title: Silent scaffolds: inhibition OF c-Jun N-terminal kinase 3 activity in cell by dominant-negative arrestin-3 mutant.
abstract: 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 that readily binds β2-adrenergic receptors as well as two mutants impaired in receptor binding, Δ7 and KNC, to directly bind JNK3 and to promote JNK3 phosphorylation in cells. Both receptor binding-deficient mutants interact with JNK3 significantly better than WT and 3A arrestin-3. WT arrestin-3 and Δ7 mutant robustly promoted JNK3 activation, whereas 3A and KNC mutants did not. Thus, receptor binding, JNK3 interaction, and JNK3 activation are three distinct arrestin functions. We found that the KNC mutant, which tightly binds ASK1, MKK4, and JNK3 without facilitating JNK3 phosphorylation, has a dominant-negative effect, competitively decreasing JNK activation by WT arrestin-3. Thus, KNC is a silent scaffold, a novel type of molecular tool for the suppression of MAPK signaling in living cells.
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Maya
authors.initials: M
authors.lastname: Breitman
authors.forename: Seunghyi
authors.initials: S
authors.lastname: Kook
authors.forename: Luis E
authors.initials: LE
authors.lastname: Gimenez
authors.forename: Britney N
authors.initials: BN
authors.lastname: Lizama
authors.forename: Maria C
authors.initials: MC
authors.lastname: Palazzo
authors.forename: Eugenia V
authors.initials: EV
authors.lastname: Gurevich
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
mesh_terms: D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D048848:MAP Kinase Kinase Kinase 5 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System; D048057:Mitogen-Activated Protein Kinase 10 / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics
chemical_list: D019390:Arrestins; C082333:arrestin3; D048057:Mitogen-Activated Protein Kinase 10; D048848:MAP Kinase Kinase Kinase 5; D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human