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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.

Pubmed ID: 23297348

Authors

  • Chen JS
  • Broadus MR
  • McLean JR
  • Feoktistova A
  • Ren L
  • Gould KL

Journal

Molecular & cellular proteomics : MCP

Publication Data

May 7, 2013

Associated Grants

  • Agency: NCI NIH HHS, Id: N.I.H. T32 CA009582
  • Agency: NCI NIH HHS, Id: NCI T32CA119925
  • Agency: NCI NIH HHS, Id: T32 CA009582
  • Agency: NCI NIH HHS, Id: T32 CA119925
  • Agency: Howard Hughes Medical Institute, Id:

Mesh Terms

  • Active Transport, Cell Nucleus
  • CDC2 Protein Kinase
  • Cell Cycle Proteins
  • Cell Nucleus
  • Karyopherins
  • Peptide Mapping
  • Phosphorylation
  • Protein Interaction Mapping
  • Protein Interaction Maps
  • Protein Processing, Post-Translational
  • Protein Tyrosine Phosphatases
  • Proteomics
  • Schizosaccharomyces
  • Schizosaccharomyces pombe Proteins