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

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 ...

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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 (SIN) called Cdc11, an essential kinase of the conserved morphogenesis-related NDR kinase network (MOR) named Shk1, and multiple Mlu1-binding factor (MBF) transcriptional regulators. In addition, we identified Sal3, a nuclear beta-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 wildtype Clp1-TAP versus Clp1-C286S-TAP, suggesting that they may directly regulate Clp1 signaling or serve as scaffolding platforms to localize Clp1 activity.

Date: Jan. 07, 2013

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