ABIN-2 forms a ternary complex with TPL-2 and NF-kappa B1 p105 and is essential for TPL-2 protein stability

Mol Cell Biol. 2004 Jun;24(12):5235-48. doi: 10.1128/MCB.24.12.5235-5248.2004.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Line
  • DNA, Complementary / genetics
  • Enzyme Activation
  • HeLa Cells
  • Humans
  • In Vitro Techniques
  • MAP Kinase Kinase Kinases / chemistry
  • MAP Kinase Kinase Kinases / genetics
  • MAP Kinase Kinase Kinases / metabolism*
  • Macromolecular Substances
  • Macrophages / metabolism
  • Membrane Glycoproteins / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Mice, Knockout
  • Molecular Sequence Data
  • NF-kappa B p50 Subunit
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • RNA, Small Interfering / genetics
  • Receptors, Cell Surface / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Solubility
  • Toll-Like Receptor 4
  • Toll-Like Receptors
  • Transcription Factors / chemistry
  • Transcription Factors / deficiency
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transfection

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • DNA, Complementary
  • Macromolecular Substances
  • Membrane Glycoproteins
  • NF-kappa B p50 Subunit
  • NFKB1 protein, human
  • Proto-Oncogene Proteins
  • RNA, Small Interfering
  • Receptors, Cell Surface
  • Recombinant Proteins
  • TLR4 protein, human
  • TNIP2 protein, human
  • Toll-Like Receptor 4
  • Toll-Like Receptors
  • Transcription Factors
  • Nfkb1 protein, mouse
  • MAP Kinase Kinase Kinases
  • MAP3K8 protein, human
  • Map3k8 protein, mouse