A CCR4 Association Factor 1, OsCAF1B, Participates in the αAmy3 mRNA Poly(A) Tail Shortening and Plays a Role in Germination and Seedling Growth

2019 ◽  
Vol 61 (3) ◽  
pp. 554-564 ◽  
Author(s):  
Jhen-Cheng Fang ◽  
Hsin-Yi Liu ◽  
Yin-Chuan Tsai ◽  
Wei-Lun Chou ◽  
Chun-Chen Chang ◽  
...  
Keyword(s):  
Transgenic Rice ◽  
Seedling Growth ◽  
Mammalian Cells ◽  
Ectopic Expression ◽  
Mrna Degradation ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Dominant Negative Mutation ◽  
Rate Limiting Step ◽  
Catabolite Repressor

Abstract Poly(A) tail (PAT) shortening, also termed deadenylation, is the rate-limiting step of mRNA degradation in eukaryotic cells. The carbon catabolite repressor 4-associated factor 1s (CAF1s) were shown to be one of the major enzymes for catalyzing mRNA deadenylation in yeast and mammalian cells. However, the functions of CAF1 proteins in plants are poorly understood. Herein, a sugar-upregulated CAF1 gene, OsCAF1B, is investigated in rice. Using gain–of–function and dominant-negative mutation analysis, we show that overexpression of OsCAF1B resulted in an accelerated α-amylase gene (αAmy3) mRNA degradation phenomenon, while ectopic expression of a form of OsCAF1B that had lost its deadenylase activity resulted in a delayed αAmy3 mRNA degradation phenomenon in transgenic rice cells. The change in αAmy3 mRNA degradation in transgenic rice is associated with the altered lengths of the αAmy3 mRNA PAT, indicating that OsCAF1B acts as a negative regulator of αAmy3 mRNA stability in rice. Additionally, we found that overexpression of OsCAF1B retards seed germination and seedling growth. These findings indicate that OsCAF1B participates in sugar-induced αAmy3 mRNA degradation and deadenylation and acts a negative factor for germination and seedling development.

scholarly journals Involvement of Myc Activity in a G1/S-Promoting Mechanism Parallel to the pRb/E2F Pathway

2000 ◽  
Vol 20 (10) ◽  
pp. 3497-3509 ◽  
Author(s):  
Eric Santoni-Rugiu ◽  
Jacob Falck ◽  
Niels Mailand ◽  
Jiri Bartek ◽  
Jiri Lukas
Keyword(s):  
Dna Synthesis ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Cyclin E ◽  
Ectopic Expression ◽  
S Phase ◽  
Dominant Negative ◽  
Kinase Activation ◽  
Cdc25a Phosphatase ◽  
In Cells

ABSTRACT The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRbΔcdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic Myc and E2F1 rescue the G1/S delay caused by pRbΔcdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRbΔcdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G1/S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G1/S-promoting mechanism regulating cyclin E-CDK2 in parallel to the “classical” pRb/E2F pathway.

scholarly journals p53-Independent Expression of p21CIP1/WAF1 in Plasmacytic Cells during G2 Cell Cycle Arrest Induced by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

2002 ◽  
Vol 70 (2) ◽  
pp. 528-534 ◽  
Author(s):  
Tsuyoshi Sato ◽  
Takeyoshi Koseki ◽  
Kenji Yamato ◽  
Keitarou Saiki ◽  
Kiyoshi Konishi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Flow Cytometric Analysis ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Actinobacillus Actinomycetemcomitans ◽  
Cytolethal Distending Toxin ◽  
Cycle Arrest

ABSTRACT The cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans has been shown to induce cell cycle arrest in the G2/M phase in HeLa cells. In the present study, the mechanism of CDT-induced cell cycle arrest was investigated by using HS-72 cells, a murine B-cell hybridoma cell line. Using flow cytometric analysis, we found that the recombinant CDT (rCDT) from A. actinomycetemcomitans induced G2 cell cycle arrest in HS-72 cells and that rCDT upregulated expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1 and the tumor suppressor protein p53. HS-72 cells transfected with the E6/E7 gene of human papillomavirus type 16, which lacked rCDT-induced accumulation of p53, exhibited expression of p21CIP1/WAF1 or G2 cell cycle arrest upon exposure to rCDT. Furthermore, ectopic expression of a dominant negative p53 mutant did not inhibit rCDT-mediated p21CIP1/WAF1 expression or G2 cell cycle arrest in HS-72 cells. These results suggest that the CDT from A. actinomycetemcomitans induces p21CIP1/WAF1 expression and G2 cell cycle arrest in B-lineage cells by p53-independent pathways. Together with additional observations made with HeLa cells and COS-1 cells cultured with the rCDT from A. actinomycetemcomitans, the results of this study indicate that CDT-induced p53 accumulation may not be required for G2 cell cycle arrest and that an increased level of p21CIP1/WAF1 may be important for sustaining G2 cell cycle arrest in several mammalian cells.

scholarly journals TRB3 Blocks Adipocyte Differentiation through the Inhibition of C/EBPβ Transcriptional Activity

2007 ◽  
Vol 27 (19) ◽  
pp. 6818-6831 ◽  
Author(s):  
Olivier Bezy ◽  
Cecile Vernochet ◽  
Stephane Gesta ◽  
Stephen R. Farmer ◽  
C. Ronald Kahn
Keyword(s):  
Mammalian Cells ◽  
Ectopic Expression ◽  
Negative Regulator ◽  
Biological Processes ◽  
Preadipocyte Differentiation ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Important Negative Regulator ◽  
Regulatory Sites

ABSTRACT TRB3 has been implicated in the regulation of several biological processes in mammalian cells through its ability to influence Akt and other signaling pathways. In this study, we investigated the role of TRB3 in regulating adipogenesis and the activity of adipogenic transcription factors. We find that TRB3 is expressed in 3T3-L1 preadipocytes, and this expression is transiently suppressed during the initial days of differentiation concomitant with induction of C/EBPβ. This event appears to be a prerequisite for adipogenesis. Overexpression of TRB3 blocks differentiation of 3T3-L1 cells at a step downstream of C/EBPβ. Ectopic expression of TRB3 in mouse fibroblasts also inhibits the C/EBPβ-dependent induction of PPARγ2 and blocks their differentiation into adipocytes. This inhibition of preadipocyte differentiation by TRB3 appears to be the result of two complementary effects. First, TRB3 inhibits extracellular signal-regulated kinase activity, which prevents the phosphorylation of regulatory sites on C/EBPβ. Second, TRB3 directly interacts with the DR1 domain of C/EBPβ in the nucleus, further inhibiting both its ability to bind its response element and its ability to transactivate the C/EBPα and a-FABP promoters. Thus, TRB3 is an important negative regulator of adipogenesis that acts at an early step in the differentiation cascade to block the C/EBPβ proadipogenic function.

scholarly journals The Mouse SKD1, a Homologue of Yeast Vps4p, Is Required for Normal Endosomal Trafficking and Morphology in Mammalian Cells

2000 ◽  
Vol 11 (2) ◽  
pp. 747-763 ◽  
Author(s):  
Tamotsu Yoshimori ◽  
Fumi Yamagata ◽  
Akitsugu Yamamoto ◽  
Noboru Mizushima ◽  
Yukiko Kabeya ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Density Lipoprotein ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Endosomal Trafficking ◽  
Dominant Negative Mutation ◽  
Epidermal Growth

The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1–localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.

scholarly journals Tristetraprolin-mediated hexokinase 2 expression regulation contributes to glycolysis in cancer cells

2019 ◽  
Vol 30 (5) ◽  
pp. 542-553 ◽  
Author(s):  
Dong Jun Kim ◽  
Mai-Tram Vo ◽  
Seong Hee Choi ◽  
Ji-Heon Lee ◽  
So Yeon Jeong ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Glucose Metabolism ◽  
Cancer Cells ◽  
Oxygen Consumption Rate ◽  
Consumption Rate ◽  
Ectopic Expression ◽  
Mrna Degradation ◽  
Negative Regulator ◽  
Extracellular Acidification ◽  
Hexokinase 2

Hexokinase 2 (HK2) catalyzes the first step of glycolysis and is up-regulated in cancer cells. The mechanism has not been fully elucidated. Tristetraprolin (TTP) is an AU-rich element (ARE)-binding protein that inhibits the expression of ARE-containing genes by enhancing mRNA degradation. TTP expression is down-regulated in cancer cells. We demonstrated that TTP is critical for down-regulation of HK2 expression in cancer cells. HK2 mRNA contains an ARE within its 3′-UTR. TTP binds to HK2 3′-UTR and enhances degradation of HK2 mRNA. TTP overexpression decreased HK2 expression and suppressed the glycolytic capacity of cancer cells, measured as glucose uptake and production of glucose-6-phosphate, pyruvate, and lactate. TTP overexpression reduced both the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) of cancer cells. Ectopic expression of HK2 in cancer cells attenuated the reduction in glycolytic capacity, ECAR, and OCR from TTP. Taken together, these findings suggest that TTP acts as a negative regulator of HK2 expression and glucose metabolism in cancer cells.

scholarly journals Multiple environmental signaling pathways control the differentiation of RORγt-expressing regulatory T cells

2019 ◽  
Author(s):  
Hind Hussein ◽  
Sébastien Denanglaire ◽  
Frédéric Van Gool ◽  
Abdulkader Azouz ◽  
Yousra Ajouaou ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Ectopic Expression ◽  
Critical Factor ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Dominant Negative Effect ◽  
Commensal Microbiota ◽  
Negative Effect ◽  
Treg Differentiation ◽  
Treg Development

AbstractRORγt-expressing Tregs form a specialized subset of intestinal CD4+ Foxp3+ cells which is essential to maintain gut homeostasis and tolerance to commensal microbiota. Recently, c-Maf emerged as a critical factor in the regulation of RORγt expression in Tregs. However, aside from c-Maf signaling, the signaling pathways involved in the differentiation of RORγt+ Tregs and their possible interplay with c-Maf in this process are largely unknown. We show that RORγt+ Treg development is controlled by positive as well as negative signals. Along with c-Maf signaling, signals derived from a complex microbiota, as well as IL-6/STAT3- and TGF-β-derived signals act in favor of RORγt+ Treg development. Ectopic expression of c-Maf did not rescue RORγt expression in STAT3-deficient Tregs, indicating the presence of additional effectors downstream of STAT3. Moreover, we show that an inflammatory IFN-γ/STAT1 signaling pathway acts as a negative regulator of RORγt+ Treg differentiation in a c-Maf independent fashion.These data thus argue for a complex integrative signaling network that finely tunes RORγt expression in Tregs. The finding that type 1 inflammation impedes RORγt+ Treg development even in the presence of an active IL-6/STAT3 pathway further suggests a dominant negative effect of STAT1 over STAT3 in this process.

scholarly journals Kinase-Inactivated ULK Proteins Inhibit Autophagy via Their Conserved C-Terminal Domains Using an Atg13-Independent Mechanism

2008 ◽  
Vol 29 (1) ◽  
pp. 157-171 ◽  
Author(s):  
Edmond Y. W. Chan ◽  
Andrea Longatti ◽  
Nicole C. McKnight ◽  
Sharon A. Tooze
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Intramolecular Interactions ◽  
Regulatory Function ◽  
Independent Mechanism ◽  
Dominant Negative Activity

ABSTRACT The yeast Atg1 serine/threonine protein kinase and its mammalian homologs ULK1 and ULK2 play critical roles during the activation of autophagy. Previous studies have demonstrated that the conserved C-terminal domain (CTD) of ULK1 controls the regulatory function and localization of the protein. Here, we explored the role of kinase activity and intramolecular interactions to further understand ULK function. We demonstrate that the dominant-negative activity of kinase-dead mutants requires a 7-residue motif within the CTD. Our data lead to a model in which the functions of ULK1 and ULK2 are controlled by autophosphorylation and conformational changes involving exposure of the CTD. Additional mapping indicates that the CTD contains other distinct regions that direct membrane association and interaction with the putative human homologue of Atg13, which we have here characterized. Atg13 is required for autophagy and Atg9 trafficking during autophagy. However, Atg13 does not bind the 7-residue dominant-negative motif in the CTD of ULK proteins nor is the inhibitory activity of the CTDs rescued by Atg13 ectopic expression, suggesting that in mammalian cells, the CTD may interact with additional autophagy proteins.

scholarly journals Global chromatin organizer SATB1 acts as a context-dependent regulator of the Wnt/Wg target genes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Praveena L. Ramanujam ◽  
Sonam Mehrotra ◽  
Ram Parikshan Kumar ◽  
Shreekant Verma ◽  
Girish Deshpande ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Salivary Glands ◽  
Mammalian Cells ◽  
Target Genes ◽  
Ectopic Expression ◽  
Negative Regulator ◽  
Present Evidence ◽  
Mammalian Cell Lines ◽  
Context Dependent ◽  
Multiple Signaling

AbstractSpecial AT-rich binding protein-1 (SATB1) integrates higher-order chromatin architecture with gene regulation, thereby regulating multiple signaling pathways. In mammalian cells SATB1 directly interacts with β-catenin and regulates the expression of Wnt targets by binding to their promoters. Whether SATB1 regulates Wnt/wg signaling by recruitment of β-catenin and/or its interactions with other components remains elusive. Since Wnt/Wg signaling is conserved from invertebrates to humans, we investigated SATB1 functions in regulation of Wnt/Wg signaling by using mammalian cell-lines and Drosophila. Here, we present evidence that in mammalian cells, SATB1 interacts with Dishevelled, an upstream component of the Wnt/Wg pathway. Conversely, ectopic expression of full-length human SATB1 but not that of its N- or C-terminal domains in the eye imaginal discs and salivary glands of third instar Drosophila larvae increased the expression of Wnt/Wg pathway antagonists and suppressed phenotypes associated with activated Wnt/Wg pathway. These data argue that ectopically-provided SATB1 presumably modulates Wnt/Wg signaling by acting as negative regulator in Drosophila. Interestingly, comparison of SATB1 with PDZ- and homeo-domain containing Drosophila protein Defective Proventriculus suggests that both proteins exhibit limited functional similarity in the regulation of Wnt/Wg signaling in Drosophila. Collectively, these findings indicate that regulation of Wnt/Wg pathway by SATB1 is context-dependent.

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