FANCG is phosphorylated at serines 383 and 387 during mitosis.
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.
Mi
Jun
J
Department of Microbiology, University of Virginia Health System, Charlottesville, VA, USA.
Qiao
Fengyu
F
Wilson
James B
JB
High
Anthony A
AA
Schroeder
Melanie J
MJ
Stukenberg
Peter T
PT
Moss
Amy
A
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Jones
Nigel J
NJ
Kupfer
Gary M
GM
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D052241:Fanconi Anemia Complementation Group G Protein; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008938:Mitosis / Q000502:physiology*; D009154:Mutation; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*
D004268:DNA-Binding Proteins; C499909:FANCG protein, human; D052241:Fanconi Anemia Complementation Group G Protein; D012694:Serine; D016203:CDC2 Protein Kinase