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1.
PLoS One ; 7(3): e33786, 2012.
Article in English | MEDLINE | ID: mdl-22442722

ABSTRACT

BACKGROUND: Necdin, a MAGE family protein expressed primarily in the nervous system, has been shown to interact with both nuclear and cytoplasmic proteins, but the mechanism of its nucleocytoplasmic transport are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a large-scale interaction screen using necdin as a bait in the yeast RRS system, and found a wide range of potential interactors with different subcellular localizations, including over 60 new candidates for direct binding to necdin. Integration of these interactions into a comprehensive network revealed a number of coherent interaction modules, including a cytoplasmic module connecting to necdin through huntingtin-associated protein 1 (Hap1), dynactin and hip-1 protein interactor (Hippi); a nuclear P53 and Creb-binding-protein (Crebbp) module, connecting through Crebbp and WW domain-containing transcription regulator protein 1 (Wwtr1); and a nucleocytoplasmic transport module, connecting through transportins 1 and 2. We validated the necdin-transportin1 interaction and characterized a sequence motif in necdin that modulates karyopherin interaction. Surprisingly, a D234P necdin mutant showed enhanced binding to both transportin1 and importin ß1. Finally, exclusion of necdin from the nucleus triggered extensive cell death. CONCLUSIONS/SIGNIFICANCE: These data suggest that necdin has multiple roles within protein complexes in different subcellular compartments, and indicate that it can utilize multiple karyopherin-dependent pathways to modulate its localization.


Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Active Transport, Cell Nucleus/physiology , Amino Acid Motifs , Animals , Cell Nucleus/genetics , Cytoplasm/genetics , HEK293 Cells , Humans , Mice , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , PC12 Cells , Protein Binding/physiology , Rats
2.
J Biol Chem ; 286(2): 1453-63, 2011 Jan 14.
Article in English | MEDLINE | ID: mdl-21068390

ABSTRACT

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Endoplasmic Reticulum/enzymology , Membrane Proteins/metabolism , Signal Transduction/physiology , Stress, Physiological/physiology , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Endoribonucleases/metabolism , HEK293 Cells , HeLa Cells , Humans , In Vitro Techniques , Membrane Proteins/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational/physiology , Protein Serine-Threonine Kinases/metabolism , Transfection , Ubiquitination/physiology
3.
Cell ; 143(5): 686-93, 2010 Nov 24.
Article in English | MEDLINE | ID: mdl-21111230

ABSTRACT

Protein phosphorylation and protein ubiquitination regulate most aspects of cell life, and defects in these control mechanisms cause cancer and many other diseases. In the past decade, protein kinases have become one of the most important classes of drug targets for the pharmaceutical industry. In contrast, drug discovery programs that target components of the ubiquitin system have lagged behind. In this Perspective, we discuss the reasons for the delay in this pipeline, the drugs targeting the ubiquitin system that have been developed, and new approaches that may popularize this area of drug discovery in the future.


Subject(s)
Drug Discovery , Protein Kinase Inhibitors/pharmacology , Ubiquitin/metabolism , Ubiquitination/drug effects , Animals , Humans , Protein Kinase Inhibitors/metabolism , Proteins/metabolism , Ubiquitin-Protein Ligases/antagonists & inhibitors
4.
Nat Genet ; 42(2): 181-5, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20081860

ABSTRACT

Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells. Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-kappaB pathway that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified. Here we report recurrent somatic mutations affecting the polycomb-group oncogene EZH2, which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27). After the recent discovery of mutations in KDM6A (UTX), which encodes the histone H3K27me3 demethylase UTX, in several cancer types, EZH2 is the second histone methyltransferase gene found to be mutated in cancer. These mutations, which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641), occur in 21.7% of GCB DLBCLs and 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro.


Subject(s)
DNA-Binding Proteins/genetics , Germinal Center/metabolism , Germinal Center/pathology , Lymphoma, Follicular/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Mutation/genetics , Transcription Factors/genetics , Adult , Aged , Amino Acid Sequence , Base Sequence , DNA Mutational Analysis , DNA-Binding Proteins/chemistry , Enhancer of Zeste Homolog 2 Protein , Exons/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genome, Human/genetics , Humans , Male , Middle Aged , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Polycomb Repressive Complex 2 , Transcription Factors/chemistry , Tyrosine/genetics
5.
Neuron ; 63(5): 585-91, 2009 Sep 10.
Article in English | MEDLINE | ID: mdl-19755102

ABSTRACT

The TrkA receptor tyrosine kinase is crucial for differentiation and survival of nerve-growth-factor-dependent neurons. Paradoxically, TrkA also induces cell death in pediatric tumor cells of neural origin, via an unknown mechanism. Here, we show that CCM2, a gene product associated with cerebral cavernous malformations, interacts with the juxtamembrane region of TrkA via its phosphotyrosine binding (PTB) domain and mediates TrkA-induced death in diverse cell types. Both the PTB and Karet domains of CCM2 are required for TrkA-dependent cell death, such that the PTB domain determines the specificity of the interaction, and the Karet domain links to death pathways. Downregulation of CCM2 in medulloblastoma or neuroblastoma cells attenuates TrkA-dependent death. Combined high expression levels of CCM2 and TrkA are correlated with long-term survival in a large cohort of human neuroblastoma patients. Thus, CCM2 is a key mediator of TrkA-dependent cell death in pediatric neuroblastic tumors.


Subject(s)
Apoptosis/physiology , Carrier Proteins/metabolism , Microfilament Proteins/metabolism , Neuroblastoma/physiopathology , Receptor, trkA/metabolism , Animals , Carrier Proteins/genetics , Cell Death/physiology , Cell Line , Cells, Cultured , Humans , Medulloblastoma/physiopathology , Mice , Microfilament Proteins/genetics , Mutation , Neuroblastoma/diagnosis , PC12 Cells , Prognosis , Rats , Receptor, Nerve Growth Factor/metabolism , Receptor, trkA/genetics , Receptor, trkB/genetics , Receptor, trkB/metabolism
6.
J Biol Chem ; 284(18): 12099-109, 2009 May 01.
Article in English | MEDLINE | ID: mdl-19269966

ABSTRACT

Clearance of misfolded proteins by endoplasmic reticulum (ER)-associated degradation (ERAD) requires concerted activity of chaperones, adaptor proteins, ubiquitin ligases, and proteasomes. RNF5 is a ubiquitin ligase anchored to the ER membrane implicated in ERAD via ubiquitination of misfolded proteins. Among RNF5-associated proteins is JNK-associated membrane protein (JAMP), a 7-transmembrane protein located within the ER membrane that facilitates degradation of misfolded proteins through recruitment of proteasomes and ERAD regulatory components. Here we demonstrate that RNF5 associates with JAMP in the ER membrane. This association results in Ubc13-dependent RNF5-mediated noncanonical ubiquitination of JAMP. This ubiquitination does not alter JAMP stability but rather inhibits its association with Rpt5 and p97. Consequently, clearance of misfolded proteins, such as CFTRDelta508 and T cell receptor alpha, is less efficient, resulting in their greater accumulation. Significantly, the RNF5 effect on JAMP is seen prior to and after ER stress response, thereby highlighting a novel mechanism to limit ERAD and proteasome assembly at the ER, to the actual ER stress response.


Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Protein Folding , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination/physiology , Carrier Proteins/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , DNA-Binding Proteins/genetics , Endoplasmic Reticulum/genetics , HeLa Cells , Humans , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Stress, Physiological/physiology , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Protein Ligases
7.
Pigment Cell Melanoma Res ; 22(2): 187-95, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19175524

ABSTRACT

The AKT/PKB pathway plays a central role in tumor development and progression and is often up-regulated in different tumor types, including melanomas. We have recently reported on the in silico approach to identify putative inhibitors for AKT/PKB. Of the reported hits, we selected BI-69A11, a compound which was shown to inhibit AKT activity in in vitro kinase assays. Analysis of BI-69A11 was performed in melanoma cells, a tumor type that commonly exhibits up-regulation of AKT. Treatment of the UACC903 human melanoma cells, harboring the PTEN mutation, with BI-69A11 caused efficient inhibition of AKT S473 phosphorylation with concomitant inhibition of AKT phosphorylation of PRAS40. Treatment of melanoma cells with BI-69A11 also reduced AKT protein expression, which coincided with inhibition of AKT association with HSP-90. BI-69A11 treatment not only caused cell death of melanoma, but also prostate tumor cell lines. Notably, the effect of BI-69A11 on cell death was more pronounced in cells that express an active form of AKT. Significantly, intra-peritoneal injection of BI-69A11 caused effective regression of melanoma tumor xenografts, which coincided with elevated levels of cell death. These findings identify BI-69A11 as a potent inhibitor of AKT that is capable of eliciting effective regression of xenograft melanoma tumors.


Subject(s)
Antineoplastic Agents/pharmacology , Benzimidazoles/pharmacology , Melanoma/enzymology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Quinolones/pharmacology , Xenograft Model Antitumor Assays , Adenosine Triphosphate/metabolism , Animals , Antineoplastic Agents/therapeutic use , Benzimidazoles/therapeutic use , Cell Death/drug effects , Cell Line, Tumor , Enzyme Activation/drug effects , HSP90 Heat-Shock Proteins/metabolism , Humans , Male , Melanoma/drug therapy , Melanoma/pathology , Mice , Mice, Nude , Models, Molecular , Phosphorylation/drug effects , Prostatic Neoplasms/pathology , Quinolones/therapeutic use , Remission Induction
8.
Mol Biol Cell ; 19(11): 5019-28, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18784250

ABSTRACT

Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER.


Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Glycoproteins/metabolism , Protein Folding , Protein Processing, Post-Translational , Animals , Caenorhabditis elegans/metabolism , Carrier Proteins/metabolism , Endoplasmic Reticulum/pathology , HeLa Cells , Humans , Membrane Glycoproteins/deficiency , Mice , Multiprotein Complexes/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Subunits/metabolism , Protein Transport
9.
Mol Cell Biol ; 25(19): 8619-30, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16166642

ABSTRACT

We report the identification and characterization of JAMP (JNK1 [Jun N-terminal kinase 1]-associated membrane protein), a predicted seven-transmembrane protein that is localized primarily within the plasma membrane and associates with JNK1 through its C-terminal domain. JAMP association with JNK1 outcompetes JNK1 association with mitogen-activated protein kinase phosphatase 5, resulting in increased and prolonged JNK1 activity following stress. Elevated expression of JAMP following UV or tunicamycin treatment results in sustained JNK activity and a higher level of JNK-dependent apoptosis. Inhibition of JAMP expression by RNA interference reduces the degree and duration of JNK activation and concomitantly the level of stress-induced apoptosis. Through its regulation of JNK1 activity, JAMP emerges as a membrane-anchored regulator of the duration of JNK1 activity in response to diverse stress stimuli.


Subject(s)
Carrier Proteins/physiology , Cell Membrane/metabolism , Gene Expression Regulation, Enzymologic , Membrane Glycoproteins/physiology , 3T3 Cells , Amino Acid Sequence , Animals , Apoptosis , Carrier Proteins/biosynthesis , Cell Line , Cell Line, Tumor , Cell Movement , DNA/metabolism , DNA, Complementary/metabolism , Dual-Specificity Phosphatases , Glycosylation , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Immunoprecipitation , MAP Kinase Signaling System , Membrane Glycoproteins/biosynthesis , Mice , Microscopy, Confocal , Molecular Sequence Data , NIH 3T3 Cells , Phosphoprotein Phosphatases/metabolism , Protein Binding , Protein Structure, Tertiary , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Subcellular Fractions/metabolism , Time Factors , Tissue Distribution , Transfection , Tunicamycin/pharmacology , Ultraviolet Rays
10.
J Neurosci ; 23(8): 3209-20, 2003 Apr 15.
Article in English | MEDLINE | ID: mdl-12716928

ABSTRACT

The nerve growth factor (NGF) family of neurotrophins binds two classes of cell-surface receptors, trk receptor tyrosine kinases and the shared p75 receptor. Rapid internalization and retrograde trafficking of neurotrophin-trk complexes have been demonstrated in a number of systems and are thought to transmit trophic signals from terminals to neuronal cell bodies. In contrast, the internalization and trafficking of neurotrophin-p75 complexes are not well understood. In this study, we used biotinylated NGF and a fluorescent-labeled anti-p75 antibody to follow the kinetics and route of ligand-induced internalization of the p75 receptor in cycling and differentiated PC12 cells. Binding of neurotrophins to p75 induced internalization at a rate approximately three times slower than that of transferrin and NGF-TrkA complexes in the same cells. The ligand-p75 complex was internalized via clathrin-coated pits into early endosomes and eventually accumulated in recycling endosomes in the cell body and vesicles colabeled by the cholera toxin B-subunit in the growth cones. Both internalized ligand and p75 were protected from proteolytic degradation and accumulated in vesicles that did not undergo acidification. Finally, NGF induced endosomal association of p75 and its MAGE interactors, necdin and NRAGE. These data suggest that signaling endosomes containing activated p75 are involved in neurotrophin signaling, and that such endosomes may be temporally and spatially distinct from those containing trk receptors.


Subject(s)
Endosomes/metabolism , Neoplasm Proteins , Receptor, trkA , Receptors, Nerve Growth Factor/metabolism , Signal Transduction/physiology , Animals , Antibodies/metabolism , Axonal Transport/physiology , Binding, Competitive/drug effects , Binding, Competitive/physiology , Biotin/chemistry , Biotinylation , Carrier Proteins/pharmacokinetics , Coated Pits, Cell-Membrane/metabolism , DNA-Binding Proteins/metabolism , Fluorescent Dyes , Iodine/chemistry , Ligands , Macromolecular Substances , Membrane Proteins/pharmacokinetics , Nerve Growth Factor/metabolism , Nerve Growth Factor/pharmacokinetics , Nerve Growth Factors/metabolism , Nerve Growth Factors/pharmacokinetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , PC12 Cells , Protein Binding/drug effects , Protein Binding/physiology , Protein Transport/physiology , Rats , Receptor, Nerve Growth Factor , Subcellular Fractions/chemistry , Transferrin/metabolism , Transferrin/pharmacokinetics
11.
J Biol Chem ; 277(51): 49101-4, 2002 Dec 20.
Article in English | MEDLINE | ID: mdl-12414813

ABSTRACT

The p75 neurotrophin receptor has been implicated in diverse aspects of neurotrophin signaling, but the mechanisms by which its effects are mediated are not well understood. Here we identify two MAGE proteins, necdin and MAGE-H1, as interactors for the intracellular domain of p75 and show that the interaction is enhanced by ligand stimulation. PC12 cells transfected with necdin or MAGE-H1 exhibit accelerated differentiation in response to nerve growth factor. Expression of these two MAGE proteins is predominantly cytoplasmic in PC12 cells, and necdin was found to be capable of homodimerization, suggesting that it may act as a cytoplasmic adaptor to recruit a signaling complex to p75. These findings indicate that diverse MAGE family members can interact with the p75 receptor and highlight type II MAGE proteins as a potential family of interactors for signaling proteins containing type II death domains.


Subject(s)
Microtubule-Associated Proteins/chemistry , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Receptors, Nerve Growth Factor/chemistry , Animals , Blotting, Western , COS Cells , Cloning, Molecular , Cytoplasm/metabolism , DNA, Complementary/metabolism , Gene Library , Mice , Microscopy, Fluorescence , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins , PC12 Cells , Phylogeny , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Rats , Receptor, Nerve Growth Factor , Receptors, Nerve Growth Factor/metabolism , Signal Transduction , Temperature , Time Factors , Tissue Distribution , Transfection
12.
J Biol Chem ; 277(12): 9812-8, 2002 Mar 22.
Article in English | MEDLINE | ID: mdl-11777929

ABSTRACT

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75(exonIII-/-) mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase(-/-) mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.


Subject(s)
Ceramides/metabolism , Hippocampus/metabolism , Nerve Growth Factor/metabolism , Neurons/metabolism , Receptors, Nerve Growth Factor/metabolism , Signal Transduction , Sphingomyelin Phosphodiesterase/metabolism , Age Factors , Amides/metabolism , Animals , Axons/metabolism , Blotting, Western , Cell Death , Cells, Cultured , Dose-Response Relationship, Drug , Hippocampus/cytology , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 8 , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Pyrones/metabolism , Rats , Rats, Wistar , Receptor, Nerve Growth Factor , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
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