Figure 4.

GST-Cdc18 is an in vitro substrate of Cdc2 kinase. (A) Precipitated GST-Cdc18 was treated with 1 mM FSBA to irreversibly inactivate the copurified protein kinases (compare lane 1, not treated GST-Cdc18, with lane 2, FSBA-treated GST-Cdc18). GST-Cdc18 treated with FSBA was mixed with active p13^suc1-Sepharose-purified Cdc2 kinase in [γ-³²P]ATP kinase assay buffer in the presence (lane 4) or absence of 250 nM GST-Rum1 (lane 3). GST-Cdc18 phosphorylation was restored by the addition of Cdc2 kinases in the reaction. The ability of Cdc2 kinases to phosphorylate GST-Cdc18 or histone H1 (right panel, lanes 5 and 6) was largely inhibited by GST-Rum1. (B) Two-dimensional tryptic phosphopeptide maps of GST-Cdc18 phosphorylated by associated kinases (left panel) or by Cdc2 kinase (right panel) were very similar. (C) Phospho-amino acid analysis of GST-Cdc18 phosphorylated by associated kinases detected only phosphothreonine.