Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant

1994 ◽  
Vol 14 (8) ◽  
pp. 5501-5509
Author(s):  
D Finley ◽  
S Sadis ◽  
B P Monia ◽  
P Boucher ◽  
D J Ecker ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Turnover ◽  
Cell Cycle Progression ◽  
Critical Role ◽  
Sole Source ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Polyubiquitin Gene

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.

scholarly journals Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.

1994 ◽  
Vol 14 (8) ◽  
pp. 5501-5509 ◽  
Author(s):  
D Finley ◽  
S Sadis ◽  
B P Monia ◽  
P Boucher ◽  
D J Ecker ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Turnover ◽  
Cell Cycle Progression ◽  
Critical Role ◽  
Sole Source ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Polyubiquitin Gene

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

scholarly journals The Diagnostic Journey of a Patient with Prader–Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 875
Author(s):  
Karlijn Pellikaan ◽  
Geeske M. van Woerden ◽  
Lotte Kleinendorst ◽  
Anna G. W. Rosenberg ◽  
Bernhard Horsthemke ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Single Nucleotide Polymorphism Array ◽  
Genetic Defect ◽  
Missense Variant ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Pituitary Hormone ◽  
Negative Effect

Prader–Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader–Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the ‘Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations’ (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype–phenotype relationship in PWS.

scholarly journals Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development

2007 ◽  
Vol 176 (5) ◽  
pp. 709-718 ◽  
Author(s):  
Chunxi Ge ◽  
Guozhi Xiao ◽  
Di Jiang ◽  
Renny T. Franceschi
Keyword(s):  
Transcriptional Activity ◽  
Mapk Pathway ◽  
Skeletal Development ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

scholarly journals c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in αT3-1 Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 839-849 ◽  
Author(s):  
Buffy S. Ellsworth ◽  
Brett R. White ◽  
Ann T. Burns ◽  
Brian D. Cherrington ◽  
Annette M. Otis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cell Model ◽  
Receptor Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Activator Protein

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in αT3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH3). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in αT3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.

scholarly journals ID: 1023 All-trans retinoic acid targets gastric cancer stem cells and inhibits patient-derived gastric carcinoma tumor growth

2017 ◽  
Vol 4 (S) ◽  
pp. 98
Author(s):  
P H Nguyen ◽  
J Giraud ◽  
C Staedel ◽  
L Chambonnier ◽  
P Dubus ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Gastric Carcinoma ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
3D Culture ◽  
All Trans Retinoic Acid

Gastric carcinoma is the third leading cause of cancer-related death worldwide. This cancer, most of the time metastatic, is essentially treated by surgery associated with conventional chemotherapy, and has a poor prognosis. The existence of cancer stem cells (CSC) expressing CD44 and a high aldehyde dehydrogenase (ALDH) activity has recently been demonstrated in gastric carcinoma and has opened new perspectives to develop targeted therapy. In this study, we evaluated the effects of all-transretinoic acid (ATRA) on CSCs in human gastric carcinoma. ATRA effects were evaluated on the proliferation and tumorigenic properties of gastric carcinoma cells from patient-derived tumors and cell lines in conventional 2D cultures, in 3D culture systems (tumorsphere assay) and in mouse xenograft models. ATRA inhibited both tumorspheres initiation and growth in vitro, which was associated with a cell-cycle arrest through the upregulation of cyclin-dependent kinase (CDK) inhibitors and the downregulation of cell-cycle progression activators. More importantly, ATRA downregulated the expression of the CSC markers CD44 and ALDH as well as stemness genes such as Klf4 and Sox2 and induced differentiation of tumorspheres. Finally, 2 weeks of daily ATRA treatment were sufficient to inhibit gastric tumor progression in vivo, which was associated with a decrease in CD44, ALDH1, Ki67 and PCNA expression in the remaining tumor cells. Administration of ATRA appears to be a potent strategy to efficiently inhibit tumor growth and more importantly to target gastric CSCs in both intestinal and diffuse types of gastric carcinoma.

scholarly journals Cernunnos influences human immunoglobulin class switch recombination and may be associated with B cell lymphomagenesis

2012 ◽  
Vol 209 (2) ◽  
pp. 291-305 ◽  
Author(s):  
Likun Du ◽  
Roujun Peng ◽  
Andrea Björkman ◽  
Noel Filipe de Miranda ◽  
Cornelia Rosner ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Class Switch Recombination ◽  
B Cell Lymphoma ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
End Joining ◽  
Class Switch

Cernunnos is involved in the nonhomologous end-joining (NHEJ) process during DNA double-strand break (DSB) repair. Here, we studied immunoglobulin (Ig) class switch recombination (CSR), a physiological process which relies on proper repair of the DSBs, in B cells from Cernunnos-deficient patients. The pattern of in vivo generated CSR junctions is altered in these cells, with unusually long microhomologies and a lack of direct end-joining. The CSR junctions from Cernunnos-deficient patients largely resemble those from patients lacking DNA ligase IV, Artemis, or ATM, suggesting that these factors are involved in the same end-joining pathway during CSR. By screening 269 mature B cell lymphoma biopsies, we also identified a somatic missense Cernunnos mutation in a diffuse large B cell lymphoma sample. This mutation has a dominant-negative effect on joining of a subset of DNA ends in an in vitro NHEJ assay. Translocations involving both Ig heavy chain loci and clonal-like, dynamic IgA switching activities were observed in this tumor. Collectively, our results suggest a link between defects in the Cernunnos-dependent NHEJ pathway and aberrant CSR or switch translocations during the development of B cell malignancies.

scholarly journals Epigenetic silencing of CDKN1A and CDKN2B by SNHG1 promotes the cell cycle, migration and epithelial-mesenchymal transition progression of hepatocellular carcinoma

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Bei Li ◽  
Ang Li ◽  
Zhen You ◽  
Jingchang Xu ◽  
Sha Zhu
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Epithelial Mesenchymal Transition ◽  
Critical Role ◽  
Luciferase Assay ◽  
Bioinformatics Analyses ◽  
Mesenchymal Transition ◽  
Rna Immunoprecipitation

Abstract Enhanced SNHG1 (small nucleolar RNA host gene 1) expression has been found to play a critical role in the initiation and progression of hepatocellular carcinoma (HCC) with its detailed mechanism largely unknown. In this study, we show that SNHG1 promotes the HCC progression through epigenetically silencing CDKN1A and CDKN2B in the nucleus, and competing with CDK4 mRNA for binding miR-140-5p in the cytoplasm. Using bioinformatics analyses, we found hepatocarcinogenesis is particularly associated with dysregulated expression of SNHG1 and activation of the cell cycle pathway. SNHG1 was upregulated in HCC tissues and cells, and its knockdown significantly inhibited HCC cell cycle, growth, metastasis, and epithelial–mesenchymal transition (EMT) both in vitro and in vivo. Chromatin immunoprecipitation and RNA immunoprecipitation assays demonstrate that SNHG1 inhibit the transcription of CDKN1A and CDKN2B through enhancing EZH2 mediated-H3K27me3 in the promoter of CDKN1A and CDKN2B, thus resulting in the de-repression of the cell cycle. Dual-luciferase assay and RNA pulldown revealed that SNHG1 promotes the expression of CDK4 by competitively binding to miR-140-5p. In conclusion, we propose that SNHG1 formed a regulatory network to confer an oncogenic function in HCC and SNHG1 may serve as a potential target for HCC diagnosis and treatment.

scholarly journals CDK4/6 inhibitors: a novel strategy for tumor radiosensitization

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Yilan Yang ◽  
Jurui Luo ◽  
Xingxing Chen ◽  
Zhaozhi Yang ◽  
Xin Mei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Clinical Studies ◽  
Cell Cycle Progression ◽  
Combined Therapy ◽  
Small Sample ◽  
Combination Strategies ◽  
Novel Strategy ◽  
Underlying Mechanisms

Abstract Recently, the focus of enhancing tumor radiosensitivity has shifted from chemotherapeutics to targeted therapies. Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are a novel class of selective cell cycle therapeutics that target the cyclin D-CDK4/6 complex and induce G1 phase arrest. These agents have demonstrated favorable effects when used as monotherapy or combined with endocrine therapy and targeted inhibitors, stimulating further explorations of other combination strategies. Multiple preclinical studies have indicated that CDK4/6 inhibitors exhibit a synergistic effect with radiotherapy both in vitro and in vivo. The principal mechanisms of radiosensitization effects include inhibition of DNA damage repair, enhancement of apoptosis, and blockade of cell cycle progression, which provide the rationale for clinical use. CDK4/6 inhibitors also induce cellular senescence and promote anti-tumor immunity, which might represent potential mechanisms for radiosensitization. Several small sample clinical studies have preliminarily indicated that the combination of CDK4/6 inhibitors and radiotherapy exhibited well-tolerated toxicity and promising efficacy. However, most clinical trials in combined therapy remain in the recruitment stage. Further work is required to seek optimal radiotherapy-drug combinations. In this review, we describe the effects and underlying mechanisms of CDK4/6 inhibitors as a radiosensitizer and discuss previous clinical studies to evaluate the prospects and challenges of this combination.

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