Progression through the cell cycle is driven by sequentially changing sets of substrate proteins getting phosphorylated by Cdk(s). This is ensured by sequentially expressed cyclin activators that confer distinct substrate specificities on Cdk(s) in different phases of the cycle. During the cell cycle, Cdk activity is modulated by opposing phosphatase activities. In budding yeast, Cdc14 acts as one of the antagonists of Cdk activity regulating a well-defined subset of Cdk substrates mainly participating in late mitotic events such as chromosome segregation, spindle disassembly, DNA damage response and cytokinesis (
Bremmer,2012;
Eissler,2014;
Kao,2014;
Powers,2017;
Mocciaro,2010;
Villoria,2017;
Manzano-Lopez,2020). Although Cdc14 homologues have been identified in a wide range of eukaryotes and their target specificities are mostly conserved (
Bremmer,2012;
Li,2015;
Vazquez-Novelle,2005), the key role played in mitotic exit is yet only confirmed in budding yeasts. For instance, the fission yeast Cdc14 homologue, Cdc14-like phosphatase 1 (Clp1; also known as Flp1;
Q9P7H1), contributes to the control of cytokinesis, but is not required for other aspects of mitotic exit or Cdk1 inactivation (
Trautmann,2004). The Caenorhabditis elegans Cdc14 homologue, cdc-14 (
P81299), does not function in mitosis, rather it is crucial for G(1)/S regulation to establish developmental cell-cycle quiescence (
Saito,2004). Among the vertebrate homologues, CDC14B (
O60729) is functionally related to yeast Cdc14 and plays major roles in functions such as the G2/M DNA damage checkpoint, DNA repair and centrosome duplication, while CDC14A (
Q9UNH5) is involved in centrosome separation and cytokinesis, suggesting that the functions of CDC14 phosphatases have been partially rewired during eukaryotic evolution but preserve important hallmarks (
Mocciaro,2010;
Powers,2017).
The yeast Cdc14 protein belongs to the dual-specificity phosphatase family (
PF00782). Cdc14 is sequestered in the nucleolus for most of the cell cycle by the nucleolar proteins Net1 and Tof2, and is only released into the nucleoplasm and cytosol during anaphase (
Visintin,1999;
Shou,1999). In anaphase, the so called Fourteen Early Anaphase Release (FEAR) network (including Cdc5, Esp1, and Slk19), and the mitotic exit network (MEN; including the Dbf2-Mob1 complex) co-ordinately trigger the release of Cdc14 from the nucleolus (
Shou,1999). Subsequently, Cdc14 performs the sequential dephosphorylation of its targets (
Bouchoux,2011). Both the molecular mechanism of (in)activation and the timing of activity during the cell cycle show remarkable differences between Cdc14 homologues (
Powers,2017;
Manzano-Lopez,2020).
In vitro, Cdc14 strongly favours dephosphorylation of phosphoserines followed by a proline, with an additional positively charged residue (preferentially lysine) in the +4 position ((S)PxK/r) which, maybe not surprisingly, conforms to a restricted version of the Cdk consensus phosphorylation motif (
Bremmer,2012;
MOD_CDK_SPxK_1). Phosphothreonines are poor Cdc14 substrates, due to a steric clash with the methyl group of a conserved alanine residue of the phosphatase active site (
Bremmer,2012;
Eissler,2014). In vitro phosphatase assays using wildtype or mutated phosphorylated target peptides clearly indicated that lysine in the +3 position enables higher catalytic efficiency than arginine (
Bremmer,2012;
Eissler,2014). Also, additional positively charged residues in the +3, +5 or +6 positions of the phosphorylated target motifs further enhance the catalytic efficiency of yeast Cdc14 (
Bremmer,2012). Based on the experimentally validated targets of Cdc14, lysine over arginine in the +4 position and the additional positively charged residues in the surrounding positions are not required for dephosphorylation by Cdc14, however, they could influence in vivo catalytic efficiency and therefore may determine the order of the different substrates getting dephosphorylated after the activation of the phosphatase.
Due to the low affinity and transient nature of the kinase/phosphatase-substrate interactions, the substrates are generally difficult to identify. The substrates of Cdc14 have been best characterized in budding yeast. The phosphatase dead mutants of yeast Cdc14 (C283S or D253A) have been declared as promising "substrate traps" and they were used to identify the potential in vivo substrates of Cdc14 in pull down and co-immunoprecipitation experiments (
Bloom,2011). These substrate trap experiments could only identify a small subset of the potential Cdc14 targets detected in high-throughput phosphoproteomic screens (
Bloom,2011;
Eissler,2014;
Powers,2017). Although many substrates of yeast Cdc14 have been validated in low-throughput experiments, the changes in the phosphorylation states of full proteins encompassing several phosphosites were mainly demonstrated by mass-dependent changes in migration within gels, therefore the dephosphorylation of individual phosphosites by Cdc14 has rarely been addressed. Nevertheless, analysis of the peptide hits of large-scale phosphoproteomic analyses of potential Cdc14 targets have confirmed the in vivo relevance of the (S)PxK/r motif (
Eissler,2014;
Powers,2017).
Fission yeast and human Cdc14 homologues showed similar residue preferences to budding yeast Cdc14 in peptide dephosphorylation assays, and the residues contributing to the active site are highly conserved, therefore the target motif was declared to be widely conserved among eukaryotes (
Bremmer,2012).
Also, the structure of the complex between a phosphatase dead mutant of Cdc14 and an S-phosphorylated Swi6p peptide provides information on the catalytic mechanism of Cdc14 (
5XW5). Furthermore, a complex structure of human CDC14B and a short substrate peptide is also available, even though the substrate peptide is too short for assessing the preference for positive residues in the positions following the (S)P (
1OHE;
Gray,2003).
Yeast Cdc14 forms a dimer, and dimerization is essential for its catalytic efficiency (
5XW4;
Kobayashi,2017). Furthermore, the non-catalytic, N-terminal domain of yeast Cdc14 (
PF14671) has a binding pocket that recognizes the PxL docking motif (
DOC_CDC14_PxL_1) on its substrates (
6G86;
6G85), enhancing target recognition and dephosphorylation of both optimal and suboptimal targets (
Kataria,2018). Although the hydrophobic pocket of Cdc14 that interacts with the PxL motif is conserved in human Cdc14 orthologues (
Kataria,2018), its functional relevance is yet to be validated outside the phylogenetic group of budding yeasts.