The tight junction-specific protein occludin is a functional target of the E3 ubiquitin-protein ligase itch.
Tight junctions create a highly selective diffusion barrier between epithelial and endothelial cells by preventing the free passage of molecules and ions across the paracellular pathway. Although the regulation of this barrier is still enigmatic, there is evidence that junctional transmembrane proteins are critically involved. Recent evidence confirms the notion that occludin, a four-pass integral plasma-membrane protein, is a functional component of the paracellular barrier. The overall hydrophilicity of occludin predicts two extracellular loops bounded by NH(2)- and COOH-terminal cytoplasmic domains. To date, the binding of the COOH terminus of occludin to intracellular proteins is well documented, but information concerning the function of the cytoplasmic NH(2) terminus is still lacking. Using yeast two-hybrid screening we have identified a novel interaction between occludin and the E3 ubiquitin-protein ligase Itch, a member of the HECT domain-containing ubiquitin-protein ligases. We have found that the NH(2)-terminal portion of occludin binds specifically to a multidomain of Itch, consisting of four WW motifs. This interaction has been confirmed by our results from in vivo and in vitro co-immunoprecipitation experiments. In addition, we provide evidence that Itch is specifically involved in the ubiquitination of occludin in vivo, and that the degradation of occludin is sensitive to proteasome inhibition.
Traweger
Andreas
A
Institute of Molecular Biology, Austrian Academy of Sciences, 5020 Salzburg, Austria.
Fang
Deyu
D
Liu
Yun-Cai
YC
Stelzhammer
Wolfgang
W
Krizbai
Istvan A
IA
Fresser
Fritz
F
Bauer
Hans-Christian
HC
Bauer
Hannelore
H
D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003546:Cysteine Endopeptidases; D003593:Cytoplasm / Q000378:metabolism*; D004594:Electrophysiology; D006801:Humans; D008025:Ligases / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D062793:Occludin; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D008565:Membrane Proteins; D009097:Multienzyme Complexes; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; D025801:Ubiquitin; C471637:Itch protein, mouse; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases