scholarly journals Suppression of TCAB1 Expression Induced Cellular Senescence by Lessening Proteasomal Degradation of p21 in Cancer Cells

2020 ◽  
Author(s):  
Jing Niu ◽  
Rui-Qi Gao ◽  
Meng-Tian Cui ◽  
Chen-Guang Zhang ◽  
Shen-Tao Li ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cancer Cells ◽  
Cellular Senescence ◽  
Proteasomal Degradation ◽  
Cell Senescence ◽  
Rapid Progression ◽  
Telomere Elongation ◽  
Protein Levels ◽  
Treatment Regimens

Abstract Background: TCAB1, a.k.a. WRAP53β or WDR79, is an important molecule for the maintenance of Cajal bodies and critically involved in telomere elongation and DNA repair. Upregulation of TCAB1 were discovered in a variety types of cancers. However, the function of TCAB1 in tumor cell senescence remains absent. Methods: The TCAB1 knockdown cell lines were constructed. The expression levels of TCAB1, p21, p16 and p53 were detected by qRT-PCR and western blotting. Staining of senescence-associated β-galactosidase was used to detect senescent cells. The ubiquitination of the p21 was analysed by immunoprecipitatation and in vivo ubiquitination assay. TCGA databases were employed to perform in silico analyses for the mRNA expression of TCAB1, p21, p16 and p53. Results: Here, we discovered that knockdown of TCAB1 induced rapid progression of cellular senescence in A549, H1299 and HeLa cells. In exploiting the mechanism underlining the role of TCAB1 on senescence, we found a significant increase of p21 at the protein levels upon TCAB1 depletion, whereas the p21 mRNA expression was not altered. We verified that TCAB1 knockdown was able to shunt p21 from proteasomal degradation by regulating the ubiquitination of p21. In rescue assays, it was demonstrated that decreasing the expression of p21 was able to attenuate the cellular senescence process induced by TCAB1 silencing. Conclusions: This study revealed the importance of TCAB1 for its biological functions in the regulation of cell senescence. Our results will be helpful to understand the mechanisms of senescence in cancer cells, which could provide clues for designing novel strategies for developing effective treatment regimens.

scholarly journals Suppression of TCAB1 expression induced cellular senescence by lessening proteasomal degradation of p21 in cancer cells

2020 ◽  
Author(s):  
Jing Niu ◽  
Rui-Qi Gao ◽  
Meng-Tian Cui ◽  
Chen-Guang Zhang ◽  
Shen-Tao Li ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cancer Cells ◽  
Cellular Senescence ◽  
Proteasomal Degradation ◽  
Cell Senescence ◽  
Rapid Progression ◽  
Telomere Elongation ◽  
Protein Levels ◽  
Treatment Regimens

Abstract Background: TCAB1, a.k.a. WRAP53β or WDR79, is an important molecule for the maintenance of Cajal bodies and critically involved in telomere elongation and DNA repair. Upregulation of TCAB1 were discovered in a variety types of cancers. However, the function of TCAB1 in tumor cell senescence remains absent.Methods: The TCAB1 knockdown cell lines were constructed. The expression levels of TCAB1, p21, p16 and p53 were detected by qRT-PCR and western blotting. Staining of senescence-associated β-galactosidase was used to detect senescent cells. The ubiquitination of the p21 was analysed by immunoprecipitatation and in vivo ubiquitination assay. TCGA databases were employed to perform in silico analyses for the mRNA expression of TCAB1, p21, p16 and p53. Results: Here, we discovered that knockdown of TCAB1 induced rapid progression of cellular senescence in A549, H1299 and HeLa cells. In exploiting the mechanism underlining the role of TCAB1 on senescence, we found a significant increase of p21 at the protein levels upon TCAB1 depletion, whereas the p21 mRNA expression was not altered. We verified that TCAB1 knockdown was able to shunt p21 from proteasomal degradation by regulating the ubiquitination of p21. In rescue assays, it was demonstrated that decreasing the expression of p21 or increasing the expression of TCAB1 were able to attenuate the cellular senescence process induced by TCAB1 silencing.Conclusions: This study revealed the importance of TCAB1 for its biological functions in the regulation of cell senescence. Our results will be helpful to understand the mechanisms of senescence in cancer cells, which could provide clues for designing novel strategies for developing effective treatment regimens.

scholarly journals Suppression of TCAB1 expression induced cellular senescence by lessening proteasomal degradation of p21 in cancer cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jing Niu ◽  
Rui-Qi Gao ◽  
Meng-Tian Cui ◽  
Chen-Guang Zhang ◽  
Shen-Tao Li ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cancer Cells ◽  
Cellular Senescence ◽  
Proteasomal Degradation ◽  
Cell Senescence ◽  
Rapid Progression ◽  
Telomere Elongation ◽  
Protein Levels ◽  
Treatment Regimens

Abstract Background TCAB1, a.k.a. WRAP53β or WDR79, is an important molecule for the maintenance of Cajal bodies and critically involved in telomere elongation and DNA repair. Upregulation of TCAB1 were discovered in a variety types of cancers. However, the function of TCAB1 in tumor cell senescence remains absent. Methods The TCAB1 knockdown cell lines were constructed. The expression levels of TCAB1, p21, p16 and p53 were detected by qRT-PCR and western blotting. Staining of senescence-associated β-galactosidase was used to detect senescent cells. The ubiquitination of the p21 was analysed by immunoprecipitation and in vivo ubiquitination assay. TCGA databases were employed to perform in silico analyses for the mRNA expression of TCAB1, p21, p16 and p53. Results Here, we discovered that knockdown of TCAB1 induced rapid progression of cellular senescence in A549, H1299 and HeLa cells. In exploiting the mechanism underlining the role of TCAB1 on senescence, we found a significant increase of p21 at the protein levels upon TCAB1 depletion, whereas the p21 mRNA expression was not altered. We verified that TCAB1 knockdown was able to shunt p21 from proteasomal degradation by regulating the ubiquitination of p21. In rescue assays, it was demonstrated that decreasing the expression of p21 or increasing the expression of TCAB1 were able to attenuate the cellular senescence process induced by TCAB1 silencing. Conclusions This study revealed the importance of TCAB1 for its biological functions in the regulation of cell senescence. Our results will be helpful to understand the mechanisms of senescence in cancer cells, which could provide clues for designing novel strategies for developing effective treatment regimens.

scholarly journals BIOM-55. DGKζ-TARGETED REGULATION OF MIR-34A IN THE PROLIFERATION AND TUMORIGENICITY OF HUMAN GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii13-ii13
Author(s):  
Wangxian Gu ◽  
Guoqing Wan ◽  
Yanjun Zheng ◽  
Xintong Yang ◽  
Peng Zhang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Precancerous Lesions ◽  
Lipid Kinase ◽  
Human Glioblastoma ◽  
Kinase Substrate ◽  
Downstream Target ◽  
Protein Levels ◽  
Type Iv

Abstract Diacylglycerol kinase (DGK) is a lipid kinase that catalyzes the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), which uses ATP as a phosphate donor. Diacylglycerol kinases ζ(DGKζ) is characterized as specific type IV due to its myristoylated alanine-rich C-kinase substrate (MARCKS), ankyrin, and PDZ binding domain. Similar to other DGKs, DGKζ is also reported to be abnormally expressed in human colorectal cancer cells, and it is indispensable for the proliferation of cancer cells. However, its implications in human glioblastoma (GBM) is largely unknown. Both the mRNA and protein levels of DGKζ were significantly higher in GBM tissues than in precancerous lesions. Knockdown of DGKζ inhibited GBM cell proliferation, cell cycle and promoted apoptosis of GBM cells. Moreover, down-regulation of DGKζ markedly reduced in vitro colony formation and in vivo tumorigenic capability. Furthermore, we confirmed that DGKζ was the downstream target of miR-34a. The expression level of DGKζ was negatively correlated with miR-34a in GBM tissues. Overexpression of DGKζ reversed the tumor suppressive roles of miR-34a in GBM cells. Taken together, DGKζ can act as a potential prognostic biomarker for GBM patients and promote the growth of GBM cells was regulated by miR-34a, and it may represent a promising therapeutic target for patients with GBM.

scholarly journals Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Xiao-ren Zhu ◽  
Shi-qing Peng ◽  
Le Wang ◽  
Xiao-yu Chen ◽  
Chun-xia Feng ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Therapeutic Target ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Human Pancreatic Cancer ◽  
Migration And Invasion ◽  
Potential Therapeutic Target ◽  
Protein Levels ◽  
Pancreatic Cancer Cells

AbstractPancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.

scholarly journals Ablation of Sirtuin 1 Deacetylase Activity Induces Pulmonary Emphysema by Inducing Cellular Senescence and Disrupting Circadian Clock in Mice (P01-022-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Xiang-Dong Wang ◽  
Kang-Quan Hu ◽  
Chun Liu ◽  
Michael McBurney
Keyword(s):  
Circadian Clock ◽  
Mrna Expression ◽  
Cellular Senescence ◽  
Pulmonary Emphysema ◽  
Clock Genes ◽  
Causal Effect ◽  
Sirtuin 1 ◽  
Wild Type ◽  
Protein Levels ◽  
Circadian Clock Genes

Abstract Objectives Sirtuin 1 (SIRT1), a NAD+-dependent protein/histone deacetylase has the capability to extend life span, delay aging, and prevent aging-related diseases. There are several reports showing that there is no significant decline in SIRT1 protein with age, indicating that SIRT1 protein levels alone may not reflect its deacetylase activity. We investigated the causal effect of systemic ablation of SIRT1 deacetylase activity on aging-related pulmonary disease development in mice. Methods We used Sirt1y/y homozygous male mice carrying a point mutation (H355Y) that ablates the deacetylase activity, along with their wild type littermates (Sirt1+/+), and followed them for 6, 10 and 18 months of age. Results Sirt1y/y homozygous mice developed severe pulmonary emphysema at the ages of 6, 10 and 18 months, with the respective incidences of 33%, 100% and 100%, while the Sirt1+/+wild-type mice only developed emphysema (13% incidence) at 18 months of age. The development of emphysema in Sirt1y/y mice was accompanied with higher protein levels of matrix metalloproteinase (MMP)-2, MMP9, and tissue inhibitor of metalloproteinase-1, and ratio of cleaved/total anti-poly (ADP-ribose) polymerase. The ablation of SIRT1 activity significantly up-regulated mRNA expression of hypoxia-inducible factor-1α and retinoic acid receptor-b, while p21 protein and phosphorylated AMPK increased and phosphorylated ribosomal S6 decreased, suggesting the association of ablation of SIRT1 activity with cellular quiescence and senescence. Additionally, the lack of SIRT1 activity down-regulated the mRNA expression of circadian clock genes (BMAL1, NPAS2, CRY1, CRY2) in the lungs of Sirt1y/y mice, as compared with that of Sirt1+/+ mice. There were no inflammatory responses in the lungs (e.g., inflammatory cell infiltrations, mRNA expressions of IL-6 and TNFα) in Sirt1y/y homozygous mice compared to Sirt1+/+ mice. Conclusions The lacking of SIRT1 enzymatic activity plays a major role in the susceptibility of organs to aging and pulmonary emphysema development by inducing cellular senescence and disrupting circadian clock genes. Funding Sources USDA/ARS (58-1950-0074) and NIFA/AFRI (2017-67017-26363).

Inhibition of Usp9x Deubiquitinase Activity by WP1130 Reduces Mcl-1 Levels and Induces Apoptosis In Cells From Patients with Plasma Cell Dyscrasia and Drug-Refractory Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3005-3005 ◽  
Author(s):  
Hanshi Sun ◽  
Vaibhav Kapuria ◽  
Luke F. Peterson ◽  
Diane Giannola ◽  
Kandarpa Malathi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cell ◽  
Proteasomal Degradation ◽  
Plasma Cell Dyscrasia ◽  
Newly Diagnosed ◽  
Down Regulation ◽  
Rapid Reduction ◽  
Protein Levels ◽  
Advanced Therapy

Abstract Abstract 3005 Multiple myeloma (MM) patients are typically treated with a number of combinations of unconventional drugs that provide excellent and sometimes durable responses. However, most patients are expected to relapse or progress and relapsed disease is usually less sensitive to chemotherapy. Therefore, drug resistance remains a key concern in MM therapy. Some important clues regarding MM drug and apoptotic resistance have been obtained from biochemical and molecular studies of pro-survival genes. Mcl-1 is one member of the Bcl-2 family with strong anti-apoptotic activity. Mcl-1 binds pro-apoptotic proteins such as Bim and Bak, forming hetero-dimers to suppress apoptosis. Specific knockdown of Mcl-1 but not other anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL) induces apoptosis in MM cells and a critical level of Mcl-1 is essential to MM cell viability. Also, drug resistant MM patients are reported to express higher levels of Mcl-1 when compared to drug naïve patients. These observations suggest that Mcl-1 plays a key role in MM drug and apoptotic resistance. Mcl-1 is unique among survival proteins as its cellular protein level is primarily controlled by its ubiquitination and proteasomal degradation. Recently Usp9x, a high MW de-ubiquitinase (DUB), was shown to bind and stabilize Mcl-1 by mediating its de-ubiquitination and loss of recognition by the proteasome. Usp9x was also shown to be highly expressed in MM patients with short term progression-free survival. Analysis of Usp9x gene expression in MM patients treated at our center supports a correlation between elevated Usp9x expression (>4-fold over normal PBMCs) and poor patient prognosis. These observations suggest that agents that inhibit Usp9x activity may be effective in controlling Mcl-1 levels and have therapeutic impact in MM patients. Here we describe a small molecule Usp9x inhibitor, WP1130, that causes a rapid reduction in Mcl-1 protein levels and stimulates apoptosis in MM cells. We further demonstrate that the anti-tumor activity of WP1130 is mediated through inhibition of Usp9x and other active DUBs in B-cell tumors. WP1130-mediated inhibition of Usp9x activity promotes Mcl-1 ubiquitination leading to its rapid (1-4 hours) proteasomal degradation with subsequent induction of apoptosis. Pretreatment of MM cells with proteasome inhibitors (MG-132 or bortezomib) prior to WP1130 treatment blocked the down-regulation of Mcl-1, demonstrating proteasome involvement in regulation of Mcl-1 levels by WP1130. Primary MM tumor cells (bone marrow aspirates) derived from newly diagnosed and advanced therapy refractory patients were also screened for their responsiveness to WP1130 by DUB activity assays. Samples were obtained from a newly diagnosed plasmacytoma/plasma cell leukemia patient and 4 MM patients refractory to the Velcade/Doxorubicin/Dexamethasone (VDD) regimen. Usp9x activity was detected in 4 of 5 patient specimens and WP1130 treatment reduced Mcl-1 protein levels and induced apoptosis only in samples with active Usp9x. Furthermore, WP1130 completely eliminated CD138+ cells after 24 hours in MM patient specimens with active Usp9x while only an 11% reduction was measured in MM cells that did not exhibit Usp9x activity. Cytogenetic prognostic predictors (del13) did not distinguish between WP1130-responsive and non-responsive cells. Our results suggest that inhibition of Usp9x activity by WP1130 is associated with Mcl-1 down-regulation in MM cells. These results also suggest that drug-refractory MM patients with elevated Usp9x activity would likely benefit from Usp9x inhibitor-based therapy. Preliminary MM tumor studies have shown that WP1130 suppresses tumor growth in vivo and can be administered safely. Together, these results suggest that WP1130-mediated Usp9x inhibition may be an effective and novel approach in treating progressive or drug-refractory MM patients. Disclosures: Jakubowiak: Millenium, Celgene, Bristol-Myers Squibb, Johnson & Johnson Ortho-Centocor: Honoraria; Millennium, Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millennium, Celgene, Centocor-Ortho Biotech: Speakers Bureau.

scholarly journals Bortezomib induces DNA hypomethylation and silenced gene transcription by interfering with Sp1/NF-κB–dependent DNA methyltransferase activity in acute myeloid leukemia

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2364-2373 ◽  
Author(s):  
Shujun Liu ◽  
Zhongfa Liu ◽  
Zhiliang Xie ◽  
Jiuxia Pang ◽  
Jianhua Yu ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Zinc Finger Protein ◽  
Human Cancer ◽  
Proteasomal Degradation ◽  
Physical Interaction ◽  
Dna Hypomethylation ◽  
Methyltransferase Activity ◽  
Protein Levels

Bortezomib reversibly inhibits 26S proteasomal degradation, interferes with NF-κB, and exhibits antitumor activity in human malignancies. Zinc finger protein Sp1 transactivates DNMT1 gene in mice and is functionally regulated through protein abundance, posttranslational modifications (ie, ubiquitination), or interaction with other transcription factors (ie, NF-κB). We hypothesize that inhibition of proteasomal degradation and Sp1/NF-κB–mediated transactivation may impair aberrant DNA methyltransferase activity. We show here that, in addition to inducing accumulation of polyubiquitinated proteins and abolishment of NF-κB activities, bortezomib decreases Sp1 protein levels, disrupts the physical interaction of Sp1/NF-κB, and prevents binding of the Sp1/NF-κB complex to the DNMT1 gene promoter. Abrogation of Sp1/NF-κB complex by bortezomib causes transcriptional repression of DNMT1 gene and down-regulation of DNMT1 protein, which in turn induces global DNA hypomethylation in vitro and in vivo and re-expression of epigenetically silenced genes in human cancer cells. The involvement of Sp1/NF-κB in DNMT1 regulation is further demonstrated by the observation that Sp1 knockdown using mithramycin A or shRNA decreases DNMT1 protein levels, which instead are increased by Sp1 or NF-κB overexpression. Our results unveil the Sp1/NF-κB pathway as a modulator of DNA methyltransferase activity in human cancer and identify bortezomib as a novel epigenetic-targeting drug.

scholarly journals PTEN deletion in luminal cells of mature prostate induces replication stress and senescence in vivo

2018 ◽  
Vol 215 (6) ◽  
pp. 1749-1763 ◽  
Author(s):  
Maxime Parisotto ◽  
Elise Grelet ◽  
Rana El Bizri ◽  
Yongyuan Dai ◽  
Julie Terzic ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cellular Senescence ◽  
Replication Stress ◽  
Cell Senescence ◽  
Pten Loss ◽  
Genetic Ablation ◽  
Adult Mice ◽  
Pten Deletion ◽  
Progressive Growth

Genetic ablation of the tumor suppressor PTEN in prostatic epithelial cells (PECs) induces cell senescence. However, unlike oncogene-induced senescence, no hyperproliferation phase and no signs of DNA damage response (DDR) were observed in PTEN-deficient PECs; PTEN loss-induced senescence (PICS) was reported to be a novel type of cellular senescence. Our study reveals that PTEN ablation in prostatic luminal epithelial cells of adult mice stimulates PEC proliferation, followed by a progressive growth arrest with characteristics of cell senescence. Importantly, we also show that proliferating PTEN-deficient PECs undergo replication stress and mount a DDR leading to p53 stabilization, which is however delayed by Mdm2-mediated p53 down-regulation. Thus, even though PTEN-deficiency induces cellular senescence that restrains tumor progression, as it involves replication stress, strategies promoting PTEN loss–induced senescence are at risk for cancer prevention and therapy.

scholarly journals Senescent Colon and Breast Cancer Cells Induced by Doxorubicin Exhibit Enhanced Sensitivity to Curcumin, Caffeine, and Thymoquinone

2020 ◽  
Vol 19 ◽  
pp. 153473541990116 ◽  
Author(s):  
Ali H. El-Far ◽  
Noureldien H. E. Darwish ◽  
Shaker A. Mousa
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Cellular Senescence ◽  
Annexin V ◽  
Mcf7 Cells ◽  
Enhanced Sensitivity ◽  
Cycle Arrest

Cellular senescence is a process of physiological growth arrest that can be induced by intrinsic or extrinsic stress signals. Some cancer therapies are associated with senescence of cancer cells with a typical cell cycle arrest. Doxorubicin (Dox) induces senescence by a p53-dependent pathway and telomere dysfunction of numerous cancers. However, cellular senescence induces suppression in proliferation activity, and these cells will remain metabolically active and play an important role in tumor relapse and development of drug resistance. In the current study, we investigated the apoptotic effect of curcumin (Cur), caffeine (Caff), and thymoquinone (TQ) on senescent colon cancer HCT116 and breast cancer MCF7 cell lines treated with Dox. Results showed typical senescence markers including decreased bromodeoxyuridine incorporation, increased accumulation of senescence-associated β-galactosidase (SA-β-gal), cell cycle arrest, and upregulation of p53, P-p53, and p21 proteins. Annexin-V analysis by flow cytometry revealed 2- to 6-fold increases in annexin-V–positive cells in Dox-treated MCF7 and HCT116 cells by Cur (15 µM), Caff (10 mM), and TQ (50 µM; P < .001). In comparison between proliferative and senescent of either HCT116 or MCF7 cells, Caff at 15 mM and TQ at 25 µM induced significant increases in apoptosis of Dox-treated cells compared with proliferative cells ( P < .001). Data revealed that Cur, Caff, and TQ potentially induced apoptosis of both proliferative and senescent HCT116 and MCF7 cells. In vivo and clinical trials are of great importance to validate this result.

scholarly journals BRF1 Protein Turnover and mRNA Decay Activity Are Regulated by Protein Kinase B at the Same Phosphorylation Sites

2006 ◽  
Vol 26 (24) ◽  
pp. 9497-9507 ◽  
Author(s):  
Don Benjamin ◽  
Martin Schmidlin ◽  
Lu Min ◽  
Brigitte Gross ◽  
Christoph Moroni
Keyword(s):  
Protein Kinase ◽  
Mrna Decay ◽  
Protein Kinase B ◽  
Proteasomal Degradation ◽  
Mrna Levels ◽  
Cell Compartment ◽  
Protein Levels ◽  
Dependent Protein

ABSTRACT BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKBα−/− cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.

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