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Silent scaffolds: inhibition of JNK3 activity in living cells by a dominant‐negative arrestin‐3 mutant
Author(s) -
Gurevich Vsevolod V,
Breitman Maya,
Gimenez Luis E,
Kook Seunghyi,
Lizama Britney N,
Palazzo Maria C,
Gurevich Eugenia V
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.761.2
Subject(s) - mutant , arrestin , chemistry , microbiology and biotechnology , luciferase , receptor , phosphorylation , mapk/erk pathway , biochemistry , g protein coupled receptor , biology , transfection , gene
We established a new in vivo arrestin‐3‐JNK3 interaction assay based on bioluminescence resonance energy transfer (BRET) between JNK3‐luciferase and Venus‐arrestins. We tested the ability of WT arrestin‐3 and its 3A mutant, both of which readily bind the β2‐adrenergic receptor, as well as two mutants impaired in receptor binding, Δ7 and KNC, to interact with JNK3 and to promote JNK3 phosphorylation in cells. Both receptor binding‐deficient mutants bind JNK3 significantly better than WT and 3A arrestin‐3. WT arrestin‐3 and the Δ7 mutant robustly promoted JNK3 activation, whereas the 3A and KNC mutants did not. Thus, receptor binding, JNK3 interaction, and JNK3 activation are three distinct functions of arrestins that can be independently manipulated by appropriate mutations. We found that the KNC mutant, which tightly binds JNK3 without facilitating its phosphorylation, functions as a dominant‐negative, competitively decreasing JNK3 activation by WT arrestin‐3. Thus, KNC is a silent scaffold, a novel type of molecular tool for selective suppression of MAPK signaling. These data provide the first demonstration that this mechanism of MAPK regulation can be used in living cells. NIH grants GM081756, GM077561, and EY011500 (VVG).