Figure 5.
UCN-01 blocks Chk1 autophosphorylation in vitro and Chk1 hyperphosphorylation in vivo, with no effect on ATR kinase in vitro or S phase activation of Chk2 in vivo. (A) Chk1 autophosphorylation was measured by incorporation of 32P from labeled ATP in the presence of 0 (lane 1), 10 nM (lane 2) 50 nM (lane 3), and 300 nM UCN-01 (lane 4) followed by SDS-PAGE and autoradiography. (B) HeLa and CHO cells were treated +/− HU (2 mM, 15 h) in the presence or absence of 300 nM UCN-01 and lysates analyzed for Chk1 by SDS-PAGE and immunoblotting. Immunoblot is overexposed to show all reduced mobility forms of Chk1 on HU treatment. (C) SDS-PAGE and autoradiography of kinase-dead Chk1 following its phosphorylation by mock (lane 1), purified ATR in the presence of 0 (lane 2), 0.5 ng/μl (lane 3), 2 ng/μl (lane 4), and 4 ng/μl DNA (lane 5) and (D) DNA-dependent phosphorylation of kinase-dead Chk1 by purified ATR in the presence of 0 (lane 1), 10 nM (lane 2) 50 nM (lane 3), and 300 nM UCN-01 (lane 4). (E) HeLa cells treated +/−HU as above in the presence or absence of 300 nM UCN-01 or 5 mM caffeine, and lysates analyzed for Chk1 by SDS-PAGE and immunoblotting. Samples in lanes 1–3 are identical to upper panel of B but at lower exposure to resolve distinct forms of Chk1 after indicated treatments. (F) HeLa cells treated +/− HU as above in presence or absence of 300 nM UCN-01 as indicated, were lysed and Chk2 analysed by IP kinase assay (upper panel) or by SDS-PAGE and immunoblotting (lower panel).