The antiinflammatory endothelial tyrosine kinase Tie2 interacts with a novel nuclear factor-kappaB inhibitor ABIN-2.
Tie2 is a receptor tyrosine kinase expressed predominantly in endothelial cells and is essential for blood vessel formation and maintenance. The receptor has potent antiinflammatory effects on endothelial cells, suppressing vascular endothelial growth factor- and tumor necrosis factor-induced expression of leukocyte adhesion molecules and procoagulant tissue factor and inhibiting vascular leakage. To delineate the signaling pathways utilized by Tie2, we performed yeast two-hybrid screening of a human endothelial cell cDNA library and identified a novel protein interacting with the intracellular domain of the receptor. This protein was found to be human A20 binding inhibitor of NF-kappaB activation-2, ABIN-2, an inhibitor of NF-kappaB-mediated inflammatory gene expression. Coexpression of Tie2 and ABIN-2 in CHO cells confirmed the interaction occurs in mammalian cells. In contrast, Tie1 did not interact with ABIN-2 in the yeast two-hybrid system or mammalian cells. Deletion analysis identified the Tie2 binding motif to be encompassed between residues 171 and 272 in ABIN-2. Interaction was dependent on Tie2 autophosphorylation but ABIN-2 was not tyrosine phosphorylated by Tie2. Furthermore, in endothelial cells the interaction was stimulated by the Tie2 ligand angiopoietin-1. Expression of ABIN-2 deletion mutants in endothelial cells suppressed the ability of angiopoietin-1 to inhibit phorbol ester-stimulated NF-kappaB-dependent reporter gene activity. These findings provide the first direct link between Tie2 and a key regulator of inflammatory responses in endothelial cells. Interaction between Tie2 and ABIN-2 may be important in the vascular protective antiinflammatory actions of Tie2.
Hughes
David P
DP
Cardiovascular Research Institute and Department Surgery, University of Leicester, UK.
Marron
Marie B
MB
Brindle
Nicholas P J
NP
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D043925:Angiogenesis Inducing Agents / Q000494:pharmacology; D042683:Angiopoietin-1; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D042787:Receptor, TIE-2; D017384:Sequence Deletion; D017385:Sequence Homology; D020798:Two-Hybrid System Techniques
C467630:ANGPT1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D043925:Angiogenesis Inducing Agents; D042683:Angiopoietin-1; C467628:Angpt1 protein, mouse; D000893:Anti-Inflammatory Agents; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; C434062:TNIP2 protein, human; D019000:Phosphotyrosine; D020794:Receptor Protein-Tyrosine Kinases; D042787:Receptor, TIE-2