Comprehensive Proteomics Analysis Reveals New Substrates and Regulators of the Fission Yeast Clp1/Cdc14 Phosphatase*

The conserved family of Cdc14 phosphatases targets cyclin-dependent kinase substrates in yeast, mediating late mitotic signaling events. To discover substrates and regulators of the Schizosaccharomyces pombe Cdc14 phosphatase Clp1, TAP-tagged Clp1, and a substrate trapping mutant (Clp1-C286S) were purified from asynchronous and mitotic (prometaphase and anaphase) cells and binding partners were identified by 2D-LC-MS/MS. Over 100 Clp1-interacting proteins were consistently identified, over 70 of these were enriched in Clp1-C286S-TAP (potential substrates) and we and others detected Cdk1 phosphorylation sites in over half (44/73) of these potential substrates. According to GO annotations, Clp1-interacting proteins are involved in many essential cellular processes including mitosis, cytokinesis, ribosome biogenesis, transcription, and trafficking among others. We confirmed association and dephosphorylation of multiple candidate substrates, including a key scaffolding component of the septation initiation network called Cdc11, an essential kinase of the conserved morphogenesis-related NDR kinase network named Shk1, and multiple Mlu1-binding factor transcriptional regulators. In addition, we identified Sal3, a nuclear β-importin, as the sole karyopherin required for Clp1 nucleoplasmic shuttling, a key mode of Cdc14 phosphatase regulation. Finally, a handful of proteins were more abundant in wild type Clp1-TAP versus Clp1-C286S-TAP, suggesting that they may directly regulate Clp1 signaling or serve as scaffolding platforms to localize Clp1 activity.

[1]  T. Toda,et al.  The NDA3 gene of fission yeast encodes β-tubulin: A cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis , 1984, Cell.

[2]  S. Moreno,et al.  Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. , 1991, Methods in enzymology.

[3]  K. Maundrell.  Thiamine-repressible expression vectors pREP and pRIP for fission yeast. , 1993, Gene.

[4]  J. Boeke,et al.  Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. , 1994, Genetics.

[5]  A. Willems,et al.  Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure , 1995, Yeast.

[6]  B. Neel,et al.  From Form to Function: Signaling by Protein Tyrosine Phosphatases , 1996, Cell.

[7]  A. Wach.  PCR‐synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae , 1996, Yeast.

[8]  D. Beach,et al.  The Cdc2 protein kinase controls Cdc10/Sct1 complex formation. , 1997, Molecular biology of the cell.

[9]  J. McIntosh,et al.  Localization of the 26S proteasome during mitosis and meiosis in fission yeast , 1998, The EMBO journal.

[10]  Angelika Amon,et al.  Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus , 1999, Nature.

[11]  K. Gould,et al.  Myb-Related Fission Yeast cdc5p Is a Component of a 40S snRNP-Containing Complex and Is Essential for Pre-mRNA Splicing , 1999, Molecular and Cellular Biology.

[12]  Anna Shevchenko,et al.  Exit from Mitosis Is Triggered by Tem1-Dependent Release of the Protein Phosphatase Cdc14 from Nucleolar RENT Complex , 1999, Cell.

[13]  K. Gould,et al.  Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[14]  G. Blobel,et al.  Karyopherins and nuclear import. , 2001, Current opinion in structural biology.

[15]  James A. DeCaprio,et al.  Feedback regulation of the MBF transcription factor by cyclin Cig2 , 2001, Nature Cell Biology.

[16]  B. Séraphin,et al.  The tandem affinity purification (TAP) method: a general procedure of protein complex purification. , 2001, Methods.

[17]  R. Deshaies,et al.  Characterization of the Net1 Cell Cycle-dependent Regulator of the Cdc14 Phosphatase from Budding Yeast* , 2001, The Journal of Biological Chemistry.

[18]  S. Moreno,et al.  Flp1, a fission yeast orthologue of the s. cerevisiae CDC14 gene, is not required for cyclin degradation or rum1p stabilisation at the end of mitosis. , 2001, Journal of cell science.

[19]  M. Tyers,et al.  Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression , 2001, Current Biology.

[20]  G. Chua,et al.  The sal3(+) gene encodes an importin-beta implicated in the nuclear import of Cdc25 in Schizosaccharomyces pombe. , 2002, Genetics.

[21]  Timothy J. Mitchison,et al.  Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT , 2002 .

[22]  A. Krapp,et al.  Mitotic Hyperphosphorylation of the Fission Yeast SIN Scaffold Protein cdc11p Is Regulated by the Protein Kinase cdc7p , 2003, Current Biology.

[23]  D. Barford,et al.  The structure of the cell cycle protein Cdc14 reveals a proline‐directed protein phosphatase , 2003, The EMBO journal.

[24]  A. Martín-Cuadrado,et al.  The endo-β-1,3-glucanase eng1p is required for dissolution of the primary septum during cell separation in Schizosaccharomyces pombe , 2003, Journal of Cell Science.

[25]  K. Gould,et al.  Fission yeast Clp1p phosphatase affects G2/M transition and mitotic exit through Cdc25p inactivation , 2004, The EMBO journal.

[26]  P. Lio’,et al.  Periodic gene expression program of the fission yeast cell cycle , 2004, Nature Genetics.

[27]  K. Gould,et al.  Sid4p-Cdc11p Assembles the Septation Initiation Network and Its Regulators at the S. pombe SPB , 2004, Current Biology.

[28]  Andrew J Link,et al.  Tandem affinity purification and identification of protein complex components. , 2004, Methods.

[29]  S. Moreno,et al.  A role for the Cdc14-family phosphatase Flp1p at the end of the cell cycle in controlling the rapid degradation of the mitotic inducer Cdc25p in fission yeast , 2004, Journal of Cell Science.

[30]  M. Balasubramanian,et al.  The Clp1p/Flp1p phosphatase ensures completion of cytokinesis in response to minor perturbation of the cell division machinery in Schizosaccharomyces pombe , 2004, Journal of Cell Science.

[31]  A. Krapp,et al.  Analysis of the S. pombe signalling scaffold protein Cdc11p reveals an essential role for the N‐terminal domain in SIN signalling , 2004, FEBS letters.

[32]  D. McCollum,et al.  The S. pombe Cdc14-like phosphatase Clp1p regulates chromosome biorientation and interacts with Aurora kinase. , 2004, Developmental cell.

[33]  Jianhua Liu,et al.  Identification of genes encoding putative nucleoporins and transport factors in the fission yeast Schizosaccharomyces pombe: a deletion analysis , 2004, Yeast.

[34]  L. Pemberton,et al.  Karyopherins: from nuclear-transport mediators to nuclear-function regulators. , 2004, Trends in cell biology.

[35]  Angelika Amon,et al.  Closing mitosis: the functions of the Cdc14 phosphatase and its regulation. , 2004, Annual review of genetics.

[36]  S. Sazer,et al.  The Fission Yeast Schizosaccharomyces pombe Has Two Importin-α Proteins, Imp1p and Cut15p, Which Have Common and Unique Functions in Nucleocytoplasmic Transport and Cell Cycle Progression , 2005, Genetics.

[37]  K. L. Gould,et al.  Ace2p controls the expression of genes required for cell separation in Schizosaccharomyces pombe. , 2005, Molecular biology of the cell.

[38]  L. Wong,et al.  Identification of cell cycle-regulated genes in fission yeast. , 2005, Molecular biology of the cell.

[39]  Sander van den Heuvel,et al.  Cell-cycle regulation. , 2005 .

[40]  M. Balasubramanian,et al.  The 14-3-3 Protein Rad24p Modulates Function of the Cdc14p Family Phosphatase Clp1p/Flp1p in Fission Yeast , 2005, Current Biology.

[41]  D. McCollum,et al.  Distinct Nuclear and Cytoplasmic Functions of the S. pombe Cdc14-like Phosphatase Clp1p/Flp1p and a Role for Nuclear Shuttling in Its Regulation , 2005, Current Biology.

[42]  J. Bähler.  Cell-cycle control of gene expression in budding and fission yeast. , 2005, Annual review of genetics.

[43]  J. Yates,et al.  Phospho-regulation of the Cdc14/Clp1 phosphatase delays late mitotic events in S. pombe. , 2006, Developmental cell.

[44]  D. McCollum,et al.  S. pombe FEAR protein orthologs are not required for release of Clp1/Flp1 phosphatase from the nucleolus during mitosis , 2006, Journal of Cell Science.

[45]  Paul Russell,et al.  Constraining G1-specific transcription to late G1 phase: the MBF-associated corepressor Nrm1 acts via negative feedback. , 2006, Molecular cell.

[46]  Andrew J Link,et al.  Role of Hcn1 and Its Phosphorylation in Fission Yeast Anaphase-promoting Complex/Cyclosome Function* , 2006, Journal of Biological Chemistry.

[47]  A. Lamond,et al.  Mitotic phosphatases: no longer silent partners. , 2006, Current opinion in cell biology.

[48]  Steven P Gygi,et al.  A probability-based approach for high-throughput protein phosphorylation analysis and site localization , 2006, Nature Biotechnology.

[49]  M. Tyers,et al.  Size control goes global. , 2007, Current opinion in biotechnology.

[50]  Ryan E. Mills,et al.  Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α* , 2007, Journal of Biological Chemistry.

[51]  K. Gould,et al.  The role of Cdc14 phosphatases in the control of cell division. , 2008, Biochemical Society transactions.

[52]  K. Bloom,et al.  Kinesin-8 molecular motors: putting the brakes on chromosome oscillations. , 2008, Trends in cell biology.

[53]  S. Elledge,et al.  A quantitative atlas of mitotic phosphorylation , 2008, Proceedings of the National Academy of Sciences.

[54]  K. Gould,et al.  The SIN Kinase Sid2 Regulates Cytoplasmic Retention of the S. pombe Cdc14-like Phosphatase Clp1 , 2008, Current Biology.

[55]  A. Bueno,et al.  Cds1 controls the release of Cdc14-like phosphatase Flp1 from the nucleolus to drive full activation of the checkpoint response to replication stress in fission yeast. , 2008, Molecular biology of the cell.

[56]  M. Balasubramanian,et al.  Schizosaccharomyces pombe Pak-related protein, Pak1p/Orb2p, phosphorylates myosin regulatory light chain to inhibit cytokinesis , 2008, The Journal of cell biology.

[57]  M. Mann,et al.  Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle. , 2008, Molecular cell.

[58]  K. Gould,et al.  Stepping into the ring: the SIN takes on contractile ring assembly. , 2008, Genes & development.

[59]  J. Yates,et al.  The Clp1/Cdc14 phosphatase contributes to the robustness of cytokinesis by association with anillin-related Mid1 , 2008, The Journal of cell biology.

[60]  G. Gould,et al.  Breaking up is hard to do – membrane traffic in cytokinesis , 2008, Journal of Cell Science.

[61]  John R Yates,et al.  Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis. , 2008, Journal of proteome research.

[62]  S. Gygi,et al.  Phosphoproteome analysis of fission yeast. , 2008, Journal of proteome research.

[63]  A. Brazma,et al.  The Fission Yeast Homeodomain Protein Yox1p Binds to MBF and Confines MBF-Dependent Cell-Cycle Transcription to G1-S via Negative Feedback , 2009, PLoS genetics.

[64]  K. Gould,et al.  A link between aurora kinase and Clp1/Cdc14 regulation uncovered by the identification of a fission yeast borealin-like protein. , 2009, Molecular biology of the cell.

[65]  Raymond J. Deshaies,et al.  Dbf2–Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis , 2009, The Journal of cell biology.

[66]  J. Yates,et al.  Phosphorylation State Defines Discrete Roles for Monopolin in Chromosome Attachment and Spindle Elongation , 2009, Current Biology.

[67]  J. J. Ward,et al.  Phospho-regulated interaction between kinesin-6 Klp9p and microtubule bundler Ase1p promotes spindle elongation. , 2009, Developmental cell.

[68]  W. Lim,et al.  Evolution of Phosphoregulation: Comparison of Phosphorylation Patterns across Yeast Species , 2009, PLoS biology.

[69]  K. Gould,et al.  Jcb: Article , 2022 .

[70]  E. Schiebel,et al.  Cdc14: a highly conserved family of phosphatases with non-conserved functions? , 2010, Journal of Cell Science.

[71]  K. Gould,et al.  Regulation of cell cycle-specific gene expression in fission yeast by the Cdc14p-like phosphatase Clp1p , 2010, Journal of Cell Science.

[72]  I. Tolic-Nørrelykke,et al.  Laser microsurgery provides evidence for merotelic kinetochore attachments in fission yeast cells lacking Pcs1 or Clr4 , 2010, Cell cycle.

[73]  L. Parker,et al.  Cdc14 Phosphatases Preferentially Dephosphorylate a Subset of Cyclin-dependent kinase (Cdk) Sites Containing Phosphoserine*♦ , 2011, The Journal of Biological Chemistry.

[74]  Trey Ideker,et al.  Cytoscape 2.8: new features for data integration and network visualization , 2010, Bioinform..

[75]  David G. Pisano,et al.  Cdc14b regulates mammalian RNA polymerase II and represses cell cycle transcription , 2011, Scientific reports.

[76]  K. Gould,et al.  SIN-Inhibitory Phosphatase Complex Promotes Cdc11p Dephosphorylation and Propagates SIN Asymmetry in Fission Yeast , 2011, Current Biology.

[77]  K. Gould,et al.  Cdk1 phosphorylation of the kinetochore protein Nsk1 prevents error-prone chromosome segregation , 2011, The Journal of cell biology.

[78]  Frank Uhlmann,et al.  A Quantitative Model for Ordered Cdk Substrate Dephosphorylation during Mitotic Exit , 2011, Cell.

[79]  J. Millar,et al.  Ringing the changes: emerging roles for DASH at the kinetochore–microtubule Interface , 2011, Chromosome Research.

[80]  Sneha Gupta,et al.  Crosstalk between NDR kinase pathways coordinates cell cycle dependent actin rearrangements , 2011, Cell Division.

[81]  Matthew Chambers,et al.  Supporting tool suite for production proteomics , 2011, Bioinform..

[82]  K. Gould,et al.  Polar opposites: Fine‐tuning cytokinesis through SIN asymmetry , 2012, Cytoskeleton.

[83]  K. Gould,et al.  The fission yeast septation initiation network (SIN) kinase, Sid2, is required for SIN asymmetry and regulates the SIN scaffold, Cdc11 , 2012, Molecular biology of the cell.

[84]  K. Gould,et al.  Multiple protein kinases influence the redistribution of fission yeast Clp1/Cdc14 phosphatase upon genotoxic stress , 2012, Molecular biology of the cell.