Comprehensive proteomics analysis reveals new substrates and regulators of the fission yeast clp1/cdc14 phosphatase

Mol Cell Proteomics. 2013 May;12(5):1074-86. doi: 10.1074/mcp.M112.025924. Epub 2013 Jan 7.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • CDC11 protein, S pombe
  • Cell Cycle Proteins
  • Karyopherins
  • Schizosaccharomyces pombe Proteins
  • CDC2 Protein Kinase
  • Clp1 protein, S. pombe
  • Protein Tyrosine Phosphatases