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 ␤-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

Dynamics of SIN Asymmetry Establishment

“…These, and other earlier findings [20], [21], [41] suggest that Sid2 phosphorylation might prime Cdc11 for dephosphorylation at other sites and Byr4 binding, making SIN an indirect activator of Byr4. Recent results suggest that such dephosphorylation events might be catalyzed by the Cdc14-like Clp1/Flp1 phosphatase, even in the absence of SIP activity [41].…”

“…If we assume that the overexpression of the SIP component, Csc1, induces higher SIP activity (if this is the only limiting factor in the complex) leading to the observed multinucleate phenotype [21], then the model predicts that SIP should have roles in removing phosphates catalyzed by Sid2 to Cdc11 (at least when it is overexpressed). Since other mitotic phosphatases, like the Cdc14 phosphatase, Clp1/Flp1 [37], [38] or the PP2A phosphatases Par1 and Pab1 [39], [40] have been associated with SIN function and recent results suggests a role for Clp1 in Cdc11 dephosphorylation [41], we cannot conclude on the exact role of SIP only by simulating single perturbations on Cdc11 phosphorylation.…”

Contractile Ring Stability in S. pombe Depends on F-BAR Protein Cdc15p and Bgs1p Transport from the Golgi Complex

“…(ii) We used a strain with the single SH3 domain deleted from Cdc15p (Roberts-Galbraith et al, 2009), hereafter called cdc15 Δ SH3 cells. (iii) We deleted the gene for the Clp1p phosphatase (Trautmann et al, 2001) that regulates Cdc15p as well as many other phosphorylated proteins (Chen et al, 2013). We followed contractile ring assembly in time lapse movies of cells expressing the myosin-II regulatory light chain, Rlc1p, tagged in the genome on the C-terminus with red fluorescent protein tdTomato.…”

“…The observation that a hypophosphorylated Cdc15p mutant accumulates in vesicle-like structures at the cell center in interphase (Roberts-Galbraith et al, 2010) suggests that dephosphorylation of Cdc15p may be required for its role in transport out of the Golgi apparatus. Given that Clp1p also acts on proteins involved in vesicular trafficking, transcription and ribosome biogenesis (Chen et al, 2013), complementation experiments with Cdc15p having mutations in its phosphorylation sites will be of interest to understand the Δ clp1 phenotype and the regulation of Cdc15p during cytokinesis.…”

Cdk1 promotes cytokinesis in fission yeast through activation of the septation initiation network

“…Thus Cdk1 is believed to inhibit the SIN, but prevention of Cdk1-mediated phosphorylation of Cdc11, the only identified Cdk1 target within the SIN, does not have a significant effect on cytokinesis, suggesting the existence of other Cdk1-targeted SIN proteins (Morrell et al. , 2004; Chen et al. , 2013).…”