Figure 3.

GST-Cdc18 purified from fission yeast is phosphorylated by an associated kinase that is inhibited by Rum1. GST, GST-Cdc18, and GST-Atf1 expression was induced for 18 h in medium lacking thiamine. GST, GST-Cdc18, and GST-Atf1 were precipitated using GSH-Sepharose and incubated with Mg²⁺ and [γ-³²P]ATP in the presence of increasing concentrations of GST-Rum1 purified from bacteria. Autoradiography of samples subjected to gel electrophoresis revealed that GST-Cdc18 became phosphorylated by a kinase that was inhibited by GST-Rum1 (upper panel). As a positive control it was confirmed that phosphorylation of histone H1 by Cdc2-Cdc13 kinase, purified from S. pombe lysates by anti-Cdc13 immunoprecipitation, was inhibited by GST-Rum1 (middle panel). As a negative control it was found that phosphorylation of GST-Atf1 by associated Spc1 kinase was insensitive to GST-Rum1 (lower panel).