Figure 5.

ATR-mediated phosphorylation of Chk1 at Ser³¹⁷ is required for reentry to the cell cycle. A, U2OS cells were transfected with control or ATR-specific siRNA. HU was added to cell culture medium on the next day as in the previous experiments. After 18 h, cell lysates were immunoblotted for the indicated antibodies. HU treatment strongly induces phosphorylation of Chk1 at both Ser³¹⁷ and Ser³⁴⁵ in control cells but not in ATR-siRNA cells. In the absence of Chk1 phosphorylation, levels of CKII were dramatically reduced. Consequently, phosphorylation and total levels of PTEN were reduced in these cells. B, ATR-mediated phosphorylation of Chk1 is critical for basal levels of CKII and PTEN after HU treatment. ATR^flox/− and ATM^−/− were generated as described previously (5). ATR^flox/− were infected with retrovirus pBabe-Cre 48 h before analysis. wt MEFs, ATR^flox/− MEFs, and ATM^−/− MEFs were treated with HU. Levels of CKII were lower in ATR^flox/− MEFs compared with normal and ATM^−/− MEFs, which were further reduced after HU treatment. Both Ser³¹⁷ and Ser³⁴⁵ of Chk1 are normally phosphorylated in ATM^−/− MEFs after HU treatment.