occur both during undamaged S phase and in response to DNA-damaging agents such as MMC. Cells arrest in S phase after induction of interstrand crosslinks.…”

“…15,17,24 Similarly, FANCG is phosphorylated at serine 7 after DNA damage and during S phase. 20 Consequently, we sought to determine whether phosphoserine 1449 followed the same paradigm. HeLa cells were synchronized at S phase using double thymidine block or treated with MMC.…”

UBE2T, the Fanconi Anemia Core Complex, and FANCD2 Are Recruited Independently to Chromatin: a Basis for the Regulation of FANCD2 Monoubiquitination

“…One way to ensure this restriction is to control the assembly of the FA core complex in response to such signals. In addition to complex assembly, some of the core complex components (such as FANCM, FANCE, and FANCG) are phosphorylated in response to DNA replication and damage (21,24,26,31,39). Such modifications may also be important for regulation.…”