scholarly journals SMURF2 phosphorylation at Thr249 modifies the stemness and tumorigenicity of glioma stem cells by regulating TGF-β receptor stability

2021 ◽  
Author(s):  
Manami Hiraiwa ◽  
Kazuya Fukasawa ◽  
Takashi Iezaki ◽  
Hemragul Sabit ◽  
Tetsuhiro Horie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Glioma Stem Cells ◽  
Self Renewal ◽  
Ubiquitin Protein Ligase ◽  
Phosphorylation Level ◽  
Ubiquitin Ligase Activity ◽  
Β Receptor ◽  
Underlying Mechanisms

AbstractGlioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma, the most malignant form of glioma. The implication and underlying mechanisms of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) on the GSC phenotypes remain unknown. We previously demonstrated that SMURF2 phosphorylation at Thr249 (SMURF2Thr249) activates its E3 ubiquitin ligase activity. Here, we demonstrate that SMURF2Thr249 phosphorylation plays an essential role in maintaining GSC stemness and tumorigenicity. SMURF2 silencing augmented the self-renewal potential and tumorgenicity of patient-derived GSCs. The SMURF2Thr249 phosphorylation level was low in human glioblastoma pathology specimens. Introduction of the SMURF2T249A mutant resulted in increased stemness and tumorgenicity of GSCs, recapitulating the SMURF2 silencing. Moreover, the inactivation of SMURF2Thr249 phosphorylation increases TGF-β receptor (TGFBR) protein stability. Indeed, TGFBR1 knockdown markedly counteracted the GSC phenotypes by SMURF2T249A mutant. These findings highlight the importance of SMURF2Thr249 phosphorylation in maintaining GSC phenotypes, thereby demonstrating a potential target for GSC-directed therapy.

scholarly journals SMURF2 phosphorylation at Thr249 modifies the stemness and tumorigenicity of glioma stem cells by regulating TGF-β receptor stability

2021 ◽  
Author(s):  
Manami Hiraiwa ◽  
Kazuya Fukasawa ◽  
Takashi Iezaki ◽  
Hemragul Sabit ◽  
Tetsuhiro Horie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Glioma Stem Cells ◽  
Self Renewal ◽  
Ubiquitin Protein Ligase ◽  
Phosphorylation Level ◽  
Ubiquitin Ligase Activity ◽  
Β Receptor ◽  
Underlying Mechanisms

Abstract Glioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma, the most malignant form of glioma. The implication and underlying mechanisms of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) on the GSC phenotypes remain unknown. We previously demonstrated that SMURF2 phosphorylation at Thr249 (SMURF2Thr249) activates its E3 ubiquitin ligase activity. Here, we demonstrate that SMURF2Thr249 phosphorylation plays an essential role in maintaining GSC stemness and tumorigenicity. SMURF2 silencing augmented the self-renewal potential and tumorgenicity of patient-derived GSCs. The SMURF2Thr249 phosphorylation level was low in human glioblastoma pathology specimens. Introduction of the SMURF2T249A mutant resulted in increased stemness and tumorgenicity of GSCs, recapitulating the SMURF2 silencing. Moreover, the inactivation of SMURF2Thr249 phosphorylation increases TGF-β receptor (TGFBR) protein stability. Indeed, TGFBR1 knockdown markedly counteracted the GSC phenotypes by SMURF2T249A mutant. These findings highlight the importance of SMURF2Thr249 phosphorylation in maintaining GSC phenotypes, thereby demonstrating a potential target for GSC-directed therapy.

scholarly journals SMURF2 phosphorylation at Thr249 modifies glioma stemness and tumorigenicity by regulating TGF-β receptor stability

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Manami Hiraiwa ◽  
Kazuya Fukasawa ◽  
Takashi Iezaki ◽  
Hemragul Sabit ◽  
Tetsuhiro Horie ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Essential Role ◽  
E3 Ubiquitin Ligase ◽  
The Self ◽  
Self Renewal ◽  
Ubiquitin Protein Ligase ◽  
Phosphorylation Level ◽  
Ubiquitin Ligase Activity ◽  
Β Receptor ◽  
Underlying Mechanisms

AbstractGlioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma, the most malignant form of glioma. The implication and underlying mechanisms of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) on the GSC phenotypes remain unknown. We previously demonstrated that SMURF2 phosphorylation at Thr249 (SMURF2Thr249) activates its E3 ubiquitin ligase activity. Here, we demonstrate that SMURF2Thr249 phosphorylation plays an essential role in maintaining GSC stemness and tumorigenicity. SMURF2 silencing augmented the self-renewal potential and tumorigenicity of patient-derived GSCs. The SMURF2Thr249 phosphorylation level was low in human glioblastoma pathology specimens. Introduction of the SMURF2T249A mutant resulted in increased stemness and tumorigenicity of GSCs, recapitulating the SMURF2 silencing. Moreover, the inactivation of SMURF2Thr249 phosphorylation increases TGF-β receptor (TGFBR) protein stability. Indeed, TGFBR1 knockdown markedly counteracted the GSC phenotypes by SMURF2T249A mutant. These findings highlight the importance of SMURF2Thr249 phosphorylation in maintaining GSC phenotypes, thereby demonstrating a potential target for GSC-directed therapy.

FANCL Ubiquitinates Beta-Catenin and Enhances Its Nuclear Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1335-1335
Author(s):  
Kim-Hien T. Dao ◽  
Michael D. Rotelli ◽  
Curtis L. Petersen ◽  
Brie R. Brown ◽  
Whitney D. Nelson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Beta Catenin ◽  
Hematopoietic Stem ◽  
Ubiquitin Ligase Activity ◽  
Blood Stem Cells ◽  
Cord Blood Stem Cells

Abstract Abstract 1335 Fanconi anemia (FA) is associated with a hereditary predisposition to bone marrow failure. The proteins encoded by the FANC genes are primarily involved in DNA repair responses through the formation of a large, multisubunit complex that has E3 ubiquitin ligase activity (Annual Review of Genetics 2009;43:223). FA hematopoietic stem cells display defective stem cell properties and limited replicative potential. However, the molecular basis for how a FA genetic background contributes to those defects remains poorly understood. Here we provide evidence that FANCL, which has E3 ubiquitin ligase activity, enhances beta-catenin activity (Figure A) and protein expression. Beta-catenin is a nuclear effector of canonical Wnt signaling. The Wnt/beta-catenin pathway is active in normal hematopoietic stem cells in the native bone marrow environment and disruption of this signaling pathway results in defective hematopoietic stem cells (Nature 2003;423:409). To test whether FANCL positively regulates beta-catenin through its ubiquitination activity, we performed cell-based ubiquitination assays. We show that FANCL functionally ubiquitinates beta-catenin (Figure B) and that ubiquitin chain extension can occur via non-lysine-48 ubiquitin linkages. Accumulating evidence reveal diverse, non-proteolytic biological roles for proteins modified by atypical ubiquitin chains (EMBO Reports 2008;9:536). Our data suggests that FANCL may enhance the protein function of beta-catenin via ubiquitination with atypical ubiquitin chains. Importantly, we demonstrate that suppression of FANCL expression in human CD34+ cord blood stem cells reduces beta-catenin expression (Figure C) and multilineage progenitor expansion. These results demonstrate a role for the FA pathway in regulating Wnt/beta-catenin signaling. Therefore, diminished Wnt/beta-catenin signaling may be an important underlying molecular defect in FA hematopoietic stem cells leading to their accelerated loss. A, LEF-TCF-luciferase reporter assay showing increasing beta-catenin activity in 293FT cells with increasing FANCL expression compared with vector-control (VC) (n=4). B, Immunoprecipitation of beta-catenin in cells transfected with vector-control or FANCL and probed for hemagglutinin (HA)-tagged ubiquitin shows increased ubiquitinated forms of beta-catenin with FANCL expression (n=4). C, shRNA suppression of FANCL expression in CD34+ cord blood stem cells results in decreased beta-catenin expression compared with a scramble control (Scr) by immunofluorescence analysis (three different shRNA constructs, n=3 for each construct). Disclosures: No relevant conflicts of interest to declare.

scholarly journals MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qiaozhi Wei ◽  
Sandra Pinho ◽  
Shuxian Dong ◽  
Halley Pierce ◽  
Huihui Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ubiquitin Ligase ◽  
Kinase Inhibitor ◽  
E3 Ubiquitin Ligase ◽  
Surface Expression ◽  
Haematopoietic Stem Cells ◽  
Receptor Signalling ◽  
Haematopoietic Cells ◽  
Ubiquitin Ligase Activity ◽  
Proliferation And Differentiation

AbstractHaematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid potential. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. Our results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.

scholarly journals STEM-04. NICARDIPINE SENSITIZES APOPTOSIS OF GLIOMA STEM CELLS INDUCED BY TEMOZOLOMIDE THROUGH INHIBITING AUTOPHAGY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi234-vi234
Author(s):  
Jun Dong ◽  
Jia Shi
Keyword(s):  
Stem Cells ◽  
Cell Apoptosis ◽  
Mtor Inhibitor ◽  
Glioma Stem Cells ◽  
Chemotherapeutic Drugs ◽  
Phosphorylation Level ◽  
Stem Cell Lines ◽  
Underlying Mechanisms ◽  
Mtor Activity ◽  
Therapy Of Hypertension

Abstract BACKGROUND Glioma stem cells (GSCs) play an important role in tumor progression and recurrence. Currently, treatments of glioma are limited mainly due to strong tolerance of GSCs against conventional chemotherapeutic drugs such as temozolomide. Nicardipine, a calcium antagonist, is commonly used in the therapy of hypertension, which is recently repurposed in the comprehensive strategies against gliomas in our previous studies. Here, we explored the cytotoxic sensitization effect of nicardipine combining temozolomide and the underlying mechanisms. METHODS Two human glioma stem cell lines were applied and treated with nicardipine, temozolomide, and combination of both, respectively. Cell viability was detected by CCK-8, cell apoptosis was analyzed by flow cytometry, and immunoblot was used to detect autophagic-releated proteins including p62, LC3 and mTOR. The mTOR agonists and inhibitors were applied to further evaluate whether nicardipine inhibits autophagy via mTOR pathway. RESULTS GSCs had strong tolerance against temozolomide while nicardipine could significantly inhibit the viaibility of GSCs and promote cell apoptosis when acting together with temozolomide, indicating that nicardipine could sensitize the toxicity of temozolomide. Furthermore, both temozolomide and nicardipine could inhibit autophagy, and the effects of nicardipine was more prominent, suggesting that nicardipine might enhance GSCs apoptosis induced by temozolomide through inhibiting autophagy. Further molecular studies showed that the cytotoxic sensitization effects of nicardipine was induced through activation of the phosphorylation level of mTOR, which was abolished with the treatment of rapamycin, a mTOR inhibitor. CONCLUSION Our results suggested that nicardipine could sensitize apoptosis of glioma stem cells induced by temozolomide through inhibiting autophagy, which was mediated by activation of mTOR activity.

scholarly journals FANCL ubiquitinates β-catenin and enhances its nuclear function

Blood ◽  
2012 ◽  
Vol 120 (2) ◽  
pp. 323-334 ◽  
Author(s):  
Kim-Hien T. Dao ◽  
Michael D. Rotelli ◽  
Curtis L. Petersen ◽  
Stefanie Kaech ◽  
Whitney D. Nelson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ubiquitin Ligase ◽  
Fanconi Anemia ◽  
E3 Ubiquitin Ligase ◽  
Chain Extension ◽  
Molecular Defect ◽  
Ubiquitin Chain ◽  
Hematopoietic Stem ◽  
Ubiquitin Ligase Activity

Abstract Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34+ stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

scholarly journals MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells

2020 ◽  
Author(s):  
Qiaozhi Wei ◽  
Sandra Pinho ◽  
Shuxian Dong ◽  
Halley Pierce ◽  
Fumio Nakahara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ubiquitin Ligase ◽  
Kinase Inhibitor ◽  
E3 Ubiquitin Ligase ◽  
Surface Expression ◽  
Haematopoietic Stem Cells ◽  
Haematopoietic Cells ◽  
Ubiquitin Ligase Activity ◽  
Proliferation And Differentiation ◽  
And Function

Abstract Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island1, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid commitment. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and function and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs, but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. These results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.

scholarly journals Id1 enhances RING1b E3 ubiquitin ligase activity through the Mel-18/Bmi-1 polycomb group complex

Oncogene ◽  
2010 ◽  
Vol 29 (43) ◽  
pp. 5818-5827 ◽  
Author(s):  
T Qian ◽  
J-Y Lee ◽  
J-H Park ◽  
H-J Kim ◽  
G Kong
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Polycomb Group ◽  
Ubiquitin Ligase Activity

scholarly journals Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

2007 ◽  
Vol 179 (5) ◽  
pp. 935-950 ◽  
Author(s):  
K.G. Suresh Kumar ◽  
Hervé Barriere ◽  
Christopher J. Carbone ◽  
Jianghuai Liu ◽  
Gayathri Swaminathan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Interferon Α ◽  
Type I ◽  
Lysosomal Degradation ◽  
Linear Motif ◽  
Site Specific ◽  
Receptor Endocytosis ◽  
Β Receptor

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

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