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1.  AIP4/Itch Regulates Notch Receptor Degradation in the Absence of Ligand 
PLoS ONE  2008;3(7):e2735.
The regulation of Notch signaling heavily relies on ubiquitination events. Drosophila Su(dx), a member of the HECT family of ubiquitin-ligases, has been described as a negative regulator of Notch signaling, acting on the post-endocytic sorting of Notch. The mammalian ortholog of Su(dx), Itch/AIP4, has been shown to have multiple substrates, including Notch, but the precise events regulated by Itch/AIP4 in the Notch pathway have not been identified yet.
Methodology/Principal Findings
Using Itch-/- fibroblasts expressing the Notch1 receptor, we show that Itch is not necessary for Notch activation, but rather for controlling the degradation of Notch in the absence of ligand. Itch is indeed required after the early steps of Notch endocytosis to target it to the lysosomes where it is degraded. Furthermore Itch/AIP4 catalyzes Notch polyubiquitination through unusual K29-linked chains. We also demonstrate that although Notch is associated with Itch/AIP4 in cells, their interaction is not detectable in vitro and thus requires either a post-translational modification, or a bridging factor that remains to be identified.
Taken together our results identify a specific step of Notch regulation in the absence of any activation and underline differences between mammalian and Drosophila Notch pathways.
PMCID: PMC2444042  PMID: 18628966
2.  Spartin activates atrophin-1-interacting protein 4 (AIP4) E3 ubiquitin ligase and promotes ubiquitination of adipophilin on lipid droplets 
BMC Biology  2010;8:72.
Spartin protein is involved in degradation of epidermal growth factor receptor and turnover of lipid droplets and a lack of expression of this protein is responsible for hereditary spastic paraplegia type 20 (SPG20). Spartin is a multifunctional protein that associates with many cellular organelles, including lipid droplets. Recent studies showed that spartin interacts with E3 ubiquitin ligases that belong to the neural precursor cell-expressed developmentally downregulated gene (Nedd4) family, including atrophin-1-interacting protein 4 (AIP4/ITCH). However, the biological importance of the spartin-AIP4 interaction remains unknown.
In this study, we show that spartin is not a substrate for AIP4 activity and that spartin's binding to AIP4 significantly increases self-ubiquitination of this E3 ligase, indicating that spartin disrupts the AIP4 autoinhibitory intramolecular interaction. Correspondingly, spartin has a seven times higher binding affinity to the WW region of AIP4 than the binding of the WW region has to the catalytic homologues of the E6-associated protein C-terminus (HECT) domain, as measured by enzyme-linked immunosorbent assay. We also show that spartin recruits AIP4 to lipid droplets and promotes ubiquitination of lipid droplet-associated protein, adipophilin, which regulates turnover of lipid droplets.
Our findings demonstrate that spartin acts as an adaptor protein that activates and recruits AIP4 E3 ubiquitin ligase to lipid droplets and by this means regulates the level of ubiquitination of adipophilin and potentially other lipid-associated proteins. We propose that this is one of the mechanisms by which spartin regulates lipid droplet turnover and might contribute to the pathology of SPG20.
PMCID: PMC2887783  PMID: 20504295
3.  Nedd4 augments the adaptive immune response by promoting ubiquitin-mediated degradation of Cbl-b in activated T cells 
Nature immunology  2008;9(12):1356-1363.
Nedd4 and Itch are E3 ubiquitin ligases that, in vitro, ubiquitinate similar targets and thus are thought to function similarly. T cells lacking Itch show spontaneous activation and T helper type 2 (TH2) polarization. To test whether the loss of Nedd4 affects T cells in the same way, we generated Nedd4+/+ and Nedd4−/−fetal liver chimeras. Nedd4−/−T cells developed normally but proliferated less, produced less interleukin 2, and provided inadequate help to B cells. Nedd4−/−T cells contain increased amounts of Cbl-b protein, and Nedd4 was required for Cbl-b poly-ubquitination induced by CD28 co-stimulation. These data demonstrate that Nedd4 promotes the conversion of naive T cells into activated T cells. We propose that Nedd4 and Itch ubiquitinate distinct target proteins in vivo.
PMCID: PMC2935464  PMID: 18931680
4.  An essential role of ubiquitination in Cbl-mediated negative regulation of the Src-family kinase Fyn 
Signal transduction  2002;2(1-2):29-39.
The Cbl family of ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent lysosomal targeting. Here, we have investigated the role of Cbl ubiquitin ligase activity in the negative regulation of a non-receptor tyrosine kinase, the Src-family kinase Fyn. Using primary embryonic fibroblasts from Cbl+/+ and Cbl−/− mice, we demonstrate that endogenous Cbl mediates the ubiquitination of Fyn and dictates the rate of Fyn turnover. By analyzing CHO-TS20 cells with a temperature-sensitive ubiquitin activating enzyme, we demonstrate that intact cellular ubiquitin machinery is required for Cbl-induced degradation of Fyn. Analyses of Cbl mutants, with mutations in or near the RING finger domain, in 293T cells revealed that the ubiquitin ligase activity of Cbl is essential for Cbl-induced degradation of Fyn by the proteasome pathway. Finally, use of a SRE-luciferase reporter demonstrated that Cbl-dependent negative regulation of Fyn function requires the region of Cbl that mediates the ubiquitin ligase activity. Given the conservation of structure between various Src-family kinases and the ability of Cbl to interact with multiple members of this family, Cbl-dependent ubiquitination could serve a general role to negatively regulate activated Src-family kinases.
PMCID: PMC2788922  PMID: 19966925
tyrosine kinase; ubiquitin; regulation; degradation
5.  Cbl-c Ubiquitin Ligase Activity Is Increased via the Interaction of Its RING Finger Domain with a LIM Domain of the Paxillin Homolog, Hic 5 
PLoS ONE  2012;7(11):e49428.
Cbl proteins (Cbl, Cbl-b and Cbl-c) are ubiquitin ligases that are critical regulators of tyrosine kinase signaling. In this study we identify a new Cbl-c interacting protein, Hydrogen peroxide Induced Construct 5 (Hic-5). The two proteins interact through a novel interaction mediated by the RING finger of Cbl-c and the LIM2 domain of Hic-5. Further, this interaction is mediated and dependent on specific zinc coordinating complexes within the RING finger and LIM domain. Binding of Hic-5 to Cbl-c leads to an increase in the ubiquitin ligase activity of Cbl-c once Cbl-c has been activated by Src phosphorylation or through an activating phosphomimetic mutation. In addition, co-transfection of Hic-5 with Cbl-c leads to an increase in Cbl-c mediated ubiquitination of the EGFR. These data suggest that Hic-5 enhances Cbl-c ubiquitin ligase activity once Cbl-c has been phosphorylated and activated. Interactions between heterologous RING fingers have been shown to activate E3s. This is the first demonstration of enhancement of ubiquitin ligase activity of a RING finger ubiquitin ligase by the direct interaction of a LIM zinc coordinating domain.
PMCID: PMC3492284  PMID: 23145173
6.  Identification of Nedd4 E3 Ubiquitin Ligase as a Binding Partner and Regulator of MAK-V Protein Kinase 
PLoS ONE  2012;7(6):e39505.
MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function.
PMCID: PMC3379983  PMID: 22745772
7.  Ndfip1 Protein Promotes the Function of Itch Ubiquitin Ligase to Prevent T Cell Activation and T Helper 2 Cell-Mediated Inflammation 
Immunity  2006;25(6):929-940.
Nedd4 family interacting protein-1 (Ndfip1) is a protein whose only known function is that it binds Nedd4, a HECT-type E3 ubiquitin ligase. Here we show that mice lacking Ndfip1 developed severe inflammation of the skin and lung and died prematurely. This condition was due to a defect in Ndfip1−/− T cells. Ndfip1−/− T cells were activated, and they proliferated and adopted a T helper 2 (Th2) phenotype more readily than did their Ndfip1+/+ counterparts. This phenotype resembled that of Itchy mutant mice, suggesting that Ndfip1 might affect the function of Itch, an E3 ubiquitin ligase. We show that T cell activation promoted both Ndfip1 expression and its association with Itch. In the absence of Ndfip1, JunB half-life was prolonged after T cell activation. Thus, in the absence of Ndfip1, Itch is inactive and JunB accumulates. As a result, T cells produce Th2 cytokines and promote Th2-mediated inflammatory disease.
PMCID: PMC2955961  PMID: 17137798
8.  HIV-1 Nef Down-Modulates C-C and C-X-C Chemokine Receptors via Ubiquitin and Ubiquitin-Independent Mechanism 
PLoS ONE  2014;9(1):e86998.
Human and Simian Immunodeficiency virus (HIV-1, HIV-2, and SIV) encode an accessory protein, Nef, which is a pathogenesis and virulence factor. Nef is a multivalent adapter that dysregulates the trafficking of many immune cell receptors, including chemokine receptors (CKRs). Physiological endocytic itinerary of agonist occupied CXCR4 involves ubiquitinylation of the phosphorylated receptor at three critical lysine residues and dynamin-dependent trafficking through the ESCRT pathway into lysosomes for degradation. Likewise, Nef induced CXCR4 degradation was critically dependent on the three lysines in the C-terminal -SSLKILSKGK- motif. Nef directly recruits the HECT domain E3 ligases AIP4 or NEDD4 to CXCR4 in the resting state. This mechanism was confirmed by ternary interactions of Nef, CXCR4 and AIP4 or NEDD4; by reversal of Nef effect by expression of catalytically inactive AIP4-C830A mutant; and siRNA knockdown of AIP4, NEDD4 or some ESCRT-0 adapters. However, ubiquitinylation dependent lysosomal degradation was not the only mechanism by which Nef downregulated CKRs. Agonist and Nef mediated CXCR2 (and CXCR1) degradation was ubiquitinylation independent. Nef also profoundly downregulated the naturally truncated CXCR4 associated with WHIM syndrome and engineered variants of CXCR4 that resist CXCL12 induced internalization via an ubiquitinylation independent mechanism.
PMCID: PMC3906104  PMID: 24489825
9.  NEDD4-1 Is a Proto-Oncogenic Ubiquitin Ligase for PTEN 
Cell  2007;128(1):129-139.
The tumor suppressor PTEN, a critical regulator for multiple cellular processes, is mutated or deleted frequently in various human cancers. Subtle reductions in PTEN expression levels have profound impacts on carcinogenesis. Here we show that PTEN level is regulated by ubiquitin-mediated proteasomal degradation, and purified its ubiquitin ligase as HECT-domain protein NEDD4-1. In cells, NEDD4-1 negatively regulates PTEN stability by catalyzing PTEN polyubiquitination. Consistent with the tumor suppressive role of PTEN, overexpression of NEDD4-1 potentiated cellular transformation. Strikingly, in a mouse cancer model and multiple human cancer samples where the genetic background of PTEN was normal but its protein levels were low, NEDD4-1 was highly expressed, suggesting aberrant upregulation of NEDD4-1 can posttranslationally suppress PTEN in cancers. Elimination of NEDD4-1 expression inhibited xenotransplanted tumor growth in a PTEN-dependent manner. Therefore, NEDD4-1 is a potential proto-oncogene that negatively regulates PTEN via ubiquitination, a paradigm analogous to that of Mdm2 and p53.
PMCID: PMC1828909  PMID: 17218260
10.  PY Motifs of Epstein-Barr Virus LMP2A Regulate Protein Stability and Phosphorylation of LMP2A-Associated Proteins 
Journal of Virology  2001;75(12):5711-5718.
Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. We have demonstrated that Nedd4 family ubiquitin-protein ligases (E3s), AIP4, WWP2/AIP2, and Nedd4, bind specifically to two PY motifs present within the LMP2A amino-terminal domain. In this study, LMP2A PY motif mutant viruses were constructed to investigate the role of the LMP2A PY motifs. AIP4 was found to specifically associate with the LMP2A PY motifs in EBV-transformed lymphoblastoid cell lines (LCLs), extending our original observation to EBV-infected cells. Mutation of both of the LMP2A PY motifs resulted in an absence of binding of AIP4 to LMP2A, which resulted in an increase in the expression of Lyn and the constitutive hyperphosphorylation of LMP2A and an unknown 120-kDa protein. In addition, there was a modest increase in the constitutive phosphorylation of Syk and an unidentified 60-kDa protein. These results indicate that the PY motifs contained within LMP2A are important in regulating phosphorylation in EBV-infected LCLs, likely through the regulation of Lyn activity by specifically targeting the degradation of Lyn by ubiquination by Nedd4 family E3s. Despite differences between PY motif mutant LCLs and wild-type LCLs, the PY motif mutants still exhibited a block in B-cell receptor (BCR) signal transduction as measured by the induction of tyrosine phosphorylation and BZLF1 expression following BCR activation. EBV-transformed LCLs with mutations in the PY motifs were not different from wild-type LCLs in serum-dependent cell growth. Protein stability of LMP1, which colocalizes with LMP2A, was not affected by the LMP2A-associated E3s.
PMCID: PMC114286  PMID: 11356981
11.  E3 Ubiquitin Ligase Cbl-b Regulates Pten via Nedd4 in T Cells Independently of Its Ubiquitin Ligase Activity 
Cell reports  2012;1(5):472-482.
E3 ubiquitin ligase Cbl-b plays a crucial role in T cell activation and tolerance induction. However, the molecular mechanism by which Cbl-b inhibits T cell activation remains unclear. Here we report that Cbl-b does not inhibit PI3-K, but rather suppresses TCR/CD28-induced inactivation of Pten. The elevated Akt activity in Cbl-b−/− T cells is therefore due to heightened Pten inactivation. Suppression of Pten inactivation in T cells by Cbl-b is achieved by impeding the association of Pten with Nedd4, which targets Pten K13 for K63-linked polyubiquitination. Consistent with this finding, introducing Nedd4 deficiency into Cbl-b−/− mice abrogates hyper-T cell responses caused by the loss of Cbl-b. Hence, our data are the first to demonstrate that Cbl-b inhibits T cell activation by suppressing Pten inactivation independently of its ubiquitin ligase activity.
PMCID: PMC3387815  PMID: 22763434
12.  Cutting Edge: The Transmembrane E3 Ligase GRAIL Ubiquitinates the Costimulatory Molecule CD40 Ligand during the Induction of T Cell Anergy1 
Activation of naive T lymphocytes is regulated through a series of discrete checkpoints that maintain unresponsiveness to self. During this multistep process, costimulatory interactions act as inducible signals that allow APCs to selectively mobilize T cells against foreign Ags. In this study, we provide evidence that the anergy-associated E3 ubiquitin ligase GRAIL (gene related to anergy in lymphocytes) regulates expression of the costimulatory molecule CD40L on CD4 T cells. Using its luminal protease-associated domain, GRAIL binds to the luminal/extracellular portion of CD40L and facilitates transfer of ubiquitin molecules from the intracellular GRAIL RING (really interesting new gene) finger to the small cytosolic portion of CD40L. Down-regulation of CD40L occurred following ectopic expression of GRAIL in naive T cells from CD40−/− mice, and expression of GRAIL in bone marrow chimeric mice was associated with diminished lymphoid follicle formation. These data provide a model for intrinsic T cell regulation of costimulatory molecules and a molecular framework for the initiation of clonal T cell anergy.
PMCID: PMC2853377  PMID: 18641297
13.  Itchy, a Nedd4 Ubiquitin Ligase, Downregulates Latent Membrane Protein 2A Activity in B-Cell Signaling 
Journal of Virology  2003;77(9):5529-5534.
Nedd4 family ubiquitin protein ligases (E3s) specifically associate with latent membrane protein 2A (LMP2A) of Epstein-Barr virus. Our previous studies analyzing LMP2A function in vitro have suggested that Nedd4 family E3s regulate LMP2A function. To determine the role of Nedd4 family E3s in LMP2A B-cell signaling, LMP2A transgenic (LMP2A+) mice were crossed with mice with the Itch-deficient (Itch−/−) background. Itchy, a mouse homologue of human AIP4, is a Nedd4 family E3 and is also the most abundant Nedd4 family E3 found in LMP2A affinity precipitates from B cells. There were significantly fewer B-cell receptor-positive B cells in spleen and bone marrow B cells in LMP2A+ Itch−/− mice than in LMP2A+ mice. In addition, LMP2A+ Itch−/− bone marrow B cells formed larger colonies in cultures treated with interleukin-7 (IL-7) than control bone marrow B cells did. Finally, there was a dramatic increase in tyrosine phosphorylation of LMP2A and Syk in IL-7-cultured LMP2A+ Itch−/− B cells. These results indicate that Nedd4 family E3s, in particular Itchy, downmodulate LMP2A activity in B-cell signaling.
PMCID: PMC153961  PMID: 12692257
14.  HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK ▿  
Molecular and Cellular Biology  2010;30(6):1541-1554.
ACK (activated Cdc42-associated tyrosine kinase) (also Tnk2) is an ubiquitin-binding protein and plays an important role in ligand-induced and ubiquitination-mediated degradation of epidermal growth factor receptor (EGFR). Here we report that ACK is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with EGFR in response to EGF stimulation. ACK interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to ACK. Although ACK binds to both Nedd4-1 and Nedd4-2 (also Nedd4L), Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK in cells. Interestingly, deletion of the sterile alpha motif (SAM) domain at the N terminus dramatically reduced the ubiquitination of ACK by Nedd4-1, while deletion of the Uba domain dramatically enhanced the ubiquitination. Use of proteasomal and lysosomal inhibitors demonstrated that EGF-induced ACK degradation is processed by lysosomes, not proteasomes. RNA interference (RNAi) knockdown of Nedd4-1, not Nedd4-2, inhibited degradation of both EGFR and ACK, and overexpression of ACK mutants that are deficient in either binding to or ubiquitination by Nedd4-1 blocked EGF-induced degradation of EGFR. Our findings suggest an essential role of Nedd4-1 in regulation of EGFR degradation through interaction with and ubiquitination of ACK.
PMCID: PMC2832494  PMID: 20086093
15.  The E3 Ubiquitin Ligase Atrophin Interacting Protein 4 Binds Directly To The Chemokine Receptor CXCR4 Via a Novel WW Domain-mediated Interaction 
Molecular Biology of the Cell  2009;20(5):1324-1339.
The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.
PMCID: PMC2649280  PMID: 19116316
16.  The Role of E3 Ubiquitin Ligase Cbl Proteins in Interleukin-2-Induced Jurkat T-Cell Activation 
BioMed Research International  2013;2013:430861.
Interleukin- (IL-) 2 is the major growth factor for T-cell activation and proliferation. IL-2 has multiple functions in the regulation of immunological processes. Although most studies focus on T-cell immunomodulation, T-cell activation by IL-2 is the foundation of priming the feedback loop. Here, we investigated the effect of MAPK/ERK and PI3K/Akt signaling pathways on IL-2-induced cell activation and the regulatory mechanisms of upstream ubiquitin ligase Cbl-b and c-Cbl. Morphological analysis of Jurkat T cells was performed by cytospin preparations with Wright-Giemsa stain. CD25 expression on Jurkat T cells was determined by flow cytometry. Changes in cell activation proteins such as p-ERK, ERK, p-Akt, Akt, and ubiquitin ligase Casitas B-cell Lymphoma (Cbl) proteins were analyzed by western blot. Following IL-2-induced activation of Jurkat T cells, p-ERK expression was upregulated, while there was no change in p-Akt, ERK, or Akt expression. Thus, the MAPK/ERK signaling pathway, but not PI3K/Akt, was involved in IL-2-induced T-cell activation. Either using PD98059 (a specific inhibitor for p-ERK) or depletion of ERK with small interfering RNA (siRNA) reduced the expression of CD25. This study also showed that ubiquitin ligase proteins Cbl-b and c-Cbl might be involved in IL-2-induced Jurkat T-cell activation by negatively regulating the MAPK/ERK signaling pathway.
PMCID: PMC3622291  PMID: 23586039
17.  Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions 
The Nedd4 (neural precursor cell-expressed developmentally downregulated gene 4) family of ubiquitin ligases (E3s) is characterized by a distinct modular domain architecture, with each member consisting of a C2 domain, 2–4 WW domains, and a HECT-type ligase domain. Of the nine mammalian members of this family, Nedd4 and its close relative, Nedd4-2, represent the ancestral ligases with strong similarity to the yeast, Rsp5. In Saccharomyces cerevisiae Rsp5 has a key role in regulating the trafficking, sorting, and degradation of a large number of proteins in multiple cellular compartments. However, in mammals the Nedd4 family members, including Nedd4 and Nedd4-2, appear to have distinct functions, thereby suggesting that these E3s target specific proteins for ubiquitylation. In this article we focus on the biology and emerging functions of Nedd4 and Nedd4-2, and review recent in vivo studies on these E3s.
PMCID: PMC2818775  PMID: 19557014
ubiquitylation; endocytosis; trafficking; receptor; signaling
18.  The E3 Ubiquitin Ligase Activity of Trip12 Is Essential for Mouse Embryogenesis 
PLoS ONE  2011;6(10):e25871.
Protein ubiquitination is a post-translational protein modification that regulates many biological conditions [1], [2], [3], [4]. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1 [5], [6]. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12mt/mt) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16 [7], [8], [9], [10]. In contrast, Trip12mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.
PMCID: PMC3196520  PMID: 22028794
19.  Inhibition of NEDD8 Conjugation Pathway by Novel Molecules: Potential Approaches to Anticancer Therapy 
Molecular oncology  2012;6(3):10.1016/j.molonc.2012.01.003.
Cancer cells can survive through the upregulation of cell cycle and the escape from apoptosis induced by numerous cellular stresses. In the normal cells, these biological cascades depend on scheduled proteolytic degradation of regulatory proteins via the ubiquitin-proteasome pathway. Therefore, interruption of regulated proteolytic pathways leads to abnormal cell-proliferation. Ubiquitin ligases called SCF complex (consisting of Skp-1, cullin, and F-box protein) or CRL (cullin-RING ubiquitin ligase) are predominant in a family of E3 ubiquitin ligases that control a final step in ubiquitination of diverse substrates. To a great extent, the ubiquitin ligase activity of the SCF complex requires the conjugation of NEDD8 to cullins, i.e. scaffold proteins. This review is anticipated to review the downregulation system of NEDD8 conjugation by several factors including a chemical compound such as MLN4924 and protein molecules (e.g. COP9 signalosome, inactive mutant of Ubc12, and NUB1/NUB1L). Since the downregulation of NEDD8 conjugation affects cell cycle progression by inhibiting the ligase activity of SCF complexes, such knowledge in the NEDD8 conjugation pathway will contribute to the more magnificent therapies that selectively suppress tumorigenesis.
PMCID: PMC3826113  PMID: 22306028
Ubiquitination; SCF complex; NEDD8; MLN4924; Ubc12; NUB1
20.  Ubiquitylation of a Melanosomal Protein by HECT-E3 Ligases Serves as Sorting Signal for Lysosomal DegradationD⃞ 
Molecular Biology of the Cell  2005;16(4):1777-1787.
The production of pigment by melanocytic cells of the skin involves a series of enzymatic reactions that take place in specialized organelles called melanosomes. Melan-A/MART-1 is a melanocytic transmembrane protein with no enzymatic activity that accumulates in vesicles at the trans side of the Golgi and in melanosomes. We show here that, in melanoma cells, Melan-A associates with two homologous to E6-AP C-terminus (HECT)-E3 ubiquitin ligases, NEDD4 and Itch, and is ubiquitylated. Both NEDD4 and Itch participate in the degradation of Melan-A. A mutant Melan-A lacking ubiquitin-acceptor residues displays increased half-life and, in pigmented cells, accumulates in melanosomes. These results suggest that ubiquitylation regulates the lysosomal sorting and degradation of Melan-A/MART-1 from melanosomes in melanocytic cells.
PMCID: PMC1073660  PMID: 15703212
21.  NOPO modulates Egr-induced JNK-independent cell death in Drosophila 
Cell Research  2011;22(2):425-431.
Tumor necrosis factor (TNF) family ligands play essential roles in regulating a variety of cellular processes including proliferation, differentiation and survival. Expression of Drosophila TNF ortholog Eiger (Egr) induces JNK-dependent cell death, while the roles of caspases in this process remain elusive. To further delineate the Egr-triggered cell death pathway, we performed a genetic screen to identify dominant modifiers of the Egr-induced cell death phenotype. Here we report that Egr elicits a caspase-mediated cell death pathway independent of JNK signaling. Furthermore, we show NOPO, the Drosophila ortholog of TRIP (TRAF interacting protein) encoding an E3 ubiquitin ligase, modulates Egr-induced Caspase-mediated cell death through transcriptional activation of pro-apoptotic genes reaper and hid. Finally, we found Bendless and dUEV1a, an ubiquitin-conjugating E2 enzyme complex, regulates NOPO-triggered cell death. Our results indicate that the Ben-dUEV1a complex constitutes a molecular switch that bifurcates the Egr-induced cell death signaling into two pathways mediated by JNK and caspases respectively.
PMCID: PMC3271591  PMID: 21844890
cTNF; JNK; NOPO; caspase; cell death
22.  Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release 
BMC Biochemistry  2009;10:12.
The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in Drosophila.
In this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues in vivo and in vitro. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1ΔPTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSHV14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1ΔPTAP L-domain mutant (YPXnL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPXnL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release.
Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPXnL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable.
PMCID: PMC2680910  PMID: 19393081
23.  The E3 Ubiquitin Ligase ITCH Negatively Regulates Canonical Wnt Signaling by Targeting Dishevelled Protein 
Molecular and Cellular Biology  2012;32(19):3903-3912.
Dishevelled (Dvl) is a key component in the canonical Wnt signaling pathway and becomes hyperphosphorylated upon Wnt stimulation. Dvl is required for LRP6 phosphorylation, which is essential for subsequent steps of signal transduction, such as Axin recruitment and cytosolic β-catenin stabilization. Here, we identify the HECT-containing Nedd4-like ubiquitin E3 ligase ITCH as a new Dvl-binding protein. ITCH ubiquitinates the phosphorylated form of Dvl and promotes its degradation via the proteasome pathway, thereby inhibiting canonical Wnt signaling. Knockdown of ITCH by RNA interference increased the stability of phosphorylated Dvl and upregulated Wnt reporter gene activity as well as endogenous Wnt target gene expression induced by Wnt stimulation. In addition, we found that both the PPXY motif and the DEP domain of Dvl are critical for its interaction with ITCH, as mutation in the PPXY motif (Dvl2-Y568F) or deletion of the DEP domain led to reduced affinity for ITCH. Consistently, overexpression of ITCH inhibited wild-type Dvl2-induced, but not Dvl2-Y568F mutant-induced, Wnt reporter activity. Moreover, the Y568F mutant, but not wild-type Dvl2, can reverse the ITCH-mediated inhibition of Wnt-induced reporter activity. Collectively, these results indicate that ITCH plays a negative regulatory role in modulating canonical Wnt signaling by targeting the phosphorylated form of Dvl.
PMCID: PMC3457526  PMID: 22826439
24.  Enigma Prevents Cbl-c-Mediated Ubiquitination and Degradation of RETMEN2A 
PLoS ONE  2014;9(1):e87116.
The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation.
PMCID: PMC3900716  PMID: 24466333
25.  USP8 links the PTEN-Akt-AIP4 pathway to the control of FLIPS stability and TRAIL sensitivity in glioblastoma multiforme 
Cancer research  2010;70(12):5046-5053.
The anti-apoptotic protein FLIPS is a key suppressor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis in human glioblastoma multiforme (GBM) cells. We previously reported that a novel phosphatase and tensin homolog (PTEN)-Akt-atrophin interacting protein 4 (AIP4) pathway regulates FLIPS ubiquitination and stability, although the means by which PTEN and Akt were linked to AIP4 activity were unclear. We here report that a second regulator of ubiquitin metabolism, the ubiquitin-specific protease (USP) 8, is a downstream target of Akt, and that USP8 links Akt to AIP4 and the regulation of FLIPS stability and TRAIL resistance. In human GBM xenografts, levels of USP8 correlated inversely with pAkt levels, and genetic or pharmacologic manipulation of Akt regulated USP8 levels in an inverse manner. Over-expression of WT USP8, but not catalytically inactive USP8, increased FLIPS ubiquitination, decreased FLIPS half-life, decreased FLIPS steady-state levels, and decreased TRAIL resistance, while siRNA-mediated suppression of USP8 levels had the opposite effects. Because high levels of the USP8 deubiquitinase correlated with high levels of FLIPS ubiquitination, USP8 appeared to control FLIPS ubiquitination through an intermediate target. Consistent with this idea, over-expression of WT USP8 decreased ubiquitination of the FLIPS E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIPS interaction, while siRNA-mediated suppression of USP8 increased AIP4 ubiquitination. Furthermore, the suppression of FLIPS levels by USP8 over-expression was reversed by introduction of siRNA targeting AIP4. These results show that USP8, a downstream target of Akt, regulates the ability of AIP4 to control FLIPS stability and TRAIL sensitivity.
PMCID: PMC2891229  PMID: 20484045
glioblastoma; TRAIL; ubiquitin; PTEN; USP8

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