The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

ABSTRACT The cell wall integrity (CWI) checkpoint in the budding yeast Saccharomyces cerevisiae coordinates cell wall construction and cell cycle progression. In this study, we showed that the regulation of Hcm1, a late-S-phase transcription factor, arrests the cell cycle via the cell wall integrity checkpoint. Although the HCM1 mRNA level remained unaffected when the cell wall integrity checkpoint was induced, the protein level decreased. The overproduction of Hcm1 resulted in the failure of the cell wall integrity checkpoint. We identified 39 Hcm1 phosphorylation sites, including 26 novel sites, by tandem mass spectrometry analysis. A mutational analysis revealed that phosphorylation of Hcm1 at S61, S65, and S66 is required for the proper onset of the cell wall integrity checkpoint by regulating the timely decrease in its protein level. Hyperactivation of the CWI mitogen-activated protein kinase (MAPK) signaling pathway significantly reduced the Hcm1 protein level, and the deletion of CWI MAPK Slt2 resulted in a failure to decrease Hcm1 protein levels in response to stress, suggesting that phosphorylation is regulated by CWI MAPK. In conclusion, we suggest that Hcm1 is regulated negatively by the cell wall integrity checkpoint through timely phosphorylation and degradation under stress to properly control budding yeast proliferation.

[1]  M. Cyert,et al.  Hcm1 integrates signals from Cdk1 and calcineurin to control cell proliferation , 2015, Molecular biology of the cell.

[2]  J. A. Benanti,et al.  Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression , 2014, The EMBO journal.

[3]  C. Wittenberg,et al.  Topology and Control of the Cell-Cycle-Regulated Transcriptional Circuitry , 2014, Genetics.

[4]  Gustav Ammerer,et al.  Genotoxic Stress Prevents Ndd1-Dependent Transcriptional Activation of G2/M-Specific Genes in Saccharomyces cerevisiae , 2013, Molecular and Cellular Biology.

[5]  C. Nombela,et al.  Activation of the yeast cell wall integrity MAPK pathway by zymolyase depends on protease and glucanase activities and requires the mucin‐like protein Hkr1 but not Msb2 , 2013, FEBS Letters.

[6]  E. Cabiscol,et al.  The FOX transcription factor Hcm1 regulates oxidative metabolism in response to early nutrient limitation in yeast. Role of Snf1 and Tor1/Sch9 kinases. , 2013, Biochimica et biophysica acta.

[7]  Lev I Levitsky,et al.  Pyteomics—a Python Framework for Exploratory Data Analysis and Rapid Software Prototyping in Proteomics , 2013, Journal of The American Society for Mass Spectrometry.

[8]  J. Eng,et al.  Comet: An open‐source MS/MS sequence database search tool , 2013, Proteomics.

[9]  T. Nühse.  Cell wall integrity signaling and innate immunity in plants , 2012, Front. Plant Sci..

[10]  G. Ammerer,et al.  Validation of regulated protein phosphorylation events in yeast by quantitative mass spectrometry analysis of purified proteins , 2012, Proteomics.

[11]  G. Mouille,et al.  Plant Cell Wall Homeostasis Is Mediated by Brassinosteroid Feedback Signaling , 2012, Current Biology.

[12]  Z. Darieva,et al.  Protein Kinase C Regulates Late Cell Cycle-Dependent Gene Expression , 2012, Molecular and Cellular Biology.

[13]  M. Smolka,et al.  Linking DNA replication checkpoint to MBF cell‐cycle transcription reveals a distinct class of G1/S genes , 2012, The EMBO journal.

[14]  John R Yates,et al.  DNA replication stress differentially regulates G1/S genes via Rad53‐dependent inactivation of Nrm1 , 2012, The EMBO journal.

[15]  D. E. Levin,et al.  Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway , 2011, Genetics.

[16]  T. Köcher,et al.  Universal and confident phosphorylation site localization using phosphoRS. , 2011, Journal of proteome research.

[17]  Patrick G. A. Pedrioli,et al.  Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases and Phosphatases in Yeast , 2010, Science Signaling.

[18]  Y. Ohya,et al.  The cell wall integrity checkpoint: coordination between cell wall synthesis and the cell cycle , 2010, Yeast.

[19]  Y. Ohya,et al.  Multiple Functional Domains of the Yeast l,3-β-Glucan Synthase Subunit Fks1p Revealed by Quantitative Phenotypic Analysis of Temperature-Sensitive Mutants , 2010, Genetics.

[20]  Bruce Stillman,et al.  Deciphering Protein Kinase Specificity through Large-scale Analysis of Materials Supplemental Deciphering Protein Kinase Specificity through Large-scale Analysis of Yeast Phosphorylation Site Motifs , 2010 .

[21]  Matthew E Ritchie,et al.  High-resolution transcription atlas of the mitotic cell cycle in budding yeast , 2010, Genome Biology.

[22]  Jesper V Olsen,et al.  Global analysis of the yeast osmotic stress response by quantitative proteomics. , 2009, Molecular bioSystems.

[23]  Wei Xiao,et al.  DNA damage-induced gene expression in Saccharomyces cerevisiae. , 2008, FEMS microbiology reviews.

[24]  Steven B. Haase,et al.  Transcription network and cyclin/CDKs: The yin and yang of cell cycle oscillators , 2008, Cell cycle.

[25]  P. Pevzner,et al.  Spectral probabilities and generating functions of tandem mass spectra: a strike against decoy databases. , 2008, Journal of proteome research.

[26]  Samuel H. Payne,et al.  A Multidimensional Chromatography Technology for In-depth Phosphoproteome Analysis*S , 2008, Molecular & Cellular Proteomics.

[27]  Joshua E. S. Socolar,et al.  Global control of cell-cycle transcription by coupled CDK and network oscillators , 2008, Nature.

[28]  Sandra Lopez-Aviles,et al.  Activation of Srk1 by the mitogen-activated protein kinase Sty1/Spc1 precedes its dissociation from the kinase and signals its degradation. , 2008, Molecular biology of the cell.

[29]  C. Nombela,et al.  The sequential activation of the yeast HOG and SLT2 pathways is required for cell survival to cell wall stress. , 2007, Molecular biology of the cell.

[30]  A. Beauvais,et al.  Homologous Subunits of 1,3-Beta-Glucan Synthase Are Important for Spore Wall Assembly in Saccharomyces cerevisiae , 2006, Eukaryotic Cell.

[31]  D. Lew,et al.  Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint. , 2006, Current opinion in microbiology.

[32]  William Stafford Noble,et al.  The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle. , 2006, Genes & development.

[33]  G. W. Hatfield,et al.  HB tag modules for PCR‐based gene tagging and tandem affinity purification in Saccharomyces cerevisiae , 2006, Yeast.

[34]  O. Bachs,et al.  Inactivation of the Cdc25 phosphatase by the stress-activated Srk1 kinase in fission yeast. , 2005, Molecular cell.

[35]  Z. Darieva,et al.  Regulation of Cell Cycle-Specific Gene Expression through Cyclin-Dependent Kinase-Mediated Phosphorylation of the Forkhead Transcription Factor Fkh2p , 2004, Molecular and Cellular Biology.

[36]  H. Kitagaki,et al.  A Temperature-Sensitive dcw1 Mutant of Saccharomyces cerevisiae Is Cell Cycle Arrested with Small Buds Which Have Aberrant Cell Walls , 2004, Eukaryotic Cell.

[37]  M. Hoyt.  A new checkpoint takes shape , 2004, Nature Cell Biology.

[38]  Y. Ohya,et al.  Dynactin is involved in a checkpoint to monitor cell wall synthesis in Saccharomyces cerevisiae , 2004, Nature Cell Biology.

[39]  Robertson Craig,et al.  TANDEM: matching proteins with tandem mass spectra. , 2004, Bioinformatics.

[40]  D. Lew,et al.  The morphogenesis checkpoint: how yeast cells watch their figures. , 2003, Current opinion in cell biology.

[41]  K. Shokat,et al.  Targets of the cyclin-dependent kinase Cdk1 , 2003, Nature.

[42]  M. Gerstein,et al.  Complex transcriptional circuitry at the G1/S transition in Saccharomyces cerevisiae. , 2002, Genes & development.

[43]  S. Ishihara,et al.  Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase, in Saccharomyces cerevisiae. , 2002, Genetics.

[44]  D. Botstein,et al.  Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF , 2001, Nature.

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

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

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

[48]  K. Kaestner,et al.  Unified nomenclature for the winged helix/forkhead transcription factors. , 2000, Genes & development.

[49]  G. Zhu,et al.  The fork head transcription factor Hcm1p participates in the regulation of SPC110, which encodes the calmodulin-binding protein in the yeast spindle pole body. , 1998, Biochimica et biophysica acta.

[50]  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.

[51]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

[52]  M. Snyder,et al.  SBF Cell Cycle Regulator as a Target of the Yeast PKC-MAP Kinase Pathway , 1997, Science.

[53]  Y. Anraku,et al.  Signaling toward yeast 1,3-beta-glucan synthesis. , 1996, Cell structure and function.

[54]  A. Futcher,et al.  Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae , 1995, Yeast.

[55]  Y. Anraku,et al.  RHO gene products, putative small GTP‐binding proteins, are importnat for activation of the CAL1/CDC43 gene product, a protein geranylgeranyltransferase in Saccharomyces cerevisiae , 1992, Yeast.

[56]  D. E. Levin,et al.  Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog , 1992, Molecular and cellular biology.

[57]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[58]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.