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1.
Eur J Hum Genet ; 30(11): 1288-1291, 2022 11.
Article in English | MEDLINE | ID: mdl-35459888

ABSTRACT

CTCF germline mutations have been related to MRD21. We report the first bilateral Wilms tumor suffered by a MRD21 patient carrying an unreported CTCF missense variant in a zinc finger domain of CTCF protein. We found that germline heterozygous variant I446K became homozygous in the tumor due to a loss of heterozygosity rearrangement affecting the whole q arm on chromosome 16. Our findings propose CTCF I446K variant as a link between MRD21 and Wilms tumor predisposition.


Subject(s)
Intellectual Disability , Kidney Neoplasms , Wilms Tumor , Humans , Wilms Tumor/genetics , Zinc Fingers/genetics , Kidney Neoplasms/genetics , Germ Cells
2.
Cancers (Basel) ; 14(8)2022 Apr 14.
Article in English | MEDLINE | ID: mdl-35454892

ABSTRACT

A suitable diagnostic classification of myeloid neoplasms and acute leukemias requires testing for a large number of molecular biomarkers. Next-generation sequencing is a technology able to integrate identification of the vast majority of them in a single test. This manuscript includes the design, analytical validation and clinical feasibility evaluation of a molecular diagnostic kit for onco-hematological diseases. It is based on sequencing of the coding regions of 76 genes (seeking single-nucleotide variants, small insertions or deletions and CNVs), as well as the search for fusions in 27 target genes. The kit has also been designed to detect large CNVs throughout the genome by including specific probes and employing a custom bioinformatics approach. The analytical and clinical feasibility validation of the Haematology OncoKitDx panel has been carried out from the sequencing of 170 patient samples from 6 hospitals (in addition to the use of commercial reference samples). The analytical validation showed sensitivity and specificity close to 100% for all the parameters evaluated, with a detection limit of 2% for SNVs and SVs, and 20% for CNVs. Clinically relevant mutations were detected in 94% of all patients. An analysis of the correlation between the genetic risk classification of AML (according to ELN 2017) established by the hospitals and that obtained by the Haematology OncoKitDx panel showed an almost perfect correlation (K = 0.94). Among the AML samples with a molecular diagnosis, established by the centers according to the WHO, the Haematology OncoKitDx analysis showed the same result in 97% of them. The panel was able to adequately differentiate between MPN subtypes and also detected alterations that modified the diagnosis (FIP1L1-PDGFRA). Likewise, the cytogenetic risk derived from the CNV plot generated by the NGS panel correlated substantially with the results of the conventional karyotype (K = 0.71) among MDS samples. In addition, the panel detected the main biomarkers of prognostic value among patients with ALL. This validated solution enables a reliable analysis of a large number of molecular biomarkers from a DNA sample in a single assay.

3.
Mod Pathol ; 35(8): 1066-1074, 2022 08.
Article in English | MEDLINE | ID: mdl-35177782

ABSTRACT

Metaplastic breast carcinomas are a rare and heterogeneous group of tumors (0.5-2%). They are mainly triple negative tumors but they present poorer chemotherapy responses and worse prognosis than other triple negative tumors. The aim of our study was to characterize the molecular profile and tumor evolution in matched (primary-relapse) tumor samples from patients with early-stage metaplastic breast carcinomas who had disease recurrence/progression. We performed genomic profiling of tumor biopsies at least from two different time points of their tumor evolution. Tumor samples were analyzed by DNA-Next Generation Sequencing (Illumina 2 x 75bp) using the Action OncoKitDX panel (Imegen-Health in Code group), which includes point mutations in 50 genes, CNVs, and fusion genes. Only pathogenic and likely pathogenic variants were considered for analysis and they were categorized following the ComPerMed criteria. We analyzed 21 matched tumor samples (8 primary and 13 relapse/progression samples). Genomic profiling of matched tumor samples revealed that mutations present in primary tumors are generally maintained in the relapse/disease progression. We did not find a significant increase in point mutations between primary and relapse/progression samples, although gene amplifications were found more frequently in relapse/progression samples. Tumor samples harbored high frequency of TP53 (100%) and TERT promoter (29%) mutations, and of MYC amplifications (80% of which in relapse/progression samples). No PI3KCA mutations were found, but PTEN variations were enriched in 38% of samples (10% mutations and 28% deletions). FGFR1 amplifications were identified in 13% of samples (primary tumor only). Neither ERBB2 nor EGFR gene amplifications were detected. The most frequent pathogenic alterations occurred in cycle regulation's genes, including TP53 and TERT promoter mutations, and MYC amplifications. Relapse/progression samples were highly enriched for MYC amplification. Larger studies are required to better characterize these tumors, and identify new strategies to improve the prognosis of these patients.


Subject(s)
Breast Neoplasms , Neoplasm Recurrence, Local , Biomarkers, Tumor/genetics , Breast Neoplasms/pathology , Female , Gene Amplification , Genomics , High-Throughput Nucleotide Sequencing , Humans , Mutation , Neoplasm Recurrence, Local/genetics
4.
Cureus ; 13(11): e19528, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34934548

ABSTRACT

The most frequently diagnosed neoplasia in the world in 2020 was breast cancer (BC). On top of its high incidence, unexpected behavior as recurrence in patients, in spite of appropriate therapies, reaches 20%-30%. We believe that some molecular characteristics of tumors may lead to this bad behavior, and we can identify them with next-generation sequencing (NGS). We made a retrospective multicentric study, conducted to molecularly characterize, by means of a custom NGS panel, cases diagnosed with treatment-refractory or treatment-resistant invasive breast carcinoma, studied in formalin-fixed paraffin-embedded (FFPE) samples. The panel included 50 genes related to tumorigenesis, cancer evolution and targeted therapies. Twelve cases were included from three centers. Alterations of driver genes were found in all of the cases, and 75% harbored mutations in TP53. Furthermore, we found alterations that could be therapeutic targets in half of the patients, such as mutations in PIK3CA (33% cases), mTOR (8.3%) or BRCA1 (8.3%). Other significant molecular alterations were: the loss of SWI-SNF complex´s components, modified genes of the MAP kinase pathway and alterations in epidermal growth factor receptor (EGFR). Not all of them are known targets but prognostic significance was found. We conclude that NGS characterization of breast cancer in FFPE samples is a reproducible technique that can provide prognostic and predictive information about our patients and therefore, constitutes, in the near future, a valuable clinical tool in the context of precision medicine.

5.
Cancers (Basel) ; 13(21)2021 Oct 24.
Article in English | MEDLINE | ID: mdl-34771502

ABSTRACT

Knowledge about genetic predisposition to pediatric cancer is constantly expanding. The categorization and clinical management of the best-known syndromes has been refined over the years. Meanwhile, new genes for pediatric cancer susceptibility are discovered every year. Our current work shares the results of genetically studying the germline of 170 pediatric patients diagnosed with cancer. Patients were prospectively recruited and studied using a custom panel, OncoNano V2. The well-categorized predisposing syndromes incidence was 9.4%. Likely pathogenic variants for predisposition to the patient's tumor were identified in an additional 5.9% of cases. Additionally, a high number of pathogenic variants associated with recessive diseases was detected, which required family genetic counseling as well. The clinical utility of the Jongmans MC tool was evaluated, showing a high sensitivity for detecting the best-known predisposing syndromes. Our study confirms that the Jongmans MC tool is appropriate for a rapid assessment of patients; however, the updated version of Ripperger T criteria would be more accurate. Meaningfully, based on our findings, up to 9.4% of patients would present genetic alterations predisposing to cancer. Notably, up to 20% of all patients carry germline pathogenic or likely pathogenic variants in genes related to cancer and, thereby, they also require expert genetic counseling. The most important consideration is that the detection rate of genetic causality outside Jongmans MC et al. criteria was very low.

6.
Int J Retina Vitreous ; 7(1): 50, 2021 Sep 03.
Article in English | MEDLINE | ID: mdl-34479642

ABSTRACT

BACKGROUND: Patients with 13q-syndrome are at risk of retinoblastoma when the RB1 gene, located in the chromosomal band 13q14.2, is deleted. This syndrome is frequently associated with congenital malformations and developmental delay, although these signs could be mild. Mosaic 13q-deletion patients have been previously reported in the literature; their phenotype is variable, and they may not be recognized. CASE PRESENTATION: Retinoblastoma diagnosed in a child with 13q-mosaicism confirmed in blood, oral mucosa, healthy retina and retinoblastoma. A second RB1 hit is present exclusively in the retinoblastoma sample (RB1 c.958C>T p.Arg320Ter). Other detected molecular events in retinoblastoma are 6p12.3pter gain and 6q25.3qter loss. Clinical examination is unremarkable except for clinodactyly of the right fifth finger. DISCUSSION AND CONCLUSIONS: We describe a case of mosaic 13q deletion syndrome affected by retinoblastoma. Molecular data obtained from the tumor analysis are similar to previous data available about this malignancy. High clinical suspicion is essential for an adequate diagnosis of mosaic cases.

7.
J Pers Med ; 11(5)2021 Apr 29.
Article in English | MEDLINE | ID: mdl-33947144

ABSTRACT

The increasing identification of driver oncogenic alterations and progress of targeted therapies addresses the need of comprehensive alternatives to standard molecular methods. The translation into clinical practice of next-generation sequencing (NGS) panels is actually challenged by the compliance of high quality standards for clinical accreditation. Herein, we present the analytical and clinical feasibility study of a hybridization capture-based NGS panel (Action OncoKitDx) for the analysis of somatic mutations, copy number variants (CNVs), fusions, pharmacogenetic SNPs and Microsatellite Instability (MSI) determination in formalin-fixed paraffin-embedded (FFPE) tumor samples. A total of 64 samples were submitted to extensive analytical validation for the identification of previously known variants. An additional set of 166 tumor and patient-matched normal samples were sequenced to assess the clinical utility of the assay across different tumor types. The panel demonstrated good specificity, sensitivity, reproducibility, and repeatability for the identification of all biomarkers analyzed and the 5% limit of detection set was validated. Among the clinical cohorts, the assay revealed pathogenic genomic alterations in 97% of patient cases, and in 82.7%, at least one clinically relevant variant was detected. The validation of accuracy and robustness of this assay supports the Action OncoKitDx's utility in adult solid tumors.

8.
Oncoimmunology ; 9(1): 1824642, 2020 10 01.
Article in English | MEDLINE | ID: mdl-33101773

ABSTRACT

Cancer-Testis antigens (CTA) are named after the tissues where they are mainly expressed: in germinal and in cancer cells, a process that mimics many gametogenesis features. Mapping accurately the CTA gene expression signature in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) is a prerequisite for downstream immune target-discovery projects. In this study, we take advantage of the use of azacitidine to treat high-risk MDS and CMML to draw the CTAs landscape, before and after treatment, using an ad hoc targeted RNA sequencing (RNA-seq) design for this group of low transcript genes. In 19 patients, 196 CTAs were detected at baseline. Azacitidine did not change the number of CTAs expressed, but it significantly increased or decreased expression in nine and five CTAs, respectively. TFDP3 and DDX53, emerged as the main candidates for immunotherapeutic targeting, as they showed three main features: i) a significant derepression on day +28 of cycle one in those patients who achieved complete remission with hypomethylating treatment (FC = 6, p = .008; FC = 2.1, p = .008, respectively), ii) similar dynamics at the protein level to what was observed at the RNA layer, and iii) to elicit significant specific cytotoxic immune responses detected by TFDP3 and DDX53 HLA-A*0201 tetramers. Our study addresses the unmet landscape of CTAs expression in MDS and CMML and revealed a previously unrecognized TFDP3 and DDX53 reactivation, detectable in plasma and able to elicit a specific immune response after one cycle of azacitidine.


Subject(s)
Myelodysplastic Syndromes , Neoplasms , Antimetabolites, Antineoplastic/therapeutic use , Azacitidine/therapeutic use , Humans , Male , Myelodysplastic Syndromes/drug therapy , Sequence Analysis, RNA , Testis , Transcription Factor DP1
9.
Cell Mol Life Sci ; 76(18): 3601-3620, 2019 Sep.
Article in English | MEDLINE | ID: mdl-30927017

ABSTRACT

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.


Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Nuclear Proteins/metabolism , Protein Phosphatase 2/metabolism , Protein Tyrosine Phosphatases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Anaphase , CDC28 Protein Kinase, S cerevisiae/metabolism , Cell Nucleus/metabolism , Chromatography, High Pressure Liquid , Humans , Mitosis , Phosphopeptides/analysis , Phosphorylation , Separase/metabolism , Tandem Mass Spectrometry
10.
Neonatology ; 115(1): 68-76, 2019.
Article in English | MEDLINE | ID: mdl-30304723

ABSTRACT

BACKGROUND: Variable responses to hypothermic neuroprotection are related to the clinical heterogeneity of encephalopathic babies; hence better disease stratification may facilitate the development of individualized neuroprotective therapies. OBJECTIVES: We examined if whole blood gene expression analysis can identify specific transcriptome profiles in neonatal encephalopathy. MATERIAL AND METHODS: We performed next-generation sequencing on whole blood RNA from 12 babies with neonatal encephalopathy and 6 time-matched healthy term babies. Genes significantly differentially expressed between encephalopathic and control babies were identified. This set of genes was then compared to the host RNA response in septic neonates and subjected to pathway analysis. RESULTS: We identified 950 statistically significant genes discriminating perfectly between healthy controls and neonatal encephalopathy. The major pathways in neonatal encephalopathy were axonal guidance signaling (p = 0.0009), granulocyte adhesion and diapedesis (p = 0.003), IL-12 signaling and production in macrophages (p = 0.003), and hypoxia-inducible factor 1α signaling (p = 0.004). There were only 137 genes in common between neonatal encephalopathy and bacterial sepsis sets. CONCLUSION: Babies with neonatal encephalopathy have striking differences in gene expression profiles compared with healthy control and septic babies. Gene expression profiles may be useful for disease stratification and for developing personalized neuroprotective therapies.


Subject(s)
Bacterial Infections/genetics , Hypoxia-Ischemia, Brain/genetics , Sepsis/genetics , Transcriptome , Bacterial Infections/diagnosis , Case-Control Studies , High-Throughput Nucleotide Sequencing , Humans , Hypoxia-Ischemia, Brain/diagnosis , Infant, Newborn , Infant, Newborn, Diseases/diagnosis , Infant, Newborn, Diseases/genetics , Sepsis/diagnosis , Signal Transduction
11.
PLoS One ; 13(12): e0207296, 2018.
Article in English | MEDLINE | ID: mdl-30517146

ABSTRACT

Nemaline Myopathy (NM) is a rare genetic disorder that encompasses a large spectrum of myopathies characterized by hypotonia and generalized muscle weakness. To date, mutations in thirteen different genes have been associated with NM. The most frequently responsible genes are NEB (50% of cases) and ACTA1 (15-25% of cases). In this report all known NM related genes were screened by Next Generation Sequencing in five Spanish patients in order to genetically confirm the clinical and histological diagnosis of NM. Four mutations in NEB (c.17779_17780delTA, c.11086A>C, c.21076C>T and c.2310+5G>A) and one mutation in ACTA1 (c.871A>T) were found in four patients. Three of the four mutations in NEB were novel. A cDNA sequencing assay of the novel variants c.17779_17780delTA, c.11086A>C and c.2310+5G>A revealed that the intronic variant c.2310+5G>A affected the splicing process. Mutations reported here could help clinicians and geneticists in NM diagnosis.


Subject(s)
Actins/genetics , Muscle Proteins/genetics , Myopathies, Nemaline/genetics , Actins/physiology , Adult , Alleles , Child , Female , Gene Frequency/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Male , Muscle Hypotonia/genetics , Muscle Proteins/physiology , Muscle Weakness/genetics , Muscle, Skeletal , Mutation , Pedigree , RNA Splicing/genetics , Spain
12.
Gigascience ; 7(5)2018 05 01.
Article in English | MEDLINE | ID: mdl-29688323

ABSTRACT

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.


Subject(s)
Cell Cycle Proteins/metabolism , Isotope Labeling/methods , Phosphoproteins/metabolism , Protein Phosphatase 2/metabolism , Proteomics/methods , Saccharomyces cerevisiae Proteins/metabolism , Saccharomycetales/metabolism , Amino Acid Sequence , Cell Cycle Proteins/chemistry , Cytokinesis , Endocytosis , Gene Ontology , Metaphase , Molecular Docking Simulation , Phosphorylation , Protein Binding , Protein Interaction Maps , Protein Phosphatase 2/chemistry , Proteome/metabolism , Reproducibility of Results , Saccharomyces cerevisiae Proteins/chemistry , Substrate Specificity
13.
Oxid Med Cell Longev ; 2017: 9175806, 2017.
Article in English | MEDLINE | ID: mdl-28808499

ABSTRACT

Aging is a multifactorial process characterized by the progressive loss of physiological functions, leading to an increased vulnerability to age-associated diseases and finally to death. Several theories have been proposed to explain the nature of aging. One of the most known identifies the free radicals produced by the mitochondrial metabolism as the cause of cellular and DNA damage. However, there are also several evidences supporting that epigenetic modifications, such as DNA methylation, noncoding RNAs, and histone modifications, play a critical role in the molecular mechanism of aging. In this review, we explore the significance of these findings and argue how the interlinked effects of oxidative stress and epigenetics can explain the cause of age-related declines.


Subject(s)
Aging , Epigenesis, Genetic , Oxidative Stress , Animals , DNA Damage , Histones/metabolism , Humans , Mitochondria/metabolism , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , RNA, Untranslated/metabolism
14.
An. pediatr. (2003. Ed. impr.) ; 85(5): e1-e7, nov. 2016. tab
Article in Spanish | IBECS | ID: ibc-157739

ABSTRACT

El concepto de medicina de precisión ha cobrado especial relevancia en los últimos tiempos debido a la creciente necesidad de desarrollar estrategias personalizadas para el diagnóstico, el tratamiento y el seguimiento de diversas enfermedades de origen genético. La medicina de precisión en Oncología, a través de la integración de los datos clínicos, anatomopatológicos y moleculares, permite obtener un conocimiento más profundo del perfil biológico tumoral de cada paciente. En este contexto ha sido fundamental la implementación de las nuevas tecnologías de secuenciación next generation sequencing (NGS) en la práctica clínica. Existe un gran abanico de técnicas de secuenciación NGS que pueden ser utilizadas en función de la aplicación que se les quiera dar. La correcta interpretación de los cambios moleculares detectados mediante estas técnicas es clave para su adecuado uso en la práctica clínica. Esta revisión tiene como objetivo repasar las diferentes tecnologías de secuenciación que se utilizan actualmente en medicina de precisión para mejorar el diagnóstico, el pronóstico y el tratamiento de pacientes oncológicos


Precision medicine is an emerging approach for the diagnosis, treatment and prognosis of genetic diseases that enables clinicians to more accurately predict which treatment strategy will be optimal in a patient. The aim of Precision Medicine in Oncology is to integrate clinical, histological, and molecular data in order to obtain a deeper knowledge about the biology and genetics of an individual's tumour. Over the last few years, the implementation of new NGS (Next Generation Sequencing) technologies into clinical practice has been essential. There is a wide variety of NGS techniques that can be used in this context. The correct interpretation of molecular changes detected by these techniques is paramount for their appropriate use. In this review, a discussion is presented on the main NGS sequencing technologies that can be used to improve the diagnosis, prognosis, and treatment of oncology patients


Subject(s)
Humans , Child , High-Throughput Nucleotide Sequencing/trends , Precision Medicine/trends , Neoplasms/genetics , Genomics/trends , Sequence Analysis/methods
15.
An Pediatr (Barc) ; 85(5): 273.e1-273.e7, 2016 Nov.
Article in Spanish | MEDLINE | ID: mdl-27349697

ABSTRACT

Precision Medicine is an emerging approach for the diagnosis, treatment and prognosis of genetic diseases that enables clinicians to more accurately predict which treatment strategy will be optimal in a patient. The aim of Precision Medicine in Oncology is to integrate clinical, histological, and molecular data in order to obtain a deeper knowledge about the biology and genetics of an individual's tumour. Over the last few years, the implementation of new NGS (Next Generation Sequencing) technologies into clinical practice has been essential. There is a wide variety of NGS techniques that can be used in this context. The correct interpretation of molecular changes detected by these techniques is paramount for their appropriate use. In this review, a discussion is presented on the main NGS sequencing technologies that can be used to improve the diagnosis, prognosis, and treatment of oncology patients.


Subject(s)
High-Throughput Nucleotide Sequencing , Neoplasms/genetics , Precision Medicine/methods , Child , Humans , Neoplasms/drug therapy
16.
PLoS Genet ; 9(12): e1003966, 2013.
Article in English | MEDLINE | ID: mdl-24339788

ABSTRACT

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.


Subject(s)
Anaphase/genetics , Cell Cycle Proteins/genetics , Mitosis/genetics , Protein Phosphatase 2/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cytoskeletal Proteins/antagonists & inhibitors , Gene Expression Regulation, Fungal , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , RNA, Small Interfering , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/metabolism , Separase/genetics
17.
J Cell Sci ; 125(Pt 12): 2875-84, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22427694

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Mitosis , Protein Phosphatase 2/metabolism , Protein Tyrosine Phosphatases/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Motifs , Cell Cycle Proteins/genetics , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Gene Expression Regulation, Fungal , Phosphorylation , Protein Binding , Protein Phosphatase 2/genetics , Protein Tyrosine Phosphatases/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
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