Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro

The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 a nd fission yeast Cds1 checkpoint kinases, is phosphorylated and activated i n response to DNA damage by ionizing radiation (IR). UV irradiation, and re plication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk 2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, wher eas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro. ATM phosphorylates the Ser-Gln/Thr-Gln (SQ /TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and T hr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-relat ed also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is p hosphorylated in an ATM-dependent manner in response to IR, but not in resp onse to UV or HU. Substitution of Thr68 with Ala reduced the extent of phos phorylation and activation of Chk2 in response to IR, and mutation of all s even SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo. Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.