Identification of a motif in the carboxyl terminus of β-arrestin2 responsible for activation of JNK3

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 -arrestin1 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 -arresting involves phosphozylation 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 -arrestin2, 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 -arrestin2, 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 HP 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.