Ubiquitin tagged dominant negative induces degradation of B-ZIP proteins.
B-ZIP transcription factors heterodimerize with dominant negative designs, termed A-ZIPs, in a dimerization specific manner and inhibit its ability to bind DNA. Different A-ZIPs produce unique phenotypes in vivo suggesting that they have distinct B-ZIP heterodimerization partners. However, the identification of the in vivo heterodimerization partners of different A-ZIPs remains problematic. To identify the in vivo heterodimerization partners, a chimeric protein containing two ubiquitin motifs at the N-terminal of the A-ZIP domain was designed. The presence of ubiquitin reduced the concentration of specific co-transfected B-ZIP proteins. The ubiquitin enhanced degradation of the B-ZIP heterodimeric partner is inhibited by the proteasome inhibitor MG-132. These ubiquitin tagged A-ZIP dominant negatives may be more active in vivo because their endogenous heterodimerization partners are degraded more efficiently. This may be a general strategy to identify protein interaction partners.
Building 37, Room 3128, Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Eric
E
Rios-Doria
Raghunath
R
Chatterjee
Jianfei
J
Zhao
Vikas
V
Rishi
Charles
C
Vinson
D000595:Amino Acid Sequence; D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism*; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D061988:Proteasome Inhibitors; D055503:Protein Multimerization; D059748:Proteolysis; D025801:Ubiquitin / Q000378:metabolism*
D050976:Basic-Leucine Zipper Transcription Factors; D022762:CCAAT-Enhancer-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde