title: Comprehensive proteomics analysis reveals new substrates and regulators of the fission yeast clp1/cdc14 phosphatase.
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.
authors.affiliation: Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, 1161 21 Avenue South, MCN B2309, Nashville, Tennessee 37232, USA.
authors.forename: Jun-Song
authors.initials: JS
authors.lastname: Chen
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Broadus
authors.forename: Janel R
authors.initials: JR
authors.lastname: McLean
authors.forename: Anna
authors.initials: A
authors.lastname: Feoktistova
authors.forename: Liping
authors.initials: L
authors.lastname: Ren
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Gould
mesh_terms: D021581:Active Transport, Cell Nucleus; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002467:Cell Nucleus / Q000201:enzymology; D028884:Karyopherins / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D011499:Protein Processing, Post-Translational; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000502:physiology*; D040901:Proteomics; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
chemical_list: C477369:CDC11 protein, S pombe; D018797:Cell Cycle Proteins; D028884:Karyopherins; D029702:Schizosaccharomyces pombe Proteins; D016203:CDC2 Protein Kinase; C439269:Clp1 protein, S. pombe; D017027:Protein Tyrosine Phosphatases