ATR phosphorylates serine 1449 of FANCA in vitro. ATR kinase was purified from HeLa cells by immunoprecipitation using anti-ATR antibody. After immunoprecipitation, beads were divided for Western blot (B) and in vitro kinase assay (A). (A) Immunoprecipitated ATR (lanes 1-3) or a control IgG immunoprecipitation (lanes 4-6) was incubated with GST (lanes 1,4), GST-FANCA C terminus (lanes 2,5), or GST-FANCA S1449A C terminus (lanes 3,6) in the presence of [γ-³²P]ATP. Products were separated by SDS-PAGE, stained with Coomassie, and dried, and resulting labeled substrates were detected by autoradiography. (B) Western blot for ATR showing immunoprecipitation of ATR with anti-ATR antibody (lane 3) but not with a control IgG immunoprecipitation (lane 2). (C) Chk1 inhibition does not inhibit phospho-FANCA S1449. HeLa cells were treated for 18 hours with 1 mM MMC (lanes 2-4). Cells in lanes 3 and 4 were additionally treated with 1 μM SB218078 or 100 μM wortmannin, respectively. Whole cell lysates were separated by SDS-PAGE and immunoblotted for phospho-FANCA S1449, total FANCA, and β-actin as a loading control. (D) In vitro kinase reaction was run as in panel A, except aliquots were incubated in the presence of wortmannin. Wortmannin inhibited ATR phosphorylation of the wild-type fusion protein. C term indicates C terminus; and IP, immunoprecipitation.