Recruitment of Thr 319-phosphorylated Ndd1p to the FHA domain of Fkh2p requires Clbkinase activity: a mechanism for CLB cluster gene activation

Abstract

Activation of the CLB gene cluster through the assembly of Mcm1p–Fkh2p complexes at target promoters is essential for mitotic entry and transition through M phase. We show that the activation of this mitotic transcriptional program is dependent on the recruitment of Ndd1p, a coactivator that performs its essential function by acting through Fkh2p. Although an essential gene,NDD1is dispensable in cells expressing a truncated form of Fkh2p lacking its C terminus. When phosphorylated on T319, Ndd1p is recruited to CLB cluster promoters by association with the forkhead-associated (FHA) domain of Fkh2p. Substitution of T319 for alanine significantly reduces recruitment of Ndd1p, resulting in loss of normal transcriptional regulation, severe impairment of cell growth, and a budding defect reminiscent of cells with a Cdk-Clbkinase deficiency. Finally, we show that phosphorylation of T319 and recruitment of Ndd1p toCLB2andSWI5promoters is dependent on Cdc28-Clbkinase activity. These data provide a model describing the activation of G2/M transcription through the phosphorylation of Ndd1p by Cdc28-Clb kinase activity.