Mutagenesis of Erythropoietin Receptor Cytoplasmic Lysines Uncouples Erythropoietin-Dependent Growth From Downstream Signal Transduction Cascades.

Abstract Abstract 3611 Poster Board III-547 Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis, stimulating growth, preventing apoptosis, and promoting differentiation of red blood cell progenitors. Fundamental to this action is the ability of EPO to bind to its cognate receptor on the cell surface, the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Erythrocyte production relies heavily on precise control of EPO-mediated cell signaling cascades. The availability of EPO-R to EPO and the activation of subsequent signaling cascades are tightly regulated by the transit of mature EPO-R to the cell surface from the Golgi, and the mechanisms by which the receptor is internalized and down-regulated. Thus, small changes in plasma membrane EPO-R levels may be reflected in the intensity and duration of downstream signaling protein activation. EPO-R is rapidly ubiquitinated and down-regulated from the cell surface upon EPO stimulation. EPO-R ubiquitination relies on more than one E3 ubiquitin ligase, the U-box containing E3 ubiquitin ligase, p33Rul, and the RING finger E3 ubiquitin ligase SCFβTrcp. The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome lead us to examine the role of EPO-R ubiquitination on cell signaling and proliferation. We hypothesized that EPO-R ubiquitination would result in down-regulation of EPO-mediated signaling cascades. This study characterized which lysines determined EPO-R ubiquitination, and assessed the ability of EPO-R lysine mutants to support EPO-mediated signaling and proliferation. Surprisingly, substitution of all EPO-R cytoplasmic lysines (EPO-R K5R) abolished the ability of EPO-R to support EPO-mediated proliferation in BaF3 cells. However, EPO-dependent phosphorylation of EPO-R, AKT, PKB and STAT5 was detected, albeit at reduced levels. While ubiquitination defects commonly prolong receptor signaling, the kinetics of EPO-R K5R-mediated signaling pathways paralleled Ba/F3-EPO-R cells. Mutation of membrane proximal lysines, K256 and K276, resulted in decreased JAK2 phosphorylation and EPO-R phosphorylation and inhibited proliferation at 0.5U/ml EPO. However, the interaction between JAK2 and EPO-R was not affected. In addition, any EPO-R cytoplasmic lysine could support signaling and proliferation at physiologically elevated EPO concentrations demonstrating significant redundancy. The requirement of EPO-R cytoplasmic lysines to promote CFU-E formation is currently being evaluated by introduction of EPO-R mutants into fetal liver cells derived from EPO-R null mice. These results suggest that the EPO-R cytoplasmic lysines play a critical role in transmitting EPO-dependent growth signals within the cell. Disclosures: No relevant conflicts of interest to declare.

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Cul4-Ddb1 ubiquitin ligases facilitate DNA replication-coupled sister chromatid cohesion through regulation of cohesin acetyltransferase Esco2

10.1101/414888 ◽

2018 ◽

Cited By ~ 1

Author(s):

Haitao Sun ◽

Jiaxin Zhang ◽

Jingjing Zhang ◽

Zhen Li ◽

Qinhong Cao ◽

...

Keyword(s):

Dna Replication ◽

Ubiquitin Ligase ◽

Sister Chromatid ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

Sister Chromatid Cohesion ◽

Vital Role ◽

Chromatid Cohesion ◽

Evolutionarily Conserved

AbstractCohesin acetyltransferases Esco1 and Esco2 play a vital role in establishing sister chromatid cohesion. How Esco1 and Esco2 are controlled to achieve this in a DNA replication-coupled manner remains unclear in higher eukaryotes. Here we show that Cul4-RING ligases (CRL4s) play a critical role in sister chromatid cohesion in human cells. Depletion of Cul4A, Cul4B or Ddb1 subunits substantially reduces normal cohesion efficiency. We also show that Mms22L, a vertebrate ortholog of yeast Mms22, is one of Ddb1 and Cul4-associated factors (DCAFs) involved in cohesion. Several lines of evidence suggest a selective interaction of CRL4s with Esco2, but not Esco1. Depletion of either CRL4s or Esco2 causes a defect in Smc3 acetylation which can be rescued by HDAC8 inhibition. More importantly, both CRL4s and PCNA act as mediators for efficiently stabilizing Esco2 on chromatin and catalyzing Smc3 acetylation. Taken together, we propose an evolutionarily conserved mechanism in which CRL4s and PCNA regulate Esco2-dependent establishment of sister chromatid cohesion.Author summaryWe identified human Mms22L as a substrate specific adaptor of Cul4-Ddb1 E3 ubiquitin ligase. Downregulation of Cul4A, Cul4B or Ddb1 subunit causes reduction of acetylated Smc3, via interaction with Esco2 acetyltransferase, and then impairs sister chromatid cohesion in 293T cells. We found functional complementation between Cul4-Ddb1-Mms22L E3 ligase and Esco2 in Smc3 acetylation and sister chromatid cohesion. Interestingly, both Cul4-Ddb1 E3 ubiquitin ligase and PCNA contribute to Esco2 mediated Smc3 acetylation. To summarise, we demonstrated an evolutionarily conserved mechanism in which Cul4-Ddb1 E3 ubiquitin ligases and PCNA regulate Esco2-dependent establishment of sister chromatid cohesion.

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The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1

eLife ◽

10.7554/elife.40009 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 24

Author(s):

Sam A Menzies ◽

Norbert Volkmar ◽

Dick JH van den Boomen ◽

Richard T Timms ◽

Anna S Dickson ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Editorial Note ◽

Hmg Coa Reductase ◽

Genome Wide ◽

Cholesterol Biosynthetic Pathway ◽

Coa Reductase ◽

Rate Limiting

Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

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Targeted Degradation of Glucose Transporters Protects against Arsenic Toxicity

Molecular and Cellular Biology ◽

10.1128/mcb.00559-18 ◽

2019 ◽

Vol 39 (10) ◽

Cited By ~ 2

Author(s):

Marco Jochem ◽

Lukas Ende ◽

Marta Isasa ◽

Jessie Ang ◽

Helena Schnell ◽

...

Keyword(s):

Cell Surface ◽

Ubiquitin Ligase ◽

Cellular Response ◽

Arsenic Exposure ◽

E3 Ubiquitin Ligase ◽

Optimal Growth ◽

Glucose Transporters ◽

Arsenic Toxicity ◽

Environmental Toxin ◽

Surprising Finding

ABSTRACTThe abundance of cell surface glucose transporters must be precisely regulated to ensure optimal growth under constantly changing environmental conditions. We recently conducted a proteomic analysis of the cellular response to trivalent arsenic, a ubiquitous environmental toxin and carcinogen. A surprising finding was that a subset of glucose transporters was among the most downregulated proteins in the cell upon arsenic exposure. Here we show that this downregulation reflects targeted arsenic-dependent degradation of glucose transporters. Degradation occurs in the vacuole and requires the E2 ubiquitin ligase Ubc4, the E3 ubiquitin ligase Rsp5, and K63-linked ubiquitin chains. We used quantitative proteomic approaches to determine the ubiquitinated proteome after arsenic exposure, which helped us to identify the ubiquitination sites within these glucose transporters. A mutant lacking all seven major glucose transporters was highly resistant to arsenic, and expression of a degradation-resistant transporter restored arsenic sensitivity to this strain, suggesting that this pathway represents a protective cellular response. Previous work suggests that glucose transporters are major mediators of arsenic import, providing a potential rationale for this pathway. These results may have implications for the epidemiologic association between arsenic exposure and diabetes.

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The Cbl E3 Ubiquitin Ligase Negatively Regulates Erythropoiesis through Expression of the Foxo3a Transcription Factor.

Blood ◽

10.1182/blood.v112.11.1374.1374 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1374-1374

Author(s):

Terri D Richmond ◽

Monica L Bailey ◽

Wallace Y Langdon ◽

Dwayne Barber

Keyword(s):

Signal Transduction ◽

Cell Signaling ◽

Ubiquitin Ligase ◽

Knockout Mice ◽

E3 Ubiquitin Ligase ◽

Parp Cleavage ◽

Signal Transduction Pathways ◽

Wild Type ◽

Erythroid Progenitors ◽

Deficient Mice

Abstract Erythropoietin (EPO) is the primary cytokine regulator of red blood cell (RBC) progenitor growth, survival and differentiation. EPO stimulation is regulated by EPO binding to its cognate ligand, the EPO receptor (EPO-R), and activating the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Intracellular signal transduction pathways regulating growth, survival and differentiation downstream of the EPO-R and JAK2 are well documented. However, relatively little is known about down-regulation of EPO-R signal transduction pathways at this time. Our laboratory has previously demonstrated that EPO stimulation leads to Cbltyrosine phosphorylation and subsequent recruitment of Crk-C3G, leading to Rap1activation. In addition, Cbl serves as an adaptor protein linking to PI 3 kinase and Rasand targets receptor tyrosine kinases for ubiquitination and proteasomal degradation. Cbl knockout mice have been generated and have defects in stem and T cell signaling pathways. Elevated platelet numbers and splenomegaly was observed, suggesting that Cbl −/− mice may have defects in megakaryocyte/erythroid progenitors or more committed cells in each lineage. The objective of this studyis to determine whether Cbl affects erythropoiesis and EPO-dependent signaling. Resting Cbl −/− mice (in the C57Bl/6 background) have increased numbers of Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid (CFU-E) cells. Furthermore, there is a 3-fold elevation of splenic CFU-E numbers. Erythroid differentiation was monitored via expression of the Transferrin Receptor (CD71) and Ter119. Cbl-deficient mice have delayed differentiation in the bone marrow with diminished CD71-Ter119+ cells. Increased apoptosis is observed in Ter119+ erythroid cells isolated from Cbl −/− mice as determined by Annexin V staining and confirmed by increased PARP cleavage. Interestingly, reactive oxygen species in wild type and Cbl-deficient mice remain unchanged. Despite normal resting hematologic parameters, serum EPO concentrations are elevated in Cbl knockout mice. Serum VEGF levels are comparable between wild type and Cbl −/− mice, suggesting that the EPO effect is specific to the erythroid lineage and not an effect of hypoxia. Notable differences in wild type and Cbl −/− mice were observed when stress erythropoiesiswas induced by phenylhydrazine-mediated anemia. Cbl-deficient mice respond with enhanced hematocrit recovery and increased reticulocyte production. EPO-dependent Aktphosphorylation is hypersensitive in Cbl −/− splenic erythroblasts. Interestingly, expression ofFoxo3a was stabilized in Cbl −/− splenic erythroblasts, suggesting that Cbl degrades Foxo3a in a direct or indirect manner. Given the importance of Foxo3a in regulating erythropoiesis, we are currently determining whether Cbl targets Foxo3a for ubiquitin-mediated degradation. These data demonstrate the remarkable homeostatic ability of the mouse to retain normal RBC concentrations in the peripheral blood despite elevated erythroid progenitors and cell signaling. Importantly, these studies are the first to phenotypically explore the effects of genetic ablation of an EPO-responsive E3 ubiquitin ligase in erythropoiesis.

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The Kaposi's Sarcoma-Associated Herpesvirus K5 E3 Ubiquitin Ligase Modulates Targets by Multiple Molecular Mechanisms

Journal of Virology ◽

10.1128/jvi.02751-06 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6573-6583 ◽

Cited By ~ 17

Author(s):

Robert E. Means ◽

Sabine M. Lang ◽

Jae U. Jung

Keyword(s):

Cell Surface ◽

Ubiquitin Ligase ◽

Kaposi's Sarcoma ◽

E3 Ubiquitin Ligase ◽

Kaposi’S Sarcoma ◽

Down Regulation ◽

Class I Mhc ◽

Mhc I ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus encodes two highly related membrane-associated, RING-CH-containing (MARCH) family E3 ubiquitin ligases, K3 and K5, that can down regulate a variety of cell surface proteins through enhancement of their endocytosis and degradation. In this report we present data that while K5 modulation of major histocompatibility complex class I (MHC-I) closely mirrors the mechanisms used by K3, alternative molecular pathways are utilized by this E3 ligase in the down regulation of intercellular adhesion molecule 1 (ICAM-1) and B7.2. Internalization assays demonstrate that down regulation of each target can occur through increased endocytosis from the cell surface. However, mutation of a conserved tyrosine-based endocytosis motif in K5 resulted in a protein lacking the ability to direct an increased rate of MHC-I or ICAM-1 internalization but still able to down regulate B7.2 in a ubiquitin-dependent but endocytosis-independent manner. Further, mutation of two acidic clusters abolished K5-mediated MHC-I degradation while only slightly decreasing ICAM-1 or B7.2 protein destruction. This same mutant abolished detectable ubiquitylation of all targets. These data indicate that while K5 can act as an E3 ubiquitin ligase to directly mediate cell surface molecule destruction, regulation of its targets occurs through multiple pathways, including ubiquitin-independent mechanisms.

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The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1

10.1101/391789 ◽

2018 ◽

Author(s):

Sam A. Menzies ◽

Norbert Volkmar ◽

Dick J. van den Boomen ◽

Richard T. Timms ◽

Anna S. Dickson ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Hmg Coa Reductase ◽

Accelerated Degradation ◽

Genome Wide ◽

Cholesterol Biosynthetic Pathway ◽

Coa Reductase ◽

Rate Limiting

ABSTRACTHMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78, independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR and Insig-1, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent ligase partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.

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New Insights into Mechanisms of Erythropoietin Receptor Mutations in Primary Familial and Congenital Polycythemia

Blood ◽

10.1182/blood.v128.22.631.631 ◽

2016 ◽

Vol 128 (22) ◽

pp. 631-631 ◽

Cited By ~ 1

Author(s):

Florence Pasquier ◽

Caroline Marty ◽

Frédérique Verdier ◽

Sarah Grosjean ◽

Claude Préhu ◽

...

Keyword(s):

Cell Surface ◽

Conflicts Of Interest ◽

Stop Codon ◽

Cell Mass ◽

Erythropoietin Receptor ◽

Functional Study ◽

Common Mechanism ◽

Wild Type ◽

Terminal Part ◽

The Impact

Abstract Primary Familial and Congenital Polycythemia (PFCP) is a non-malignant pathology of the erythroid lineage, characterized by an isolated increase of the red cell mass without evolution into myelofibrosis or acutisation. Around twenty constitutive non-sense and missense mutations located in the exon 8 of the erythropoietin receptor (EPOR) gene have been described so far. They all lead to the truncation of the C-terminal part of the protein and the loss of several cytoplasmic tyrosines. The erythropoietin (EPO) hypersensitivity of the PFCP erythroid progenitors is usually explained by the disappearance of these negative signaling regulation and internalization domains (Figure 1a). Nonetheless, relatively few functional studies have been carried out. We therefore investigated the mechanism of EPOR mutations in PFCP. We identified and extensively studied a new constitutive heterozygous frameshift EPOR mutation, p.Gln434Profs*11, which generates a new 11 amino acid (AA) C-terminal tail and a STOP codon at position 444, leading to the truncation of 63 AA of the wild-type receptor (Figure 1c). The primary progenitor cells displayed a major hypersensitivity to EPO, similar to Polycythemia Vera (PV) patients, as well as a spontaneous and persistent JAK2 and STAT5 phosphorylation, compared to the control cells. To study the mechanism of this new EPOR mutant, Ba/F3 cells were transduced with different retroviruses encoding either the HA-tagged wild-type EPOR (EPOR WT)or a truncated receptor at position 444, p.Gln444* (EPOR STOP) or the frameshift EPOR p.Gln434Profs*11mutation (EPOR FS), identical to the patient's mutation (Figure 2). As observed in primary cells, EPOR FS conferred a spontaneous STAT5 phosphorylation and a 4- to 5-fold EPO hypersensitivity to Ba/F3 cells (IC50 of 0.003 U/mL vs 0.01 U/mL) compared to both EPOR WT and EPOR STOP. As expected, the loss of negative regulatory domains in the C-terminal part of the receptor induced a persistent STAT5 activation in EPOR FS and EPOR STOP Ba/F3 cells. Moreover, EPOR FS was more stable (half-life of 120 minutes vs 60 minutes) and displayed a higher level of localization at the cell surface (more than 2-fold), compared to EPOR WT and EPOR STOP. However, no modification of the EPOR FS internalization pattern was observed during 125I-EPO labeling experiments and cytometry analysis. Furthermore, a dileucine motif, known to be a potential clathrin-dependent endocytosis site, is lost in the new C-terminal tail of EPOR FS mutant, yet its abrogation in EPOR WT and EPOR STOP did not modify the phenotype of Ba/F3 cells. Therefore, unlike previous reports, the major EPO hypersensitivity induced by EPOR p.Gln434Profs*11 cannot be explained by the receptor truncation, but rather by the appearance of a new C-terminal tail that confers spontaneous signaling. We wondered if this model could be extended to other EPOR mutations already described in PFCP (Figures 1a-b and 2). We therefore measured the impact on Ba/F3 cells proliferation of the frameshift EPOR p.Pro438Metfs*6 and its non-sense mutant counterpart, p.Pro443*, which retains the tyrosine at position 426, a binding site for the negative signaling regulators SOCS3 and CIS. EPO-hypersensitivity (4- to 5-fold) was only induced by EPOR p.Pro438Metfs*6, suggesting a common mechanism for the frameshift EPOR mutations. Interestingly, two proximal non-sense mutations, EPOR p.Glu399* and p.Glu425*, lacking 7 of the 8 cytoplasmic tyrosines that compose EPOR negative regulatory and internalization domains, were also able to confer a high EPO hypersensitivity to Ba/F3 cells. To our knowledge this is the first extensive functional study of EPOR mutations in PFCP. We highlighted that this pathology is much more complex than expected, since different mechanisms are involved in the EPO hypersensitivity phenotype, according to the type of EPOR mutation. Indeed, extensive truncations are sufficient by themselves to confer the EPO hypersensitivity phenotype due to the loss of all negative regulatory and internalization domains, whereas more distal truncations induced by frameshift mutants confer EPO hypersensitivity because of the appearance of a new C-terminal tail. The latter, by increasing EPOR stability at the cell surface, may cause pre-activation of both receptor and JAK2, constitutive signaling and hypersensitivity to EPO close to that of JAK2V617F-positive PVs. Disclosures No relevant conflicts of interest to declare.

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HELZ2 Promotes K63-Linked Polyubiquitination of c-Myc to Induce Retinoblastoma Tumorigenesis

10.21203/rs.3.rs-866845/v1 ◽

2021 ◽

Author(s):

Hanjun Dai ◽

wen ZENG ◽

WEIJUAN ZENG ◽

MING YAN ◽

ping jiang ◽

...

Keyword(s):

Cell Proliferation ◽

Zinc Finger ◽

Ubiquitin Ligase ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

Transcriptional Regulators ◽

Zinc Finger Domain ◽

Retinoblastoma Cell ◽

Animal Survival

Abstract Retinoblastoma is a rare ocular tumor in children that originates in the retina. Several core transcriptional regulators maintain the expansion of retinoblastoma tumors, including c-Myc. Here, we demonstrated that Helicase with zinc finger domain 2 (HELZ2) promoted retinoblastoma tumorigenesis by targeting c-Myc. HELZ2-deficient inhibited retinoblastoma cell proliferation, whereas overexpression of HELZ2 promoted retinoblastoma cell proliferation. In addition, high levels of HELZ2 promoted xenograft retinoblastoma tumorigenesis and inhibited animal survival. Mechanistically, HELZ2 interacted with c-Myc and promoted its K63-linked polyubiquitination. We indicated that HELZ2 promoted the interaction between E3 ubiquitin ligase HUWE1 and c-Myc, and HELZ2-mediated K63-linked polyubiquitination and activation of c-Myc were dependent on HUWE1. Taken together, HELZ2 plays a critical role in the regulation of retinoblastoma tumorigenesis by enhancing the activity of c-Myc.

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SIX1 Is a Novel Effector Gene in CALM-AF10 Leukemia

Blood ◽

10.1182/blood-2019-130178 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5035-5035

Author(s):

Waitman K. Aumann ◽

Catherine P. Lavau ◽

Amanda Harrington ◽

Amanda E. Conway ◽

Daniel S. Wechsler

Keyword(s):

Nuclear Export ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Critical Role ◽

Leukemia Cell ◽

The Self ◽

Hek293 Cells ◽

Hematopoietic Progenitors ◽

Self Renewal ◽

Hoxa Genes

Background: The CALM-AF10 translocation is detected in ~10% of T-cell acute lymphoblastic leukemias (T-ALLs), and in some acute myeloid leukemias (AMLs). CALM-AF10 leukemias are characterized by high expression of proleukemic HOXA genes, which serve a critical role in hematopoiesis. We hypothesized that identification of novel CALM-AF10 effector genes may yield new therapeutic targets in this difficult to treat leukemia. We took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal (NES) in CALM. Using next generation sequencing, we determined that, SIX1, similar to HOXA genes, is increased in CALM-AF10 leukemias and decreased in response to CRM1 inhibition with Leptomycin B (LMB). Design/Methods: RT-qPCR and Chromatin Immunoprecipitation were performed using both bone marrow progenitors and murine embryonic fibroblasts (MEFs) transduced with CALM-AF10 or an empty vector, with and without LMB. The ability of SIX1 to enhance self-renewal of hematopoietic progenitors was examined by measuring the colony-forming ability of transduced fetal liver hematopoietic progenitor cells. CRISPR-Cas9 was used to silence SIX1 in Human Embryonic Kidney 293 (HEK293) cells. Results: RT-qPCR confirmed overexpression of SIX1 in both CALM-AF10 transduced MEFs and CALM-AF10 leukemias, with decreased SIX1 expression observed in the presence of LMB. ChIP analysis showed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver cells was sufficient to increase the self-renewal potential of colony-forming progenitors. SIX1 was successfully knocked out in HEK293 cells without a significant effect on HEK293 proliferation. Conclusions: The SIX1 homeobox gene is highly expressed during development and its expression is silenced post-embryogenesis. Increased SIX1 expression has been reported in numerous solid tumors. We have determined that SIX1 is upregulated in CALM-AF10 leukemias, and increases the self-renewal potential of hematopoietic progenitors. Using CRISPR-Cas9 to silence SIX1, we have demonstrated that SIX1 is not essential for cell survival, and that its inhibition may impair CALM-AF10 leukemia cell proliferation. Thus, SIX1 may play a pathogenic role in leukemogenesis and is a potential therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

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Novel regulatory roles of Mff and Drp1 in E3 ubiquitin ligase MARCH5–dependent degradation of MiD49 and Mcl1 and control of mitochondrial dynamics

Molecular Biology of the Cell ◽

10.1091/mbc.e16-04-0208 ◽

2017 ◽

Vol 28 (3) ◽

pp. 396-410 ◽

Cited By ~ 37

Author(s):

Edward Cherok ◽

Shan Xu ◽

Sunan Li ◽

Shweta Das ◽

W. Alex Meltzer ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

Mitochondrial Morphology ◽

Ubiquitinated Proteins ◽

Terminal Domain ◽

Degradation Rates ◽

And Control

MARCH5, an OMM-associated E3 ubiquitin ligase, controls mitochondrial function. Despite its importance, the mechanism and factors controlling MARCH5 activity are largely unknown. Here we report that the MARCH5 C-terminal domain plays a critical role in degradation of MARCH5 substrates, likely by facilitating release of ubiquitinated proteins from the OMM. We also found that the mitochondrial fission proteins Drp1 and Mff negatively regulate MARCH5’s activity toward MiD49 and Mcl1. Knockouts of either Drp1 or Mff led to reduced expression, shorter half-lives, and increased ubiquitination of MiD49 and Mcl1. Effects of Mff and Drp1 depletion on degradation rates and ubiquitination of Mcl1 and MiD49 were eliminated in Drp1−/−/MARCH5−/− and Mff−/−/MARCH5−/− cells. Our data show that it is not mitochondrial morphology per se but rather Mff and Drp1 that directly control MARCH5. Consistently, we find that Mff is an integral component of the MARCH5/p97/Npl4 complex, which is also controlled by MARCH5’s C-terminal domain. Furthermore, not only mitochondrial fission but also fusion is regulated through Mff and Drp1 protein activities. Thus, in addition to their canonical roles in mitochondrial fission, Mff and Drp1 also act as regulatory factors that control mitochondrial fission and fusion.

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