Cdc2 Research Articles

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.

Sesquiterpenes with inhibitory activity against CDC25 phosphatases from the soft coral Pseudopterogorgia rigida

Twenty one sesquiterpenes were isolated from the organic extract of the Caribbean gorgonian Pseudopterogogia rigida. Among them, seven bisabolanes (1, 2, 9–11, 13 and 14), six cadinanes (15–20) and one sesquiterpene featuring a new tricyclic carbon skeleton (21) are new natural products, while two (6 and 7) are reported for the first time from a natural source. The structures of the new compounds were established by detailed analyses of their NMR and MS data. The isolated metabolites were evaluated for their inhibitory activity against CDC25 phosphatases, which represent possible targets related to cancer therapy. Compounds 4 and 18 were the most active with IC50 values of 1.88 and 3.44 μM, respectively, against the CDC25A isoform.

Differential Protein Expression in Small Intestinal Neuroendocrine Tumors and Liver Metastases.

Small intestinal neuroendocrine tumors (SI-NETs) are often detected after they have become metastatic. Using a novel protein array, we identified pathways important in SI-NET metastasis development in surgically resected patients. Paired primary tumors and liver metastases from 25 patients undergoing surgical resection for metastatic SI-NETs were harvested. Extracted proteins were separated by sodium dodecyl sulfate gel and multiplex immunoblots were performed with 136 antibodies. Significant Analysis of Microarray was used to select for differentially expressed proteins. A tissue microarray was constructed from 27 archived specimens and stained by immunohistochemistry. Comparing primary SI-NETs with matched normal small-bowel mucosa, 9 proteins were upregulated and cyclin E was downregulated. The SI-NET liver metastases demonstrated upregulation of P-ERK and p27 but downregulation of CDK2 and CDC25B. When comparing primary SI-NET with their paired liver metastases, cyclin E demonstrated a significant upregulation in the liver metastasis. Tissue microarray demonstrated higher p38 expression and lower Cdc 25b expression in SI-NETs versus liver metastases and confirmed higher expression of p27 in liver metastases versus normal liver. Few studies have compared protein expression in paired primary and metastatic SI-NETs. Our findings reveal changes in a limited number of proteins, suggesting that these may be targets for therapy.

Cdc20 mutation promotes development of colon cancer in APCmin/+ mice

Objective To demonstrate the relationship between Cdc20 mutation and the promotion of colon cancer via Cdc20loxp/+ APCmin/+ villin-cre+/- compound mutant mice. Methods Cdc20loxp/+ APCmin/+ villin-cre+/- compound mutant mice and APCmin/+ mutant mice were generated by mice mating strategy. The colon tumors of two group mice were compared by phenotypic analysis and histology analysis. Results Phenotypic analysis showed that the number of tumors in Cdc20loxp/+ APCmin/+ villin-cre+/- compound mutant mice group and APCmin/+ mutant mice group was 1.2±0.5 and 1.6±0.5, respectively (t=0.215, P=0.588), and the maximum diameter of tumors was (2.7±0.3) cm and (2.5±0.2) cm, respectively (t=0.568, P=0.575). Pathologic type of Cdc20loxp/+ APCmin/+ villin-cre+/- compound mutant mice was adenocarcinoma, while that of APCmin/+ mice was tubular adenoma. Conclusion Cdc20 carrying a null allele can accelerate the promotion of colon cancer in APCmin/+ mice without influence on the tumor number and size. Key words: Colon neoplasms; Mice, mutant strains; Gene, Cdc20; Gene, APC

Inhibitory effect of miR-204 on corneal epithelial cell proliferation in vitro

Background As the main cellular constituent of corneal epithelium, corneal epithelial cells play critical roles in regulating and controlling the migration, proliferation and differentiation of cells during the repair of damage. MicroRNA (miRNA) is endogenously expressed small non-coding RNAs, which participates in a variety of biological processes. Previous studies demonstrated that miR-204 is highly expressed in normal corneal epithelium, but its function is still unclear. Objective This study was to investigate the function and mechanism of miR-204 in corneal epithelial cell proliferation. Methods Corneal epithelial tissue was collected during the corneal refractive surgery from the patients with refractive error under the informed consent, and human corneal epithelial cells(HCECs)were cultured and passaged. The relative expressing levels of miR-204 mRNA in the normal corneal epithelium and HCECs were detected by real time quantitative PCR. Cultured HCECs were evenly divided into three groups. The liposome with miR-204 mimic was transfected into the cells of the miR-204 mimic group, and blank liposome was transfected in the cells of the positive control group, and regularly cultured cells served as the normal control group. Cell proliferation capability was evaluated by colony-forming assay, and the percentage of the cells in different cell cycles was analyzed by flow cytometry. Western blot assay was employed to detect the expression levels of p-RB, E2F1, p27, CyclinA, CDC2, p-CDC2 and CDK2 proteins in the cells. Results The relative expression levels of miR-204 mRNA were 1.077±0.268 in the normal corneal epithelium and 0.041±0.018 in the HCECs, showing a significant difference between them (t=7.700, P<0.001). The cloning cell number was evidently decreased in the miR-204 mimic group in comparison with the positive control group and normal control group. The percentage of cells in the G1 phase was 47.75% in the miR-204 mimic group, which was significantly higher than 37.23% in the positive control group and 40.72% in the normal control group. The expression levels of E2F1 and p27 proteins in the cells were elevated (t=14.87, 25.11; both at P<0.01) and those of CDK2 and p-CDC2 proteins were decreased (t=5.39, 10.65; both at P<0.01) in the miR-204 mimic group in comparison with the positive control group. Conclusions The overexpression of miR-204 in the normal corneas probably is associated with non-proliferation status of corneal epithelial cells. Transfection of miR-204 into corneal epithelial cells can inhibit the proliferation of corneal epithelial cells probably by up-regulating the expression of E2F1 and p27 and suppressing the expression of CDK2/CyclinA and p-CDC2/CyclinA, which lead to cell arrest in G1 phase. Key words: [Key words] Epithelial cells, corneal; MicroRNAs; Proliferation; Cyclins; Gene expression; Humans; miR-204

Flexibility vs. robustness in cell cycle regulation of timing of M-phase entry in Xenopus laevis embryo cell-free extract.

During the cell cycle, cyclin dependent kinase 1 (CDK1) and protein phosphatase 2A (PP2A) play major roles in the regulation of mitosis. CDK1 phosphorylates a series of substrates triggering M-phase entry. Most of these substrates are dephosphorylated by PP2A. To allow phosphorylation of CDK1 substrates, PP2A is progressively inactivated upon M-phase entry. We have shown previously that the interplay between these two activities determines the timing of M-phase entry. Slight diminution of CDK1 activity by the RO3306 inhibitor delays M-phase entry in a dose-dependent manner in Xenopus embryo cell-free extract, while reduction of PP2A activity by OA inhibitor accelerates this process also in a dose-dependent manner. However, when a mixture of RO3306 and OA is added to the extract, an intermediate timing of M-phase entry is observed. Here we use a mathematical model to describe and understand this interplay. Simulations showing acceleration and delay in M-phase entry match previously described experimental data. CDC25 phosphatase is a major activator of CDK1 and acts through CDK1 Tyr15 and Thr14 dephosphorylation. Addition of CDC25 activity to our mathematical model was also consistent with our experimental results. To verify whether our assumption that the dynamics of CDC25 activation used in this model are the same in all experimental variants, we analyzed the dynamics of CDC25 phosphorylation, which reflect its activation. We confirm that these dynamics are indeed very similar in control extracts and when RO3306 and OA are present separately. However, when RO3306 and OA are added simultaneously to the extract, activation of CDC25 is slightly delayed. Integration of this parameter allowed us to improve our model. Furthermore, the pattern of CDK1 dephosphorylation on Tyr15 showed that the real dynamics of CDK1 activation are very similar in all experimental variants. The model presented here accurately describes, in mathematical terms, how the interplay between CDK1, PP2A and CDC25 controls the flexible timing of M-phase entry.

Measuring Activity and Specificity of Protein Phosphatases.

Reversible protein phosphorylation plays essential roles in coordinating cell division and many other biological processes. Cell cycle regulation by opposing kinase and protein phosphatase activities is often complex and major challenges exist in identifying the direct substrates of these enzymes and the specific sites at which they act. While cell cycle kinases are known to exhibit strict substrate specificities important for coordinating the complex events of cell division, phosphatases have only recently been recognized to exert similarly precise regulatory control over cell cycle events through timely dephosphorylation of specific substrates. The molecular determinants for substrate recognition by many phosphatases that function in cell division are still poorly delineated. To understand phosphatase specificity, it is critical to employ methods that monitor the dephosphorylation of individual phosphorylation sites on physiologically relevant substrates. Here, using the cell cycle phosphatase Cdc14 as an example, we describe two methods for studying phosphatase specificity, one using synthetic phosphopeptide substrates and the other using intact phosphoprotein substrates. These methods are useful for targeted characterization of small substrate sets and are also adaptable to large-scale applications for global specificity studies.

Sulforaphane Induces Cell Death Through G2/M Phase Arrest and Triggers Apoptosis in HCT 116 Human Colon Cancer Cells.

Sulforaphane (SFN), an isothiocyanate, exists exclusively in cruciferous vegetables, and has been shown to possess potent antitumor and chemopreventive activity. However, there is no available information that shows SFN affecting human colon cancer HCT 116 cells. In the present study, we found that SFN induced cell morphological changes, which were photographed by contrast-phase microscopy, and decreased viability. SFN also induced G2/M phase arrest and cell apoptosis in HCT 116 cells, which were measured with flow cytometric assays. Western blotting indicated that SFN increased Cyclin A, cdk 2, Cyclin B and WEE1, but decreased Cdc 25C, cdk1 protein expressions that led to G2/M phase arrest. Apoptotic cell death was also confirmed by Annexin V/PI and DAPI staining and DNA gel electrophoresis in HCT 116 cells after exposure to SFN. The flow cytometric assay also showed that SFN induced the generation of reactive oxygen species (ROS) and Ca[Formula: see text] and decreased mitochondria membrane potential and increased caspase-8, -9 and -3 activities in HCT 116 cell. Western blotting also showed that SFN induced the release of cytochrome c, and AIF, which was confirmed by confocal microscopy examination. SFN induced ER stress-associated protein expression. Based on those observations, we suggest that SFN may be used as a novel anticancer agent for the treatment of human colon cancer in the future.

Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.

A novel approach to the expression and purification of recombinant Xenopus Cdc25C

The Cdc25 family encodes dual specificity protein phosphatases that play critical roles in cell cycle progression. Activation of the Cdc25C represents a primary driver for meiosis progression in Xenopus oocytes. Given its central role in meiosis the Xenopus Cdc25C has been studied extensively, however purification of the recombinant protein is difficult thus preventing better characterization of its function. Here we describe methods to overcome these difficulties resulting in the production of high purity and yield recombinant Xenopus Cdc25C. We use a synthetic Xenopus Cdc25C gene that was codon optimized for expression in E. coli. We further combine an N-terminal His-tag with a C-terminal Strep-tag II, to isolate extremely pure full-length Cdc25C protein. The recombinant Xenopus Cdc25C is active both in vitro using a phosphatase assay and in vivo when injected into Xenopus oocytes. This new approach should be applicable to the purification of other members of the Cdc25 gene family.

Vnd and its target gene twine are required for cell cycle progression during embryonic nervous system development in Drosophila melanogaster

Twine is a homolog of CDC25 phosphatase regulating cell cycle progression. To elucidate a possible role of twine during embryonic neurogenesis, its expression levels and patterns were determined. twine was strongly expressed in the embryonic brain, and segmentally expressed along the ventral nerve cord during stage 12–14 in which neuroblasts and ganglionic mother cells are rapidly dividing. To examine whether twine is involved in the nervous system development, immunohistochemistry using neuron-specific antibodies was performed. The results showed the reduction of dorsal clusters in the peripheral nervous system and disorganization of neurons in the central nervous system. Moreover, consistent with our microarray data, real-time PCR results show that expression level of twine is proportional to that of vnd, suggesting that Vnd functions as an activator to regulate twine expression. This relationship is also confirmed by analyzing the expression patterns of twine in vnd loss-of-function and gain-of-function mutant background. Immunostaining using anti-phospho-histone H3 and anti-BrdU antibodies revealed that Twine regulates cell cycle progression in the developing embryonic nervous system. Down-regulation of vnd also caused a significant reduction of mitotic cells in embryonic neuroblasts, suggesting a role for vnd-twine in mitosis-associated nervous system development.

Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displayingin vitroefficacy against melanoma cells

CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5-9, 21, 24, and 25) were identified with Ki values for CDC25A, -B and -C ranging from 0.01 to 4.4 μM. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds.

FgCDC14 regulates cytokinesis, morphogenesis, and pathogenesis in Fusarium graminearum.

Members of Cdc14 phosphatases are common in animals and fungi, but absent in plants. Although its orthologs are conserved in plant pathogenic fungi, their functions during infection are not clear. In this study, we showed that the CDC14 ortholog is important for pathogenesis and morphogenesis in Fusarium graminearum. FgCDC14 is required for normal cell division and septum formation and FgCdc14 possesses phosphatase activity with specificity for a subset of Cdk-type phosphorylation sites. The Fgcdc14 mutant was reduced in growth, conidiation, and ascospore formation. It was defective in ascosporogenesis and pathogenesis. Septation in Fgcdc14 was reduced and hyphal compartments contained multiple nuclei, indicating defects in the coordination between nuclear division and cytokinesis. Interestingly, foot cells of mutant conidia often differentiated into conidiogenous cells, resulting in the production of inter-connected conidia. In the interphase, FgCdc14-GFP localized to the nucleus and spindle-pole-body. Taken together, our results indicate that Cdc14 phosphatase functions in cell division and septum formation in F. graminearum, likely by counteracting Cdk phosphorylation, and is required for plant infection.

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

The location of proteins within eukaryotic cells is often critical for their function and relocation of proteins forms the mainstay of regulatory pathways. To assess the importance of protein location to cellular homeostasis, we have developed a methodology to systematically create binary physical interactions between a query protein and most other members of the proteome. This method allows us to rapidly assess which of the thousands of possible protein interactions modify a phenotype. As proof of principle we studied the kinetochore, a multiprotein assembly that links centromeres to the microtubules of the spindle during cell division. In budding yeast, the kinetochores from the 16 chromosomes cluster together to a single location within the nucleus. The many proteins that make up the kinetochore are regulated through ubiquitylation and phosphorylation. By systematically associating members of the proteome to the kinetochore, we determine which fusions affect its normal function. We identify a number of candidate kinetochore regulators, including the phosphatase Cdc14. We examine where within the kinetochore Cdc14 can act and show that the effect is limited to regions that correlate with known phosphorylation sites, demonstrating the importance of serine phospho-regulation for normal kinetochore homeostasis.

Abstract 4559: Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor

Abstract Although several nuclear regulatory proteins may be targeted by estrogen, our efforts have focused on the redox nuclear respiratory factor-1 (NRF-1), because our recent study showed that NRF-1 gene expression significantly increases with the progression of breast tumor grades. NRF-1 overexpression supported in vitro tumor formation. To determine if NRF-1 is required for 17 beta estradiol (E2)-induced neoplastic phenotype, we have generated NRF-1 mutants by site directed mutagenesis. We have generated stable clones that express NRF-1 mutant. The in vitro tumor formation was detected by anchorage independent growth and 3D spheroid assays. NRF-1 overexpression enhanced in vitro tumor spheroid formation, cell migration and cell invasion. Our flow cytometry analysis showed that overexpression of wild-type NRF-1 increased the percent of transformed MCF-10A cells in S phase compared to vector alone. Dominant negative NRF-1 protein showed reduction in the tumor formation. In addition, shRNA targeting NRF-1 resulted in the inhibition of anchorage independent growth of MCF-10A cells in both vehicle control and E2-treated cells. Since NRF-1 is a substrate of the kinase AKT, we determined whether NRF-1 phosphorylation was increased in MCF-10A cells after treatment with a carcinogenic regimen of E2. We observed more than a 2-fold increase in phospho-NRF-1 in E2 treated (100pg/ml for 30 min) MCF-10A cells and phosphorylation of NRF-1 was inhibited by co-treatment with either biological (CAT or MnSOD) or chemical (20μM ebselen) ROS modifiers. These results suggest that E2-induced phosphorylation of NRF-1 is influenced by ROS signaling messengers. We found that E2 treated MCF-10A cells showed increased NRF-1 binding to the promoters of Cdc2, PRC1, PCNA, Cyclin B1, and CDC25C genes. NRF-1 binding induced by E2 treatment was inhibited by the overexpression of CAT and MnSOD. NRF-1 phosphorylation site specific to kinase AKT, Thr-109, was mutated to Asp (D) or Ala (A), and the NRF-1 acetylation site specific to acetyl-transferase PCAF, Lys- 89, was mutated to Gln (glutamine)-Q or Ala (A). We examine the effect of one of the NRF-1 mutants on E2-induced in vitro tumor formation. We observed that the expression of NRF-1 (T109A) phosphodeficient mutant (NRF-1PMT) significantly inhibited E2-induced cell transformation, whereas empty vector did not influence E2-induced colony formation. Together these findings support the idea that NRF-1 may play an important role in E2-induced malignant transformation of breast epithelial cells. Thus, our data is consistent with the hypothesis that in addition to the estrogen receptor activity, NRF-1 activation contributes to the susceptibility to develop malignant phenotype in response to exposure to estrogen. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR Citation Format: Lazaro Mesa, Jayanta Das, Alok Deoraj, Victor Okoh, Deodutta Roy. Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4559. doi:10.1158/1538-7445.AM2015-4559

Cdc14 phosphatase: warning, no delay allowed for chromosome segregation!

Cycling events in nature start and end to restart again and again. In the cell cycle, whose purpose is to become two where there was only one, cyclin-dependent kinases (CDKs) are the beginning and, therefore, phosphatases must play a role in the ending. Since CDKs are drivers of the cell cycle and cancer cells uncontrollably divide, much attention has been put into knocking down CDK activity. However, much less is known on the consequences of interfering with the phosphatases that put an end to the cell cycle. We have addressed in recent years the consequences of transiently inactivating the only master cell cycle phosphatase in the model yeast Saccharomyces cerevisiae, Cdc14. Transient inactivation is expected to better mimic the pharmacological action of drugs. Interestingly, we have found that yeast cells tolerate badly a relatively brief inactivation of Cdc14 when cells are already committed into anaphase, the first cell cycle stage where this phosphatase plays important roles. First, we noticed that the segregation of distal regions in the chromosome arm that carries the ribosomal DNA array was irreversibly impaired, leading to an anaphase bridge (AB). Next, we found that this AB could eventually be severed by cytokinesis and led to two different types of genetically compromised daughter cells. All these previous studies were done in haploid cells. We have now recently expanded this analysis to diploid cells and used the advantage of making hybrid diploids to study chromosome rearrangements and changes in the ploidy of the surviving progeny. We have found that the consequences for the genome integrity were far more dramatic than originally envisioned.

Cdc14 Early Anaphase Release, FEAR, Is Limited to the Nucleus and Dispensable for Efficient Mitotic Exit.

Cdc14 phosphatase is a key regulator of exit from mitosis, acting primarily through antagonism of cyclin-dependent kinase, and is also thought to be important for meiosis. Cdc14 is released from its sequestration site in the nucleolus in two stages, first by the non-essential Cdc Fourteen Early Anaphase Release (FEAR) pathway and later by the essential Mitotic Exit Network (MEN), which drives efficient export of Cdc14 to the cytoplasm. We find that Cdc14 is confined to the nucleus during early mitotic anaphase release, and during its meiosis I release. Proteins whose degradation is directed by Cdc14 as a requirement for mitotic exit (e.g. the B-type cyclin, Clb2), remain stable during mitotic FEAR, a result consistent with Cdc14 being restricted to the nucleus and not participating directly in mitotic exit. Cdc14 released by the FEAR pathway has been proposed to have a wide variety of activities, all of which are thought to promote passage through anaphase. Proposed functions of FEAR include stabilization of anaphase spindles, resolution of the rDNA to allow its segregation, and priming of the MEN so that mitotic exit can occur promptly and efficiently. We tested the model for FEAR functions using the FEAR-deficient mutation net1-6cdk. Our cytological observations indicate that, contrary to the current model, FEAR is fully dispensable for timely progression through a series of anaphase landmarks and mitotic exit, although it is required for timely rDNA segregation. The net1-6cdk mutation suppresses temperature-sensitive mutations in MEN genes, suggesting that rather than activating mitotic exit, FEAR either inhibits the MEN or has no direct effect upon it. One interpretation of this result is that FEAR delays MEN activation to ensure that rDNA segregation occurs before mitotic exit. Our findings clarify the distinction between FEAR and MEN-dependent Cdc14 activities and will help guide emerging quantitative models of this cell cycle transition.

The Transient Inactivation of the Master Cell Cycle Phosphatase Cdc14 Causes Genomic Instability in Diploid Cells of Saccharomyces cerevisiae.

Genomic instability is a common feature found in cancer cells . Accordingly, many tumor suppressor genes identified in familiar cancer syndromes are involved in the maintenance of the stability of the genome during every cell division and are commonly referred to as caretakers. Inactivating mutations and epigenetic silencing of caretakers are thought to be the most important mechanisms that explain cancer-related genome instability. However, little is known of whether transient inactivation of caretaker proteins could trigger genome instability and, if so, what types of instability would occur. In this work, we show that a brief and reversible inactivation, during just one cell cycle, of the key phosphatase Cdc14 in the model organism Saccharomyces cerevisiae is enough to result in diploid cells with multiple gross chromosomal rearrangements and changes in ploidy. Interestingly, we observed that such transient loss yields a characteristic fingerprint whereby trisomies are often found in small-sized chromosomes, and gross chromosome rearrangements, often associated with concomitant loss of heterozygosity, are detected mainly on the ribosomal DNA-bearing chromosome XII. Taking into account the key role of Cdc14 in preventing anaphase bridges, resetting replication origins, and controlling spindle dynamics in a well-defined window within anaphase, we speculate that the transient loss of Cdc14 activity causes cells to go through a single mitotic catastrophe with irreversible consequences for the genome stability of the progeny.

A role for the budding yeast separase, Esp1, in Ty1 element retrotransposition.

Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast esp1-1 separase mutation. We identified 161 genes that when mutated aggravate esp1-1 growth and 44 genes that upon increased dosage are detrimental to esp1-1 viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the esp1-1 genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the SUF16 tRNA gene are both reduced in an esp1-1 strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition – one to remove cohesin and the second to target Ty1-IN to chromatin.

LATS1 and LATS2 phosphorylate CDC26 to modulate assembly of the tetratricopeptide repeat subcomplex of APC/C.

In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C.