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

Abstract

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

  • Abbreviations:
    GPCR
    G-protein-coupled receptor
    GRK
    G-protein-coupled receptor kinase
    MAPK
    mitogen-activated protein 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
    • Received March 13, 2001.
    • Revision received April 19, 2001.
    Table of Contents

    This Article

    1. The Journal of Biological Chemistry 276, 27770-27777.
    1. All Versions of this Article:
      1. M102264200v1
      2. 276/30/27770 (most recent)

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