Figure 2.
Rad3 and ATM phosphorylate a peptide derived from Chk1 at serine-345. (a) Rad3 phosphorylates serine-345 of Chk1. Unfused GST, a kinase dead form of GST-Rad3 (GST-Rad3KD) and active GST-Rad3 were expressed and purified from fission yeast and tested with the substrates GST-Chk1330–366 (wild type and S345A mutant) and GST-Chk1273–412 expressed and purified from bacteria. GST-Rad3 phosphorylated GST-Chk1273–412 and GST-Chk1330–366, but failed to phosphorylate GST-Chk1330–366 that contained the S345A mutation. (b) ATM phosphorylates serine-345 of Chk1. ATM was purified from HeLa cells and incubated with [Δ-32P]ATP and wild type (WT) or S345A mutant GST-Chk1330–366. GST and GST-HsCds11–91 were use as negative and positive controls, respectively.