Cell Cycle: New Functions for Cdc14 Family Phosphatases (original) (raw)
Related papers
Global Analysis of Cdc14 Phosphatase Reveals Diverse Roles in Mitotic Processes
Journal of Biological Chemistry, 2011
Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1, which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A), which allow binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared with the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events.
Journal of Biological Chemistry, 2001
In the budding yeast Saccharomyces cerevisiae, the multifunctional protein Net1 is implicated in regulating the cell cycle function of the Cdc14 protein phosphatase. Genetic and cell biological data suggest that during interphase and early mitosis Net1 holds Cdc14 within the nucleolus where its activity is suppressed. Upon its transient release from Net1 at late anaphase, active Cdc14 promotes exit from mitosis by dephosphorylating targets in the nucleus and cytoplasm. In this paper we present evidence supporting the proposed role of Net1 in regulating Cdc14 and exit from mitosis. We show that the NH 2-terminal fragment Net1(1-600) directly binds Cdc14 in vitro and is a highly specific competitive inhibitor of its activity (K i ؍ 3 nM) with five different substrates including the physiologic targets Swi5 and Sic1. An analysis of truncation mutants indicates that the Cdc14 binding site is located within a segment of Net1 containing residues 1-341. We propose that Net1 inhibits by occluding the active site of Cdc14 because it acts as a competitive inhibitor, binds to a site located within the catalytic domain (residues 1-374), binds with reduced affinity to a Cdc14 C283S mutant in which an active site Cys is replaced, and is displaced by tungstate, a transition state analog known to bind in the catalytic site of protein-tyrosine phosphatases.
Scientific reports, 2018
Cdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species. Moreover, in yeast, Cdc14 is regulated by changes in its subcellular location and by phosphorylation events. In contrast, little is known about the regulation of human Cdc14 phosphatases. Here, we have studied how the human Cdc14A orthologue is regulated during the cell cycle. We found that Cdc14A is phosphorylated on Ser411, Ser453 and Ser549 by Cdk1 early in mitosis and becomes dephosphorylated during late mitotic stages. Interestingly, in vivo a...
The role of Cdc14 phosphatases in the control of cell division
Biochemical Society Transactions, 2008
The periodicity of CDKs (cyclin-dependent kinases) regulates most cell cycle transitions including cytokinesis. High Cdk1 activity promotes cytoskeletal rearrangements necessary for cell division while at the same time ensuring that cytokinesis does not begin before the separation of sister chromatids during anaphase. The conserved Cdc14 (cell division cycle 14)-family of phosphatases reverses Cdk phosphorylation events and therefore Cdc14 phosphatases promote the process of cytokinesis. Here, we review the elucidated roles of Cdc14 phosphatases in cytokinesis and the current outstanding questions regarding their function in this process.
2018
Mitotic phosphatases play crucial roles in anaphase regulation and mitotic exit by annulling the kinase-mediated protein phosphorylation. In budding yeast Saccharomyces cerevisiae, Cdc14 (cell division cycle 14) phosphatase antagonizes key Cdk1 (cyclin-dependent kinase 1) functions to drive cells out of mitosis. Despite the presence of highly conserved catalytic domains, human CDC14s are dispensable for cell cycle progression. Nevertheless, to decipher the molecular mechanisms of human CDC14s functions, we have investigated the knockout cellular models aided by various imaging and proteomics approaches. Phenotypic analyses of the generated hCDC14A knockout in human retinal pigment epithelium (hTERT-RPE1) cells have exhibited the occurrence of longer primary cilia upon serum starvation. The intermediate longer cilia in haploid-insufficient cells, as well as the extended cilia observed upon siRNA-mediated acute depletion of hCDC14A, have further confirmed the phenotype. Primary cilia ...
The EMBO journal, 1994
We show that the fission yeast dis2 protein phosphatase, which is highly similar to mammalian type 1 phosphatase, is a phosphoprotein containing phosphoserine (phospho-S) and threonine (phospho-T). It has several phosphorylation sites, two of which locate in the C-terminus. Phospho-T was abolished in the alanine substitution mutant at the C-terminal T316, which is conserved as a residue in the cdc2 consensus, TPPR, in a number of type 1-like phosphatases. In G2-arrested cdc2-L7 cells, the degree of T316 phosphorylation was reduced, whereas it was enhanced in metaphase-arrested nuc2-663 mutant cells. Phospho-T was produced in dis2 by fission yeast cdc2 kinase, but not in the substitution mutant A316, indicating that the T316 residue was the site for cdc2 kinase in vitro. Phosphatase activity of wild type dis2 was reduced by incubation with cdc2 kinase, but that of mutant dis2-A316 was not. Phosphorylation of T316 hence has a potential significance in cell cycle control in conjunction...
The chromosome complement of the human fungal pathogen Candida albicans is unusually unstable, suggesting that the process of nuclear division is error prone. The Cdc14 phosphatase plays a key role in organising the intricate choreography of mitosis and cell division. In order to understand the role of Cdc14 in C. albicans we used quantitative proteomics to identify proteins that physically interact with Cdc14. To distinguish genuine Cdc14-interactors from proteins that bound non-specifically to the affinity matrix, we used a substrate trapping mutant combined with mass spectrometry analysis using Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC). The results identified 126 proteins that interact with Cdc14 of which 80% have not previously been identified as Cdc14 interactors in C. albicans or S. cerevisiae. In this set, 55 proteins are known from previous research in S. cerevisiae and S. pombe to play roles in the cell cycle, regulating the attachment of the mitotic spindle to kinetochores, mitotic exit, cytokinesis, licensing of DNA replication by reactivating pre-replication complexes, and DNA repair. Five Cdc14-interacting proteins with previously unknown functions localised to the Spindle Pole Bodies (SPBs). Thus, we have greatly increased the number of proteins that physically interact with Cdc14 in C. albicans. Candida albicans is normally a harmless commensal of the skin, urogenital and gastrointestinal tracts. However, in otherwise healthy individuals it can be responsible for debilitating and recurrent mucosal infections. In immu-nocompromised and other classes of vulnerable patients it causes life-threatening bloodstream infections 1-3. It is normally diploid and lacks the capability to undergo meiosis. Although lacking a sexual cycle, C. albicans can undergo a parasexual cycle in which two diploid cells mate to form a tetraploid, which then sheds chromosome during subsequent mitotic divisions to regain either diploid or aneuploidy states 4. Stresses, such as exposure to the antifungal drug fluconazole, result in ploidy changes, loss of heterozygosity and whole chromosome and segmental aneuploidy (reviewed in 5-8). Such genome plasticity is thought to be a major generator of diversity in the absence of a sexual cycle and has been shown to be adaptive. Recently, in response to fluconazole or passage through mice it has been shown that diploids can be reduced by chromosome loss to generate mating-competent haploids 9. This genome plasticity occurs through non-disjunction events in mitosis, which implies a high error rate in the intricately choreographed events of mitosis. A key player in orchestrating mitosis and cell division is Cdc14, a dual specificity, proline-and serine-directed phosphatase (for reviews see 10-12). In the budding yeast Saccharomyces cerevisiae after activation by the Fourteen Early Anaphase Release (FEAR) and Mitotic Exit Network (MEN) pathways it ensures irreversible exit from mitosis by directing the destruction of G2 cyclins and disassembly of the spindle. After mitotic exit it relocates to the bud neck and activates cytokinesis. Finally, in the G1 of the next cycle it reactivates DNA origins, which were inactivated after firing in the previous cell cycle 13. Previously, CaCdc14 has been shown not to be essential for viability, but it is required for normal polarized growth of hyphae and regulation of CaCdc14 has been shown to be necessary for the inhibition of cell separation characteristic of hyphal growth 14 .
Human Cdc14A Phosphatase Modulates the G2/M Transition through Cdc25A and Cdc25B
Journal of Biological Chemistry, 2010
The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.