Fig. 3. Serine 345 is phosphorylated and required for activation of Chk1 kinase activity in response to DNA damage. (A) A serine 345 phosphoantibody recognized Chk1 but not Chk1S345A after exposure to CPT. NW556 (Chk1) and NW492 (S345A) were grown to mid-log phase, treated with CPT and lysed. Lysate was incubated with F-7 anti-HA antibody, subjected to SDS-PAGE, and immunoblotted with S345 phosphoantibody or Y-11 anti-HA antibody. (B) Serine 345 phosphorylation is required for activation of Chk1 kinase activity in response to CPT. Chk1S345D is not constitutively activated. Immunoprecipitated Chk1, Chk1D155A, Chk1S345A and Chk1S345D were assayed for their ability to phosphorylate a peptide substrate. All values are from triplicate immunoprecipitations except Chk1+CPT and S345D+CPT, which are derived from two immunoprecipitations. (C) Full activation of Chk1 requires the checkpoint pathway protein Rad3. Chk1 was prepared as in B from a wild-type strain, NW223, and from a strain with a deletion of the rad3 protein, NW246, and assayed as above. All values are from triplicate immunoprecipitations; the data shown represents the mean, with error bars showing the standard deviation.