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Fig. 1. Characterization of Cdk1, the catalytic subunit of the mitotic kinase from Ustilago maydis. (A) Scheme of the Cdk1 protein. Cdk1 contains all motifs typical for members of the Cdc2 family. This includes Tyr¹⁵, which is the putative Wee1 phosphorylation site; a PSTAIRE domain; and Thr¹⁶⁷, which is the putative CAK phosphorylation site. (B) Alignment of selected members of the fungal Cdc2 family. Accession numbers for these proteins are: S. pombe Cdc2: AAA35293; U. maydis Cdk1: AY260971; S. cerevisiae Cdc28: NP009718, Candida albicans Cdc28: P43063. (C) Identification of the Cdk1 molecule. Lysates were prepared from exponential cultures of wild-type FB1 (cdk1), and tagged-strain TAU17 (cdk1-1) cells. The whole cell lysates were separated by SDS-PAGE and immunoblotted with anti-PSTAIRE (upper panel) or anti-FLAG (lower panel) antibodies. (D) The Cdk1 protein binds Suc1. The same lysates used in C were incubated with Suc1-beads to pull down Cdc2-like proteins. Precipitates were immunoblotted with anti-PSTAIRE and anti-FLAG antibodies. (E) Immunoprecipitation of Cdk1 tagged with the FLAG epitope. Lysates from FB1 (cdk1) and TAU17 (cdk1-1) cells were subjected to immunoprecipitation with the anti-FLAG M2 antibody. Crude extract or immunoprecipitates (FLAG IP) were immunoblotted with anti-PSTAIRE. (F) H1-histone kinase activity of the immunoprecipitates recovered in E.

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