Identification of a motif in the carboxyl terminus of beta -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-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.
Miller
W E
WE
Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Box 3821, Durham, North Carolina 27710, USA.
McDonald
P H
PH
Cai
S F
SF
Field
M E
ME
Davis
R J
RJ
Lefkowitz
R J
RJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins; D019390:Arrestins / Q000737:chemistry*; D019556:COS Cells; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D015151:Immunoblotting; D048057:Mitogen-Activated Protein Kinase 10; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D000071557:beta-Arrestins
C120363:ASK1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D019390:Arrestins; D010940:Plant Proteins; D018343:Receptors, Adrenergic, beta-2; D000071557:beta-Arrestins; D048057:Mitogen-Activated Protein Kinase 10; D011505:Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases