Identification of a Motif in the Carboxyl Terminus of β-Arrestin2 Responsible for Activation of JNK3

William E Miller,Patricia H Mcdonald,Sheng F Cai,Michael E Field,Roger J Davis,Robert J Lefkowitz

doi:10.1074/jbc.m102264200

William E Miller, Patricia H Mcdonald + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m102264200

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Abstract

Accumulating evidence indicates that the beta-arrestins act as scaffold molecules that couple G-protein-coupled receptors to mitogen-activated protein (MAP) kinase signaling pathways. Recently, we identified the c-Jun N-terminal kinase 3 (JNK3) as a beta-arrestin2-interacting protein in yeast-two hybrid and co-immunoprecipitation studies. Beta-arrestin2 acts as a scaffold to enhance signaling to JNK3 stimulated by overexpression of the MAP3 kinase ASK1 or by agonist activation of the angiotensin 1A receptor. Whereas beta-arrestin2 is a very strong activator of JNK3 signaling, beta-arrestin1 is very weak in this regard. The data also indicate that the specific step enhanced by beta-arrestin2 involves phosphorylation of JNK3 by the MAP2 kinase MKK4. We reasoned that defining the region (or domain) in beta-arrestin2 responsible for high level JNK3 activation would provide insight into the mechanism by which beta-arrestin2 enhances the activity of this signaling pathway. Using chimeric beta-arrestins, we have determined that sequences in the carboxyl-terminal region of beta-arrestin2 are important for the enhancement of JNK3 phosphorylation. More detailed analysis of the carboxyl-terminal domains of the beta-arrestins indicated that beta-arrestin2, but not beta-arrestin1, contains a sequence (RRSLHL) highly homologous to the conserved docking motif present in many MAP kinase-binding proteins. Replacement of the beta-arrestin2 RRS residues with the corresponding KP residues present in beta-arrestin1 dramatically reduced both JNK3 interaction and enhancement of JNK3 phosphorylation. Conversely, replacement of the KP residues in beta-arrestin1 with RRS significantly increased both JNK3 binding and enhancement of JNK3 phosphorylation. These results delineate a mechanism by which beta-arrestin2 functions as a scaffold protein in the JNK3 signaling pathway and implicate the conserved docking site in beta-arrestin2 as an important factor in binding JNK3 and stimulating the phosphorylation of JNK3 by MKK4.

Highlights

Initial evidence implicating the ␤-arrestins as adapter molecules indicated that the ␤-arrestins directly interact with the heavy chain of clathrin, the clathrin adapter protein (AP-2), and the N-ethylmaleimide sensitive fusion protein (NSF) to promote endocytosis of activated G-protein-coupled receptors (GPCRs) [11,12,13]
Overexpression of ASK1 or stimulation of the angiotensin 1A receptor (AT1aR) leads to a marked increase in Jun N-terminal kinase 3 (JNK3) phosphorylation that requires ␤-arprotein kinase; MAP2K, MAPK kinase; MAP3K, MAP2K kinase; ERK, extracellular-regulated kinase, JNK, c-Jun N-terminal kinase; AP2, adapter protein 2; NSF, N-ethylmaleimide-sensitive fusion protein; ASK1, apoptosis-stimulating kinase 1; AT1A, angiotensin 1A; PCR, polymerase chain reaction; HA, hemagglutinin; iso, isoproterenol; PAGE, polyacrylamide gel electrophoresis
We present evidence in this study that specific residues in the carboxyl terminus of ␤-arrestin2 facilitate the assembly of a complex that contains JNK3. ␤-Arrestin2 appears to act as a scaffold protein to enhance signaling through the mitogen-activated protein (MAP) kinase cascade resulting in the robust phosphorylation of JNK3

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—COS-7 African Green Monkey cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified essential medium supplemented with 10% fetal bovine serum and penn/strep (Life Technologies, Inc.). For the ␤-arrestin1/2 chimera, DNA encoding a minimal KOZAK sequence and amino acids 1–184 of ␤-arrestin and DNA encoding amino acids 186 – 410 of ␤-arrestin with a carboxyl-terminal FLAG tag were PCR amplified and cloned by three way ligation into the EcoRV sites of pcDNA3. ␤-arrestin1(RRS) mutant, DNA encoding a minimal KOZAK sequence and amino acids 1–184 of ␤-arrestin and DNA encoding amino acids 185– 418 of ␤-arrestin (containing RRS in place of KP at residues 195–196) with a carboxyl-terminal FLAG tag were PCR amplified and cloned by three way ligation into the EcoRV sites of pcDNA3. For the ␤-arrestin (KP) mutant, DNA encoding a minimal KOZAK sequence and amino acids 1–185 of ␤-arrestin and DNA encoding amino acids 186 – 410 of ␤-arrestin (containing KP in place of RRS at residues 196 –198) with a carboxyl-terminal FLAG tag were PCR amplified and cloned by three way ligation into the EcoRV sites of pcDNA3. Presence of cellsurface receptors was determined by flow cytometry (Duke University Flow Cytometry Facility)

RESULTS

DISCUSSION