ABSTRACT
RELL1 and RELL2 are two newly identified RELT homologues that bind to the TNF receptor family member RELT. The expression of RELL1 at the mRNA level is ubiquitous, whereas expression of RELL2 mRNA is more restricted to particular tissues. RELT, RELL1, and RELL2 co-localized with one another at the plasma membrane. The three proteins interacted with one another as demonstrated by in vitro co-immunoprecipitation experiments. We propose that RELL1 and RELL2 be considered RELT family members based on their similar amino acid sequences and on their ability to physically interact with one another. OSR1 was identified through a yeast two-hybrid screen utilizing the intracellular portion of RELL1 as bait, and OSR1 was shown to interact with the three RELT family members by in vitro co-immunoprecipitation experiments. Additionally, OSR1 phosphorylated the RELT family members in an in vitro kinase assay. These results report two novel homologues of RELT that interact with RELT and are phosphorylated by the OSR1 kinase.
Subject(s)
Protein Serine-Threonine Kinases/physiology , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Sequence Homology, Amino Acid , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Chlorocebus aethiops , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Multigene Family , Organ Specificity/genetics , Phosphorylation , Protein Structure, Tertiary , Two-Hybrid System TechniquesABSTRACT
Receptor-interacting protein 2 (RIP2) is a member of the RIP kinase family that has been shown to be crucially involved in inflammation, innate and adaptive immune responses. The physiological and pathological roles of RIP2 are mediated through its involvement in multiple NF-kappaB activation pathways, including those triggered by tumor necrosis factor (TNF), interleukin 1 (IL-1), Toll-like receptor 2 (TLR2), TLR3, TLR4 and Nod1. In this report, we identified the LIM-domain-containing protein TRIP6 as a RIP2-interacting protein in yeast two-hybrid screens. In mammalian cells, TRIP6 interacts with RIP2 in a TNF- or IL-1-dependent manner. Overexpression of TRIP6 potentiates RIP2-mediated NF-kappaB activation in a dose-dependent manner. The LIM domains of TRIP6 are responsible for its interaction with RIP2. TRIP6 also interacts with TRAF2, a protein that is crucially involved in TNF signaling, as well as the IL-1 receptor, TLR2 and Nod1. Overexpression of TRIP6 potentiates NF-kappaB activation by TNF, IL-1, TLR2 or Nod1, whereas a dominant negative mutant or RNA-interference construct of TRIP6 inhibits NF-kappaB activation by TNF, IL-1, TLR2 or Nod1. Moreover, TRIP6 also potentiates RIP2- and Nod1-mediated ERK activation. These data have established a physical and functional association between TRIP6 and RIP2, and suggest that RIP2's involvement in multiple NF-kappaB and ERK activation pathways is mediated through TRIP6.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/physiology , Transcription Factors/physiology , ATPases Associated with Diverse Cellular Activities , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Binding Sites/genetics , Cell Line , DNA-Binding Proteins/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression/drug effects , Humans , Interleukin-1/pharmacology , LIM Domain Proteins , Membrane Glycoproteins/genetics , NF-kappa B/genetics , Nod1 Signaling Adaptor Protein , Proteasome Endopeptidase Complex , Protein Binding/drug effects , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Proteins/metabolism , Proto-Oncogene Proteins/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2 , Receptor-Interacting Protein Serine-Threonine Kinases , Receptors, Cell Surface/genetics , Toll-Like Receptor 2 , Toll-Like Receptor 3 , Toll-Like Receptor 4 , Toll-Like Receptors , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Two-Hybrid System Techniques , ets-Domain Protein Elk-1ABSTRACT
AIM: NAG6 gene is a novel tumor related gene identified recently. This study was designed to examine the expression of this gene in gastric cancer and corresponding normal tissues, and to investigate its role in the occurrence and development of gastric cancer, also to study if the genetic structure of NAG6 was altered in gastric cancer. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis and dot hybridization were used to compare the expression level of NAG6 gene in 42 cases of gastric cancer tissues with their corresponding normal tissues of the same patients respectively. In addition, restriction fragment length polymorphism (RFLP) analysis was adopted to study if the genetic structure of NAG6 was altered in gastric carcinomas. RESULTS: The expression of NAG6 in 57.1% gastric cancer tissues (25/42) was absent by RT-PCR analysis. The down-regulation rate of NAG6 in gastric cancer tissues was significantly higher than that in corresponding normal tissues (P<0.01). However no correlation between the down-regulation of NAG6 and lymph-node and/or distance metastasis was found in this study (P>0.05). Dot hybridization confirmed the results of RT-PCR. Furthermore, the results of EcoRI RFLP analysis of NAG6 gene demonstrated that 3 of 7 cases of gastric cancer showed loss of 5 kb fragment in comparison with their corresponding normal tissues. CONCLUSION: NAG6 gene is significantly down regulated in gastric cancer. The loss of genetic materials may be the cause of down-regulation of NAG6 expression. This seems to suggest that NAG6 may represent a candidate of putative tumor suppressor gene at 7q31-32 loci associated with gastric carcinoma. The down-regulation of this gene may play a role in occurrence and development of this disease, however it may not be associated with lymph node and/or distance metastasis.
Subject(s)
Neoplasm Proteins/metabolism , Polymorphism, Restriction Fragment Length , Stomach Neoplasms/genetics , Adult , Aged , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Humans , Male , Middle Aged , Neoplasm Proteins/genetics , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Tissue DistributionABSTRACT
Toll-like receptor-3 is critically involved in host defense against viruses through induction of type I interferons (IFNs). Recent studies suggest that a Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) and two protein kinases (TANK-binding kinase-1 (TBK1) and IkappaB kinase (IKK)-epsilon) are critically involved in Toll-like receptor-3-mediated IFN-beta production through activation of IFN regulatory factor (IRF)-3 and IRF-7. In this study, we demonstrate that TRIF interacts with both IRF-7 and IRF-3. In addition to TBK1 and IKKepsilon, our results indicate that IKKbeta can also phosphorylate IRF-3 and activate the IFN-stimulated response element. TRIF-induced IRF-3 and IRF-7 activation was mediated by TBK1 and its downstream kinases IKKbeta and IKKepsilon. TRIF induced NF-kappaB activation through an IKKbeta- and tumor necrosis factor receptor-associated factor-6-dependent (but not TBK1- and IKKepsilon-dependent) pathway. In addition, TRIF also induced apoptosis through a RIP/FADD/caspase-8-dependent and mitochondrion-independent pathway. Furthermore, our results suggest that the TRIF-induced IFN-stimulated response element and NF-kappaB activation and apoptosis pathways are uncoupled and provide a molecular explanation for the divergent effects induced by the adapter protein TRIF.
Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Apoptosis , Interferons/metabolism , NF-kappa B/metabolism , Response Elements , Animals , Blotting, Western , Caspase 8 , Caspases/metabolism , Cell Line , DNA Fragmentation , DNA-Binding Proteins/metabolism , Gene Library , Genes, Reporter , Genetic Vectors , Humans , I-kappa B Kinase , Interferon Regulatory Factor-3 , Interferon Regulatory Factor-7 , Models, Biological , Models, Genetic , Phosphorylation , Plasmids/metabolism , Precipitin Tests , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Proteins/metabolism , Sendai virus/genetics , Signal Transduction , TNF Receptor-Associated Factor 6 , Transcription Factors/metabolism , Transfection , Two-Hybrid System TechniquesABSTRACT
High in normal (HIN)-1 is a secreted protein highly expressed in normal breast epithelium and down-regulated in breast carcinomas. By searching GenBank expressed sequence tag databases, we identified HIN-2, a protein homologous to HIN-1. HIN-2 is identical with a recently identified protein called uteroglobin-related protein 1 (UGRP1). Northern blot analysis demonstrated that UGRP1 is specifically expressed by lung, but not by the other tissues examined. By in situ hybridization experiments, UGRP1 was shown to be expressed by lung Clara-like cells in the bronchial epithelium and to be up-regulated in cystic fibrosis. In a mammalian expression system, secreted recombinant UGRP1 was copurified with apolipoprotein A-I. Using a retroviral vector-mediated expression cloning approach, we identified macrophage scavenger receptor with collagenous structure (MARCO) as a receptor for UGRP1. Northern blot and in situ hybridization experiments indicated that MARCO is expressed by alveolar macrophages in the lung. UGRP1 also bound to bacteria and yeast. LPS, a previously identified MARCO ligand, competed with UGRP1 for binding to MARCO and bacteria. Our findings suggest that UGRP1-MARCO is a ligand-receptor pair that is probably involved in inflammation and pathogen clearance in the lung.
Subject(s)
Carrier Proteins , Lung/metabolism , Receptors, Immunologic/chemistry , Receptors, Immunologic/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Tumor Suppressor Proteins , Amino Acid Sequence , Cell Line , Cloning, Molecular , Collagen/chemistry , Cytokines/chemistry , Cytokines/genetics , Cytokines/metabolism , Genes, Tumor Suppressor , Humans , Ligands , Listeria monocytogenes/metabolism , Lung/chemistry , Macrophages, Alveolar/metabolism , Molecular Sequence Data , Organ Specificity/genetics , Pseudomonas aeruginosa/metabolism , Receptors, Immunologic/biosynthesis , Receptors, Immunologic/genetics , Receptors, Scavenger , Saccharomyces cerevisiae/metabolism , Secretoglobins , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transfection , Tumor Cells, Cultured , U937 Cells , UteroglobinABSTRACT
The Toll/interleukin-1 receptor (TIR) family members play important roles in host defense. These receptors signal through TIR domain-containing adapter proteins. In this report, we identified a novel TIR domain-containing adapter protein designated as TIRP. Co-immunoprecipitation experiments suggest that TIRP is associated with IL-1 receptors. TIRP also interacts with kinase-inactive mutants of IRAK and IRAK-4, IRAK-2, IRAK-M, and TRAF6. Overexpression of TIRP activates NF-kappaB and potentiates IL-1 receptor-mediated NF-kappaB activation. A dominant negative mutant of TIRP inhibits IL-1- but not tumor necrosis factor-triggered NF-kappaB activation. Moreover, TIRP-mediated NF-kappaB activation is inhibited by dominant negative mutants of IRAK, IRAK-2, TRAF6, and IKKbeta. Our findings suggest that TIRP is involved in IL-1-triggered NF-kappaB activation and functions upstream of IRAK, IRAK-2, TRAF6, and IKKbeta
