E3 Ubiquitin Ligase RNF125 Activates Interleukin-36 Receptor Signaling and Contributes to Its Turnover

Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R.

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IL-3 and Retinoic Acid-Induced Myeloid Differentiation of Hematopoietic Progenitors Is Negatively Regulated by E3 Ubiquitin Ligase Rnf41 (FLRF).

Blood ◽

10.1182/blood.v106.11.3160.3160 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3160-3160

Author(s):

Xin Jing ◽

Luis Dabul ◽

Ronald G. Nachtman ◽

Roland Jurecic

Keyword(s):

Retinoic Acid ◽

Steady State ◽

Ligand Binding ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Myeloid Differentiation ◽

Hematopoietic Progenitors ◽

Induced Differentiation ◽

Gm Csf ◽

Steady State Levels

Abstract The proliferation and differentiation of hematopoietic cells is regulated by interaction of diverse group of cytokines with corresponding receptors. Regulation of cytokine signaling occurs at multiple levels, including the regulation of the amount of receptors present before and after ligand binding. However, the mechanisms that govern pre-determined steady-state amount of cytokine receptors are poorly understood. Previously we have reported that new E3 ubiquitin ligase Rnf41 (FLRF/Nrdp1) regulates cytokine-induced differentiation of hematopoietic progenitors through negative regulation of steady-state receptor levels. Increased levels of Rnf41 protein significantly attenuate differentiation of multipotent progenitors in response to IL-3, Epo and GM-CSF, due to a significant and constitutive decrease in the steady-state amount of IL-3, Epo and GM-CSF receptors. Immunoprecipitation and Western analysis of proteins from several types of hematopoietic progenitors has confirmed that Rnf41 protein associates with IL-3, Epo and GM-CSF receptors, and has shown that Rnf41-mediated down-regulation of receptors is independent of the ligand binding. Thus, by regulating steady-state amounts of IL-3, Epo and GM-CSF receptors Rnf41 could be maintaining optimal levels of signaling necessary for proper lineage commitment and differentiation of HSC and progenitors. Retinoic acid (RA) is known to augment IL-3 and GM-CSF induced differentiation of progenitors into myeloid lineages. Surprisingly, instead of improving the RA treatment further suppressed IL-3 and GM-CSF-induced myeloid differentiation of hematopoietic progenitors over-expressing Rnf41. The RA functions through RARα and RXR receptors, whihc act as transcription factors and interact with specific DNA targets as hetero- (RAR-RXR) or homodimers (RXR-RXR). Interestingly, Western analysis has shown that hematopoietic progenitors over-expressing Rnf41 exhibit a reduction in the steady-state levels of RARα receptors, whereas levels of RXR receptors remain unchanged. Moreover, the treatment of hematopoietic progenitors over-expressing Rnf41 with RA leads to even further decrease of RARα receptor levels. The transient over-expression of Rnf41 in BaF3 cell line also decreases steady-state levels of RARα receptors, while RXR receptor levels remain unchanged. Protein IP with α-Rnf41, α-RARα or α-RXR antibodies has shown that endogenous Rnf41 protein associates with RARα receptors in hematopoietic progenitors. Taken together, these results suggest that Rnf41 influences RA-mediated myeloid differentiation of hematopoietic progenitors by negatively regulating the levels of RARα receptors. Several studies have reported that IL-3 and GM-CSF-induced myeloid differentiation of hematopoietic progenitors leads to activation of RARa through induction of the Jak2/Stat5 pathway. These studies have defined a previously unknown cytokine-RAR interaction during myelopoiesis and suggested that RAR activation might be a critical downstream event following IL-3 and GM-CSF signaling during myeloid differentiation. Combined together the existing data suggest a model in which Rnf41 negatively regulates myeloid differentiation of hematopoietic progenitors by independently regulating steady state levels of IL-3, GM-CSF and RAR receptors, and thus impacting cytokine and RA signaling. Ongoing studies with RA agonists and antagonists are aimed at unraveling further the role of Rnf41 in regulation of RA signaling during progenitor cell differentiation.

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Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10941-10953.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10941-10953 ◽

Cited By ~ 748

Author(s):

Donna D. Zhang ◽

Shih-Ching Lo ◽

Janet V. Cross ◽

Dennis J. Templeton ◽

Mark Hannink

Keyword(s):

Oxidative Stress ◽

Steady State ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Adaptor Protein ◽

Bzip Transcription Factor ◽

Critical Determinant ◽

Ubiquitin Ligase Complex ◽

Steady State Levels

ABSTRACT The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.

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Inhibition of retroviral replication by anti-sense RNA

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4758-4762.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4758-4762

Author(s):

R Y To ◽

S C Booth ◽

P E Neiman

Keyword(s):

Steady State ◽

Cultured Cells ◽

Host Cells ◽

Rna Molecules ◽

Sense Orientation ◽

Steady State Levels ◽

Rna Inhibition ◽

Retroviral Replication ◽

Retroviral Infections ◽

Neomycin Resistance

We tested the effect of anti-sense RNA on the replication of avian retroviruses in cultured cells. The replication of a recombinant retrovirus carrying a neomycin resistance gene (neor) in the anti-sense orientation was blocked when the cells expressed high steady-state levels of RNA molecules with neor in sequence in the sense was blocked when the cells expressed high steady-state levels of RNA molecules with neor sequences in the sense orientation, i.e., complementary to the viral sequence. Viral DNA bearing neor sequences was not detected specifically in host cells where this anti-sense RNA inhibition of viral replication occurred. These observations suggest that anti-sense RNA inhibition may be a useful strategy for the inhibition of retroviral infections.

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TRIM65 in White Matter Lesions, Innate Immunity and Tumor

Current Molecular Pharmacology ◽

10.2174/1874467214666210203211603 ◽

2021 ◽

Vol 14 ◽

Author(s):

Bingxue Liu ◽

Yunlian Tang ◽

Ping Yang ◽

Chu Wu ◽

Yue Huang

Keyword(s):

Signal Transduction ◽

Innate Immunity ◽

White Matter ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

White Matter Lesions ◽

Intracellular Signal Transduction ◽

Cellular Processes ◽

Tripartite Motif ◽

Intracellular Signal

Abstract: Most of the tripartite motif (TRIM) family proteins have E3 ubiquitin ligase activities and also have a variety of functions in cellular processes such as intracellular signal transduction, apoptosis, innate immunity and carcinogenesis. TRIM65 is a member of the TRIM family. More and more evidences have shown a unique and importance of TRIM65 protein in the occurrence and development of some diseases. In this review, the importance on TRIM65 in white matter lesions, innate immunity and the effect of tumors was mainly reviewed.

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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 E3 ubiquitin ligase mind bomb 1 ubiquitinates and promotes the degradation of survival of motor neuron protein

Molecular Biology of the Cell ◽

10.1091/mbc.e13-01-0042 ◽

2013 ◽

Vol 24 (12) ◽

pp. 1863-1871 ◽

Cited By ~ 32

Author(s):

Deborah Y. Kwon ◽

Maria Dimitriadi ◽

Barbara Terzic ◽

Casey Cable ◽

Anne C. Hart ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Ubiquitin Ligase ◽

Cultured Cells ◽

Muscular Atrophy ◽

E3 Ubiquitin Ligase ◽

Proteasome Inhibition ◽

Protein Levels ◽

Smn Protein ◽

Survival Of Motor Neuron

Spinal muscular atrophy is an inherited motor neuron disease that results from a deficiency of the survival of motor neuron (SMN) protein. SMN is ubiquitinated and degraded through the ubiquitin proteasome system (UPS). We have previously shown that proteasome inhibition increases SMN protein levels, improves motor function, and reduces spinal cord, muscle, and neuromuscular junction pathology of spinal muscular atrophy (SMA) mice. Specific targets in the UPS may be more efficacious and less toxic. In this study, we show that the E3 ubiquitin ligase, mind bomb 1 (Mib1), interacts with and ubiquitinates SMN and facilitates its degradation. Knocking down Mib1 levels increases SMN protein levels in cultured cells. Also, knocking down the Mib1 orthologue improves neuromuscular function in Caenorhabditis elegans deficient in SMN. These findings demonstrate that Mib1 ubiquitinates and catalyzes the degradation of SMN, and thus represents a novel therapeutic target for SMA.

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An orthogonal c-Cbl recognition mode targets LynA for rapid degradation and builds specificity into the LynA checkpoint

10.1101/550053 ◽

2019 ◽

Author(s):

Ben F. Brian ◽

Myra G. Nunez ◽

Kathryn L. Schwertfeger ◽

Tanya S. Freedman

Keyword(s):

Steady State ◽

Ubiquitin Ligase ◽

Biochemical Analysis ◽

Cell Activation ◽

Critical Factor ◽

Myeloid Cell ◽

State Level ◽

Cell Specificity ◽

Steady State Levels ◽

Insert Region

AbstractThe activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is critical factor regulating myeloid-cell activation. In a previous paper (Freedman et al., 2015) we showed in macrophages that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation, functioning as a rheostat regulating ITAM signaling. We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic and biochemical analysis, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via tyrosine 32 in its unique insert region. This orthogonal mode of c-Cbl recognition depresses the steady-state level of macrophage LynA. Mast cells, however, express little c-Cbl and have correspondingly high steady-state levels of LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression therefore builds cell specificity into the LynA checkpoint.

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