MEK1 Binds Directly to  Arrestin1, Influencing Both Its Phosphorylation by ERK and the Timing of Its Isoprenaline-stimulated Internalization

Abstract

{beta}arrestin is a multifunctional signal scaffold protein. Using SPOT immobilised peptide arrays, coupled with scanning alanine substitution and mutagenesis, we show that the MAPKK, MEK1 interacts directly with {beta}arrestin1. Asp26 and Asp29 in the N-terminal domain of {beta}arrestin1 are critical for its binding to MEK1, while Arg47 and Arg49 in the N-terminal domain of MEK1 are critical for its binding to {beta}arrestin1. Wild-type FLAG-tagged {beta}arrestin1 co-immunopurifies with MEK1 in HEKB2 cells while the Asp26Ala:Asp29:Ala mutant does not. ERK-dependent phosphorylation at Ser412 was compromised in the Asp26Ala:Asp29:Ala-{beta}arrestin1mutant. A 25-mer N-stearoylated {beta}arrestin1 peptide that encompassed the N-domain MEK1 binding site, blocked {beta}arrestin1/MEK1 association in HEK cells and recapitulated the altered phenotype seen with the Asp26Ala:Asp29:Ala-{beta}arrestin1 in compromising the ERK-dependent phosphorylation of {beta}arrestin1. In addition, the MEK disruptor peptide promoted the ability of {beta}arrestin1 to co-immunoprecipitate with endogenous cSrc and clathrin, facilitating the isoprenaline-stimulated internalisation of the {beta}2-adrenergic receptor