TOR complex 1 negatively regulates NDR kinase Cbk1 to control cell separation in budding yeast.

The target of rapamycin (TOR) signalling pathway plays a key role in the coordination between cellular growth and the cell cycle machinery in eukaryotes. The underlying molecular mechanisms by which TOR might regulate events after anaphase remain unknown. We show for the first time that one of the 2 TOR complexes in budding yeast, TORC1, blocks the separation of cells following cytokinesis by phosphorylation of a member of the NDR (nuclear Dbf2-related) protein-kinase family, the protein Cbk1. We observe that TORC1 alters the phosphorylation pattern of Cbk1 and we identify a residue within Cbk1 activation loop, T574, for which a phosphomimetic substitution makes Cbk1 catalytically inactive and, indeed, reproduces TORC1 control over cell separation. In addition, we identify the exocyst component Sec3 as a key substrate of Cbk1, since Sec3 activates the SNARE complex to promote membrane fusion. TORC1 activity ultimately compromises the interaction between Sec3 and a t-SNARE component. Our data indicate that TORC1 negatively regulates cell separation in budding yeast by participating in Cbk1 phosphorylation, which in turn controls the fusion of secretory vesicles transporting hydrolase at the site of division.

PP2A-Cdc55 phosphatase regulates actomyosin ring contraction and septum formation during cytokinesis.

Eukaryotic cells divide and separate all their components after chromosome segregation by a process called cytokinesis to complete cell division. Cytokinesis is highly regulated by the recruitment of the components to the division site and through post-translational modifications such as phosphorylations. The budding yeast mitotic kinases Cdc28-Clb2, Cdc5, and Dbf2-Mob1 phosphorylate several cytokinetic proteins contributing to the regulation of cytokinesis. The PP2A-Cdc55 phosphatase regulates mitosis counteracting Cdk1- and Cdc5-dependent phosphorylation. This prompted us to propose that PP2A-Cdc55 could also be counteracting the mitotic kinases during cytokinesis. Here we show that in the absence of Cdc55, AMR contraction and the primary septum formation occur asymmetrically to one side of the bud neck supporting a role for PP2A-Cdc55 in cytokinesis regulation. In addition, by in vivo and in vitro assays, we show that PP2A-Cdc55 dephosphorylates the chitin synthase II (Chs2 in budding yeast) a component of the Ingression Progression Complexes (IPCs) involved in cytokinesis. Interestingly, the non-phosphorylable version of Chs2 rescues the asymmetric AMR contraction and the defective septa formation observed in cdc55∆ mutant cells. Therefore, timely dephosphorylation of the Chs2 by PP2A-Cdc55 is crucial for proper actomyosin ring contraction. These findings reveal a new mechanism of cytokinesis regulation by the PP2A-Cdc55 phosphatase and extend our knowledge of the involvement of multiple phosphatases during cytokinesis.

Hog1 activation delays mitotic exit via phosphorylation of Net1.

Adaptation to environmental changes is crucial for cell fitness. In Saccharomyces cerevisiae, variations in external osmolarity trigger the activation of the stress-activated protein kinase Hog1 (high-osmolarity glycerol 1), which regulates gene expression, metabolism, and cell-cycle progression. The activation of this kinase leads to the regulation of G1, S, and G2 phases of the cell cycle to prevent genome instability and promote cell survival. Here we show that Hog1 delays mitotic exit when cells are stressed during metaphase. Hog1 phosphorylates the nucleolar protein Net1, altering its affinity for the phosphatase Cdc14, whose activity is essential for mitotic exit and completion of the cell cycle. The untimely release of Cdc14 from the nucleolus upon activation of Hog1 is linked to a defect in ribosomal DNA (rDNA) and telomere segregation, and it ultimately delays cell division. A mutant of Net1 that cannot be phosphorylated by Hog1 displays reduced viability upon osmostress. Thus, Hog1 contributes to maximizing cell survival upon stress by regulating mitotic exit.

Cdc14 activation requires coordinated Cdk1-dependent phosphorylation of Net1 and PP2A-Cdc55 at anaphase onset.

Exit from mitosis and completion of cytokinesis require the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. In budding yeast, Cdc14 phosphatase is a key mitotic regulator that is activated in anaphase to counteract Cdk activity. In metaphase, Cdc14 is kept inactive in the nucleolus, where it is sequestered by its inhibitor, Net1. At anaphase onset, downregulation of PP2ACdc55 phosphatase by separase and Zds1 protein promotes Net1 phosphorylation and, consequently, Cdc14 release from the nucleolus. The mechanism by which PP2ACdc55 activity is downregulated during anaphase remains to be elucidated. Here, we demonstrate that Cdc55 regulatory subunit is phosphorylated in anaphase in a Cdk1-Clb2-dependent manner. Interestingly, cdc55-ED phosphomimetic mutant inactivates PP2ACdc55 phosphatase activity towards Net1 and promotes Cdc14 activation. Separase and Zds1 facilitate Cdk-dependent Net1 phosphorylation and Cdc14 release from the nucleolus by modulating PP2ACdc55 activity via Cdc55 phosphorylation. In addition, human Cdk1-CyclinB1 phosphorylates human B55, indicating that the mechanism is conserved in higher eukaryotes.

PP2A Functions during Mitosis and Cytokinesis in Yeasts.

Protein phosphorylation is a common mechanism for the regulation of cell cycle progression. The opposing functions of cell cycle kinases and phosphatases are crucial for accurate chromosome segregation and exit from mitosis. Protein phosphatases 2A are heterotrimeric complexes that play essential roles in cell growth, proliferation, and regulation of the cell cycle. Here, we review the function of the protein phosphatase 2A family as the counteracting force for the mitotic kinases. We focus on recent findings in the regulation of mitotic exit and cytokinesis by PP2A phosphatases in S. cerevisiae and other fungal species.

Dual Regulation of the mitotic exit network (MEN) by PP2A-Cdc55 phosphatase.

Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A(Cdc55) phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2A(Cdc55) phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2A(Cdc55) regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2A(Cdc55) initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2A(Cdc55) functions affect the regulation of various MEN components, contributing to mitotic exit.

De novo heterozygous mutations in SMC3 cause a range of Cornelia de Lange syndrome-overlapping phenotypes.

Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations, and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS-like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published. Here, we report the efforts of an international research and clinical collaboration to provide clinical comparison of 16 patients with CdLS-like features caused by mutations in SMC3. Modeling of the mutation effects on protein structure suggests a dominant-negative effect on the multimeric cohesin complex. When compared with typical CdLS, many SMC3-associated phenotypes are also characterized by postnatal microcephaly but with a less distinctive craniofacial appearance, a milder prenatal growth retardation that worsens in childhood, few congenital heart defects, and an absence of limb deficiencies. While most mutations are unique, two unrelated affected individuals shared the same mutation but presented with different phenotypes. This work confirms that de novo SMC3 mutations account for ∼ 1%-2% of CdLS-like phenotypes.

Zds1 regulates PP2A(Cdc55) activity and Cdc14 activation during mitotic exit through its Zds_C motif.

At anaphase onset, highly active mitotic cyclin-dependent kinase (Cdk) is inactivated to promote exit from mitosis and completion of cytokinesis. The budding yeast Cdc14p phosphatase is a key mitotic regulator that counteracts cyclin-dependent kinase (Cdk) activity during mitotic exit. Separase, together with Zds1p, promotes the downregulation of the protein phosphatase 2A in conjunction with its Cdc55p regulatory subunit (PP2A(Cdc55)) in early anaphase, enabling accumulation of phosphorylated forms of Net1p and release of Cdc14p from the nucleolus. Here we show that the C-terminal domain of Zds1p, called the Zds_C motif, is required for Zds1-induced release of Cdc14p, and the N-terminal domain of the protein might be involved in regulating this activity. More interestingly, Zds1p physically interacts with Cdc55p, and regulates its localization through the Zds_C motif. Nevertheless, expression of the Zds_C motif at endogenous levels cannot induce timely release of Cdc14p from the nucleolus, despite the proper (nucleolar) localization of Cdc55p. Our results suggest that the activity of PP2A(Cdc55) cannot be modulated solely through regulation of its localization, and that an additional regulatory step is probably required. These results suggest that Zds1p recruits PP2A(Cdc55) to the nucleolus and induces its inactivation by an unknown mechanism.

Disruption of NIPBL/Scc2 in Cornelia de Lange Syndrome provokes cohesin genome-wide redistribution with an impact in the transcriptome.

Cornelia de Lange syndrome (CdLS) is a rare disease affecting multiple organs and systems during development. Mutations in the cohesin loader, NIPBL/Scc2, were first described and are the most frequent in clinically diagnosed CdLS patients. The molecular mechanisms driving CdLS phenotypes are not understood. In addition to its canonical role in sister chromatid cohesion, cohesin is implicated in the spatial organization of the genome. Here, we investigate the transcriptome of CdLS patient-derived primary fibroblasts and observe the downregulation of genes involved in development and system skeletal organization, providing a link to the developmental alterations and limb abnormalities characteristic of CdLS patients. Genome-wide distribution studies demonstrate a global reduction of NIPBL at the NIPBL-associated high GC content regions in CdLS-derived cells. In addition, cohesin accumulates at NIPBL-occupied sites at CpG islands potentially due to reduced cohesin translocation along chromosomes, and fewer cohesin peaks colocalize with CTCF.

The regulation of Net1/Cdc14 by the Hog1 MAPK upon osmostress unravels a new mechanism regulating mitosis.

During evolution, cells have developed a plethora of mechanisms to optimize survival in a changing and unpredictable environment. In this regard, they have evolved networks that include environmental sensors, signaling transduction molecules and response mechanisms. Hog1 (yeast) and p38 (mammals) stress-activated protein kinases (SAPKs) are activated upon stress and they drive a full collection of cell adaptive responses aimed to maximize survival. SAPKs are extensively used to learn about the mechanisms through which cells adapt to changing environments. In addition to regulating gene expression and metabolism, SAPKs control cell cycle progression. In this review, we will discuss the latest findings related to the SAPK-driven regulation of mitosis upon osmostress in yeast.

SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast.

Background: Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. Results: We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. Conclusions: We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome.

Cell cycle activation of the Swi6p transcription factor is linked to nucleocytoplasmic shuttling.

The control of the subcellular localization of cell cycle regulators has emerged as a crucial mechanism in the regulation of cell division. In the present work, we have characterized the function of the karyopherin Msn5p in the control of the cell cycle of Saccharomyces cerevisiae. Phenotypic analysis of the msn5 mutant revealed an increase in cell size and a functional interaction between Msn5p and the cell cycle transcription factor SBF (composed of the Swi4p and Swi6p proteins), indicating that Msn5p is involved in Start control. In fact, we have shown that the level of Cln2p protein is drastically reduced in an msn5 mutant. The effect on CLN2 expression is mediated at a transcriptional level, Msn5p being necessary for proper SBF-dependent transcription. On the contrary, loss of MSN5 has no effect on the closely related transcription factor MBF (composed of the Mbp1p and Swi6p proteins). Regulation of SBF by Msn5p is exerted by control of the localization of the regulatory subunit Swi6p. Swi6p shuttles between the nucleus and the cytoplasm during the cell cycle, and we have found that Msn5p is required for Swi6p export from the nucleus during the G(2)-M phase. What is more important, we have demonstrated that export of Swi6p to the cytoplasm is required for SBF activity, providing evidence for a functional switch of Swi6p linked to its nucleocytoplasmic shuttling during the cell cycle.

Regulation of Mitotic Exit in Saccharomyces cerevisiae.

The Mitotic Exit Network (MEN) is an essential signaling pathway, closely related to the Hippo pathway in mammals, which promotes mitotic exit and initiates cytokinesis in the budding yeast Saccharomyces cerevisiae. Here, we summarize the current knowledge about the MEN components and their regulation.

A New Methodology for the Quantification of In Vivo Cdc14 Phosphatase Activity.

The phosphatase Cdc14 has a pivotal function in the mitotic exit of Saccharomyces cerevisiae. During interphase, Cdc14 remains inactive in the nucleolus bound to the inhibitor Net1. Cdc14 activation occurs in the metaphase to anaphase transition and it is promoted by at least two signaling pathways called FEAR (CdcFourteen Early Anaphase Release) and MEN (Mitotic Exit Network). These two pathways act in parallel and target the phosphorylation of Net1, thus decreasing Net1 affinity for Cdc14. The activity of Cdc14 can be used as a readout to assay functional interactions of different components of the mitotic exit signaling pathways.

Functional connection between the Clb5 cyclin, the protein kinase C pathway and the Swi4 transcription factor in Saccharomyces cerevisiae.

The rsf12 mutation was isolated in a synthetic lethal screen for genes functionally interacting with Swi4. RSF12 is CLB5. The clb5 swi4 mutant cells arrest at G(2)/M due to the activation of the DNA-damage checkpoint. Defects in DNA integrity was confirmed by the increased rates of chromosome loss and mitotic recombination. Other results suggest the presence of additional defects related to morphogenesis. Interestingly, genes of the PKC pathway rescue the growth defect of clb5 swi4, and pkc1 and slt2 mutations are synthetic lethal with clb5, pointing to a connection between Clb5, the PKC pathway, and Swi4. Different observations suggest that like Clb5, the PKC pathway and Swi4 are involved in the control of DNA integrity: there is a synthetic interaction between pkc1 and slt2 with rad9; the pkc1, slt2, and swi4 mutants are hypersensitive to hydroxyurea; and the Slt2 kinase is activated by hydroxyurea. Reciprocally, we found that clb5 mutant is hypersensitive to SDS, CFW, latrunculin B, or zymolyase, which suggests that, like the PKC pathway and Swi4, Clb5 is related to cell integrity. In summary, we report numerous genetic interactions and phenotypic descriptions supporting a close functional relationship between the Clb5 cyclin, the PKC pathway, and the Swi4 transcription factor.

Mitotic Exit Function of Polo-like Kinase Cdc5 Is Dependent on Sequential Activation by Cdk1.

To complete mitosis, Saccharomyces cerevisiae needs to activate the mitotic phosphatase Cdc14. Two pathways contribute to Cdc14 regulation: FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network). Cdc5 polo-like kinase was found to be an important mitotic exit component. However, its specific role in mitotic exit regulation and its involvement in Cdc14 release remain unclear. Here, we provide insight into the mechanism by which Cdc5 contributes to the timely release of Cdc14. Our genetic and biochemical data indicate that Cdc5 acts in parallel with MEN during anaphase. This MEN-independent Cdc5 function requires active separase and activation by Cdk1-dependent phosphorylation. Cdk1 first phosphorylates Cdc5 to activate it in early anaphase, and then, in late anaphase, further phosphorylation of Cdc5 by Cdk1 is needed to promote its MEN-related functions.

Functional distinction between Cln1p and Cln2p cyclins in the control of the Saccharomyces cerevisiae mitotic cycle.

Cln1p and Cln2p are considered as equivalent cyclins on the basis of sequence homology, regulation, and functional studies. Here we describe a functional distinction between the Cln1p and Cln2p cyclins in the control of the G1/S transition. Inactivation of CLN2, but not of CLN1, leads to a larger-than-normal cell size, whereas overexpression of CLN2, but not of CLN1, results in smaller-than-normal cells. Furthermore, mild ectopic expression of CLN2, but not of CLN1, suppresses the lethality of swi4swi6 and cdc28 mutant strains. In the absence of Cln1p, the kinetics of budding, initiation of DNA replication, and activation of the Start-transcription program are not affected; by contrast, loss of Cln2p causes a delay in bud emergence. A primary role for Cln2p but not for Cln1p in budding is reinforced by the observation that only the cln2 mutation is synthetic lethal with a cdc42 mutation, and only the cln2 mutant strain is hypersensitive to latrunculin B. In addition, we found that Cln1p showed a more prominent nuclear staining than Cln2p. Finally, chimeric proteins composed of Cln1p and Cln2p revealed that Cln2p integrity is required for its functional specificity.

Severe ipsilateral musculoskeletal involvement in a Cornelia de Lange patient with a novel NIPBL mutation.

Cornelia de Lange Syndrome (CdLS) is a congenital autosomal dominant (NIPBL, SMC3 and RAD21) or X-linked (SMC1A and HDAC8) disorder characterized by facial dysmorphism, pre and postnatal growth retardation, developmental delay and/or intellectual disability, and multiorgan involvement. Musculoskeletal malformations are usually bilateral and affect mainly the upper limbs; the range goes from brachyclinodactyly to severe reduction defects. Instead lower extremities are usually less and mildly involved. Here, we report on a 3-year-old Senegalese boy with typical craniofacial CdLS features, pre and postnatal growth retardation, atrial septal defect, developmental delay and right ipsilateral limb malformations, consistent with oligodactyly of the 3rd and 4th fingers, tibial agenesis and fibula hypoplasia. Exome sequencing and Sanger sequencing showed a novel missense mutation in NIPBL gene (c.6647A>G; p.(Tyr2216Cys)), which affects a conserved residue located within NIPBL HEAT repeat elements. Pyrosequencing analysis of NIPBL gene, disclosed similar levels of wild-type and mutated alleles in DNA and RNA samples from all tissues analyzed (oral mucosa epithelial cells, peripheral blood leukocytes and fibroblasts). These findings indicated the absence of somatic mosaicism, despite of the segmental asymmetry of the limbs, and confirmed biallelic expression for NIPBL transcripts, respectively. Additionally, conditions like Split-hand/foot malformation with long-bone deficiency secondary to duplication of BHLHA9 gene have been ruled out by the array-CGH and MLPA analysis. To our knowledge, this is the first CdLS patient described with major ipsilateral malformations of both the upper and lower extremities, that even though this finding could be due to a random event, expands the spectrum of limb reduction defects in CdLS.

Lack of the Glc7 phosphatase regulatory subunit Ypi1 activates the morphogenetic checkpoint.

Ypi1 is an essential regulator of the Saccharomyces cerevisiae Glc7 protein phosphatase. Although lack of Ypi1 results in a dramatic blockage in the G2/M cell cycle transition, with abnormally shaped large buds and short spindles, the molecular bases for this phenotype are still obscure. We report here that depletion of Ypi1 results in stabilization of the Pds1 securin, suggesting the activation of a G2/M checkpoint. Depletion of Ypi1 in cells deleted for MAD1/MAD2 or RAD9 still resulted in G2/M blockage, in spite that these cells lack key components of the spindle assembly and DNA damage checkpoints signaling, respectively. In contrast, deletion of SWE1, which encodes a protein kinase required for the morphogenesis checkpoint signaling, allowed passage through G2/M and recovery of normal cell morphology, although the cells did not proliferate. Depletion of Ypi1 caused stabilization of the Swe1 kinase, persistent phosphorylation of protein kinase Cdc28 at Y19, a landmark for morphogenesis checkpoint activation, and depletion of the Cdc11 septin, which explains the failure to form properly assembled septin rings at the bud necks. Deletion of SWE1 restored normal Cdc11 levels in the absence of Ypi1. These results demonstrate that Ypi1 plays an important role in the morphogenesis checkpoint, possibly by regulating Swe1.