Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1

In response to DMA damage, eukaryotic cells use a system of checkpoint cont rols to delay cell-cycle progression. Checkpoint delays provide time for re pair of damaged DNA before its replication in S phase and before segregatio n of chromatids in M phase(1). The Cds1 (Chk2) tumour-suppressor protein(2) has been implicated in certain checkpoint responses in mammalian cells. It directly phosphorylates and inactivates the mitosis-inducing phosphatase C dc25 in vitro and is required to maintain the G2 arrest that is observed in response to gamma-irradiation(3-5). Cds1 also directly phosphorylates p53 in vitro at a site that is implicated in its stabilization, and is required for stabilization of p53 and induction of p53-dependent transcripts in viv o upon gamma-ionizing radiation(5-7). Thus, Cds1 functions in both the G1 a nd G2 checkpoint responses. Like Cds1, the checkpoint protein kinase ATM (a taxiate-tangiectasia-mutated) is required for correct operation of both the G1 and G2 damage checkpoints. ATM is necessary for phosphorylation and act ivation of Gds1 in vivo(4) and can phosphorylate Cds1 in vitro(8), although evidence that the sites that are phosphorylated by ATM are required for ac tivation is lacking, Here we show that threonine 68 of Cds1 is the preferre d site of phosphorylation by ATM in vitro, and is the principal irradiation -induced site of phosphorylation in vivo. The importance of this phosphoryl ation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G 1 arrest in response to ionising radiation.