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

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, NDD1 is 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-Clb kinase deficiency. Finally, we show that phosphorylation of T319 and recruitment of Ndd1p to CLB2 and SWI5 promoters is dependent on Cdc28-Clb kinase activity. These data provide a model describing the activation of G2/M transcription through the phosphorylation of Ndd1p by Cdc28-Clb kinase activity.

[1]  B. Futcher.  Transcriptional regulatory networks and the yeast cell cycle. , 2002, Current opinion in cell biology.

[2]  L. Breeden,et al.  Cyclin transcription: Timing is everything , 2000, Current Biology.

[3]  Uttam Surana,et al.  The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae , 1991, Cell.

[4]  Mike Tyers,et al.  Mechanisms that help the yeast cell cycle clock tick: G2 cyclins transcriptionally activate G2 cyclins and repress G1 cyclins , 1993, Cell.

[5]  S. V. Van Doren,et al.  The FHA domain mediates phosphoprotein interactions. , 2000, Journal of cell science.

[6]  J. Gautier,et al.  Phosphorylation of MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance complex. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[7]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .

[8]  J. Heierhorst,et al.  Role of the N-terminal Forkhead-associated Domain in the Cell Cycle Checkpoint Function of the Rad53 Kinase* 210 , 2001, The Journal of Biological Chemistry.

[9]  Peter C. Hollenhorst,et al.  Mechanisms controlling differential promoter-occupancy by the yeast forkhead proteins Fkh1p and Fkh2p: implications for regulating the cell cycle and differentiation. , 2001, Genes & development.

[10]  K. Nasmyth,et al.  A new role for MCM1 in yeast: cell cycle regulation of SW15 transcription. , 1991, Genes & development.

[11]  P Bucher,et al.  The FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors. , 1995, Trends in biochemical sciences.

[12]  Lukas Endler,et al.  Forkhead-like transcription factors recruit Ndd1 to the chromatin of G2/M-specific promoters , 2000, Nature.

[13]  B. Kemp,et al.  FHA domain boundaries of the Dun1p and Rad53p cell cycle checkpoint kinases , 2000, FEBS letters.

[14]  K. Nasmyth,et al.  Loading of an Mcm Protein onto DNA Replication Origins Is Regulated by Cdc6p and CDKs , 1997, Cell.

[15]  D Kindelberger,et al.  Cell cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a ternary complex factor , 1995, Molecular and cellular biology.

[16]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[17]  L. Johnston,et al.  The forkhead protein Fkh2 is a component of the yeast cell cycle transcription factor SFF , 2000, The EMBO journal.

[18]  David Lydall,et al.  NDD1, a High-Dosage Suppressor ofcdc28-1N, Is Essential for Expression of a Subset of Late-S-Phase-Specific Genes in Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[19]  Peter C. Hollenhorst,et al.  Forkhead genes in transcriptional silencing, cell morphology and the cell cycle. Overlapping and distinct functions for FKH1 and FKH2 in Saccharomyces cerevisiae. , 2000, Genetics.

[20]  Mike Tyers,et al.  The fork'ed path to mitosis , 2000, Genome Biology.

[21]  Michael Ruogu Zhang,et al.  Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. , 1998, Molecular biology of the cell.

[22]  S. Reed,et al.  Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins , 1993, The Journal of cell biology.

[23]  A. Shevchenko,et al.  Forkhead transcription factors, Fkh1p and Fkh2p, collaborate with Mcm1p to control transcription required for M-phase , 2000, Current Biology.

[24]  D. Durocher,et al.  The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. , 2000, Molecular cell.

[25]  R. Treisman,et al.  Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element , 1992, Cell.

[26]  J. Haber,et al.  Saccharomyces forkhead protein Fkh1 regulates donor preference during mating-type switching through the recombination enhancer. , 2002, Genes & development.

[27]  T. Vernet,et al.  A family of yeast expression vectors containing the phage f1 intergenic region. , 1987, Gene.

[28]  C. Martínez-A,et al.  Forkhead transcription factors contribute to execution of the mitotic programme in mammals , 2001, Nature.

[29]  D. Botstein,et al.  Two yeast forkhead genes regulate the cell cycle and pseudohyphal growth , 2000, Nature.

[30]  S. Dalton,et al.  Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Gerald R. Fink,et al.  Guide to yeast genetics and molecular biology , 1993 .

[32]  M. Tsai,et al.  Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53. , 1999, Journal of molecular biology.

[33]  A. Amon,et al.  Regulation of B‐type cyclin proteolysis by Cdc28–associated kinases in budding yeast , 1997, The EMBO journal.

[34]  K Nasmyth,et al.  Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. , 1993, The EMBO journal.

[35]  Daniel Durocher,et al.  The FHA domain , 2002, FEBS letters.

[36]  A. Nordheim,et al.  Mcm1 is required to coordinate G2-specific transcription in Saccharomyces cerevisiae , 1995, Molecular and cellular biology.