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

βArrestin 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 βarrestin1. Asp26 and Asp29 in the N-terminal domain of βarrestin1 are critical for its binding to MEK1, whereas Arg47 and Arg49 in the N-terminal domain of MEK1 are critical for its binding to βarrestin1. Wild-type FLAG-tagged βarrestin1 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-βarrestin1 mutant. A cell-permeable, 25-mer N-stearoylated βarrestin1 peptide that encompassed the N-domain MEK1 binding site blocked βarrestin1/MEK1 association in HEK cells and recapitulated the altered phenotype seen with the D26A/D29A-βarrestin1 in compromising the ERK-dependent phosphorylation of βarrestin1. In addition, the MEK disruptor peptide promoted the ability of βarrestin1 to co-immunoprecipitate with endogenous c-Src and clathrin, facilitating the isoprenaline-stimulated internalization of the β2-adrenergic receptor.