MEK1 binds directly to βarrestin1, influencing both its phosphorylation by ERK and the timing of its isoprenaline-stimulated internalization

ßArrestin is a multifunctional signal scaffold protein. Using SPOT immobilized peptide arrays, coupled with scanning alanine substitution and mutagenesis, we show that the MAPK kinase, MEK1, interacts directly with ßarrestin1. Asp²⁶ and Asp²⁹ in the N-terminal domain of ßarrestin1 are critical for its binding to MEK1, whereas Arg⁴⁷ and Arg⁴⁹ in the N-terminal domain of MEK1 are critical for its binding to ßarrestin1. Wildtype FLAG-tagged ßarrestin1 co-immunopurifies with MEK1 in HEKB2 cells, whereas the D26A/D29A mutant does not. ERK-dependent phosphorylation at Ser⁴¹² was compromised in the D26A/D29A-ßarrestin1 mutant. A cell-permeable, 25-mer N-stearoylated ßarrestin1 peptide that encompassed the N-domain MEK1 binding site blocked ßarrestin1/ MEK1 association in HEK cells and recapitulated the altered phenotype seen with the D26A/D29A-ßarrestin1 in compromising the ERK-dependent phosphorylation of ßarrestin1. In addition, the MEK-disruptor peptide promoted the ability of ßarrestin1 to co-immunoprecipitate with endogenous c-Src and clathrin, facilitating the isoprenaline-stimulated internalization of the ß ₂-adrenergic receptor. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.