Figure 4. The FHA/BRCT domains of Nbs1 are important for ATM-mediated phosphorylation of CtIP.

A. The CtIP-7A-CDK mutant does not show defects in ATM and checkpoint activation. U2OS cells stably expressing HA-CtIP WT or 7A-CDK mutant, with endogenous CtIP silenced, were treated with or without IR (10 Gy) and allowed to recover for 1 h. Cells were then collected and lysed for Western blot analysis with indicated antibodies. B. U2OS cells stably expressing control vector MKO, sh-Mre11 or sh-Nbs1 were treated with IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. C. Schematic drawing of human Nbs1 protein domains [FHA, BRCT and Mre11- and ATM interacting domains (i.d.)], with indicated phospho-binding sites on the FHA/BRCT domains [31], [32]. D. U2OS cells stably expressing C-terminus Myc-tagged Nbs1 alleles, Nbs1-WT and Nbs1-RRHK (R28A, R43A, H45A and K160M) or empty vector with endogenous Nbs1 silenced by shRNAs were treated with or without IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. E. Purified GST-CtIP WT and GST-CtIP-12A-CDK from Sf9 insect cells were analyzed by SDS-PAGE with Coomassie blue staining. F. CtIP expressed in insect cells is phosphorylated by CDKs. Purified GST-CtIP WT and 12A-CDK mutant protein from insect cells were treated without or with lambda phosphatase (ptase), and anti-CtIP Western blot analysis was performed. G and H. Baculovirus-expressed and purified CtIP-WT and CtIP-12A-CDK mutant proteins (100 ng) were used as substrates for in vitro ATM kinase assays with or without addition of purified Nbs1-WT or Nbs1-RRHK mutant proteins (100 ng and 500 ng). The radiolabeled CtIP was visualized following SDS-PAGE.