FANCG Is Phosphorylated at Serines 383 and 387 during Mitosis

Abstract

Fanconi anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and high incidence of leukemia and solid tumors. Eight genes have been cloned, with the accompanying protein products participating in at least two complexes, which appear to be functionally dependent upon one another. Previous studies have described chromatin localization of the FA core complex, except at mitosis, which is associated with phosphorylation of the FANCG protein (F. Qiao, A. Moss, and G. M. Kupfer, J. Biol. Chem. 276:23391-23396, 2001). The phosphorylation of FANCG at serine 7 by using mass spectrometry was previously mapped. The purpose of this study was to map the phosphorylation sites of FANCG at mitosis and to assess their functional importance. Reasoning that a potential kinase might be cdc2, which was previously reported to bind to FANCC, we showed that cdc2 chiefly phosphorylated a 14-kDa fragment of the C-terminal half of FANCG. Mass spectrometry analysis demonstrated that this fragment contains amino acids 374 to 504. Kinase motif analysis demonstrated that three amino acids in this fragment were leading candidates for phosphorylation. By using PCR-directed in vitro mutagenesis we mutated S383, S387, and T487 to alanine. Mutation of S383 and S387 abolished the phosphorylation of FANCG at mitosis. These results were confirmed by use of phosphospecific antibodies directed against phosphoserine 383 and phosphoserine 387. Furthermore, the ability to correct FA-G mutant cells of human or hamster (where S383 and S387 are conserved) origin was also impaired by these mutations, demonstrating the functional importance of these amino acids. S387A mutant abolished FANCG fusion protein phosphorylation by cdc2. The FA pathway, of which FANCG is a part, is highly regulated by a series of phosphorylation steps that are important to its overall function.

Fanconi anemia signaling network regulates the spindle assembly checkpoint

“…The aneuploidy and multinucleation consistently observed in FA-deficient cells (20)(21)(22)(23)30) are suggestive of abnormal cell division (31), but the role of the FA pathway in mitosis has previously not been well defined. Several publications revealed a biochemical interaction between FA proteins and the key mitotic cyclin-dependent kinase CDK1 (32,33), while other work established an essential role for CDK1 in the SAC (34). A previous study in a murine model highlighted the functional cross-talk between the SAC pathway and FANCD1, a breast cancer tumor suppressor subsequently discovered to be an FA protein (35).…”

Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

“…29 MDA-MB-157-FANCD2 was generated by transducing MDA-MB-157 cells with a retroviral supernatant of a wild-type FANCD2 cDNA pMMP construct 30 and subsequent selection in puromycin as previously described. 31 Fibroblast cell lines PD20 and PD733, isolated from unrelated hypomorphic FANCD2-deficient patients and PD20-3-15 (chromosome 3p-complemented PD20), 32 were grown as previously described. 20 Mitomycin C (MMC; Sigma, UK) exposure (50 nmol/L), when used, was for 18 hours.…”

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.…”