ABIN-2 forms a ternary complex with TPL-2 and NF-kappa B1 p105 and is essential for TPL-2 protein stability.
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.
Lang
V
V
Division of Immune Cell Biology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Symons
A
A
Watton
S J
SJ
Janzen
J
J
Soneji
Y
Y
Beinke
S
S
Howell
S
S
Ley
S C
SC
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D020930:MAP Kinase Kinase Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008264:Macrophages / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D052002:NF-kappa B p50 Subunit; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014157:Transcription Factors / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C495345:TLR4 protein, human; C434062:TNIP2 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014157:Transcription Factors; C073701:Nfkb1 protein, mouse; D020930:MAP Kinase Kinase Kinases; C069937:MAP3K8 protein, human; C489843:Map3k8 protein, mouse