Abstract
NF-kappa B1 p105 forms a high-affinity, stoichiometric interaction with TPL-2, a MEK kinase essential for TLR4 activation of the ERK mitogen-activated protein kinase cascade in lipopolysaccharide (LPS)-stimulated macrophages. Interaction with p105 is required to maintain TPL-2 metabolic stability and also negatively regulates TPL-2 MEK kinase activity. Here, affinity purification identified A20-binding inhibitor of NF-kappa B 2 (ABIN-2) as a novel p105-associated protein. Cotransfection experiments demonstrated that ABIN-2 could interact with TPL-2 in addition to p105 but preferentially formed a ternary complex with both proteins. Consistently, in unstimulated bone marrow-derived macrophages (BMDMs), a substantial fraction of endogenous ABIN-2 was associated with both p105 and TPL-2. Although the majority of TPL-2 in these cells was complexed with ABIN-2, the pool of TPL-2 which could activate MEK after LPS stimulation was not, and LPS activation of TPL-2 was found to correlate with its release from ABIN-2. Depletion of ABIN-2 by RNA interference dramatically reduced steady-state levels of TPL-2 protein without affecting levels of TPL-2 mRNA or p105 protein. In addition, ABIN-2 increased the half-life of cotransfected TPL-2. Thus, optimal TPL-2 stability in vivo requires interaction with ABIN-2 as well as p105. Together, these data raise the possibility that ABIN-2 functions in the TLR4 signaling pathway which regulates TPL-2 activation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Amino Acid Sequence
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Animals
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Base Sequence
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Line
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DNA, Complementary / genetics
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Enzyme Activation
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HeLa Cells
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Humans
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In Vitro Techniques
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MAP Kinase Kinase Kinases / chemistry
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MAP Kinase Kinase Kinases / genetics
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MAP Kinase Kinase Kinases / metabolism*
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Macromolecular Substances
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Macrophages / metabolism
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Membrane Glycoproteins / metabolism
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Mice
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Mice, Inbred BALB C
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Mice, Knockout
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Molecular Sequence Data
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NF-kappa B p50 Subunit
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Proto-Oncogene Proteins / chemistry
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism*
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RNA, Small Interfering / genetics
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Receptors, Cell Surface / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Signal Transduction
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Solubility
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Toll-Like Receptor 4
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Toll-Like Receptors
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Transcription Factors / chemistry
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Transcription Factors / deficiency
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transfection
Substances
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Adaptor Proteins, Signal Transducing
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Carrier Proteins
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DNA, Complementary
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Macromolecular Substances
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Membrane Glycoproteins
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NF-kappa B p50 Subunit
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NFKB1 protein, human
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Proto-Oncogene Proteins
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RNA, Small Interfering
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Receptors, Cell Surface
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Recombinant Proteins
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TLR4 protein, human
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TNIP2 protein, human
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Toll-Like Receptor 4
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Toll-Like Receptors
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Transcription Factors
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Nfkb1 protein, mouse
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MAP Kinase Kinase Kinases
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MAP3K8 protein, human
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Map3k8 protein, mouse