Figure 6. Phosphorylation at serine 331 is mediated by CHK1.

A). Recombinant GST-CHK1 specifically phosphorylates wild type peptide encompassing serine 331. In vitro CHK1 kinase assay was conducted with indicated concentrations of either wild type or S331A peptides.Radioactivity of blank controls (reaction systems without peptide substrate) was subtracted from counts to calculate the mole amount of phosphate groupincorporated by peptides . Only wild type FANCD2 peptide was significantly phosphorylated. Points, means; bars,SE. n=3

B). CHK1 specifically phosphorylates peptide substrates as compared to CHK2. In vitro CHK1 or CHK2 kinase assay was conducted with wild type FANCD2 peptide substrates at a concentration of 0.2 mM or 30 μM CHKtide substrates as the positive control. The kinase activity of CHK1 with wild type FANCD2 peptide substrate was adjusted for differing enzymatic potency between CHK1 and CHK2 by multiplying the ratio of CHKtide phosphorylated by CHK2 to that by CHK1.. The result indicates that CHK1 displays roughly 8 fold more relative activity than CHK2 at the FANCD2 peptide. Columns, means; bars, SE.n=3

C). In vivo inhibition of CHK1 eliminates phosphorylation of FANCD2 at serine 331. HeLa whole cell extracts made from cells undergoing the indicated treatment (16 hours) were separated by SDS-PAGE and immunoblotted against phospho-serine 331 FANCD2, FANCA, and phospho-serine 1449 FANCA. Lane 1 to 4 from left to right are 1): Hela cells without treatment; 2): Hela cells treated with 1 μM MMC; 3): Hela treated with 1 uM MMC plus 5 μM CHK1 inhibitor, SB-218078; 4): HeLa cells treated with 1 uM MMC plus 1 mM CHK1 inhibitor. CHK1 inhibitor effectively inhibited inducible expression of phospho-FANCD2 while phospho-FANCA remains intact.