MEK1 binds directly to betaarrestin1, influencing both its phosphorylation by ERK and the timing of its isoprenaline-stimulated internalization.
betaArrestin 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 betaarrestin1. Asp(26) and Asp(29) in the N-terminal domain of betaarrestin1 are critical for its binding to MEK1, whereas Arg(47) and Arg(49) in the N-terminal domain of MEK1 are critical for its binding to betaarrestin1. Wild-type FLAG-tagged betaarrestin1 co-immunopurifies with MEK1 in HEKB2 cells, whereas the D26A/D29A mutant does not. ERK-dependent phosphorylation at Ser(412) was compromised in the D26A/D29A-betaarrestin1 mutant. A cell-permeable, 25-mer N-stearoylated betaarrestin1 peptide that encompassed the N-domain MEK1 binding site blocked betaarrestin1/MEK1 association in HEK cells and recapitulated the altered phenotype seen with the D26A/D29A-betaarrestin1 in compromising the ERK-dependent phosphorylation of betaarrestin1. In addition, the MEK disruptor peptide promoted the ability of betaarrestin1 to co-immunoprecipitate with endogenous c-Src and clathrin, facilitating the isoprenaline-stimulated internalization of the beta(2)-adrenergic receptor.
Meng
Dong
D
Neuroscience and Molecular Pharmacology, Faculty of Biomedical and Life Sciences, Wolfson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Lynch
Martin J
MJ
Huston
Elaine
E
Beyermann
Michael
M
Eichhorst
Jenny
J
Adams
David R
DR
Klussmann
Enno
E
Klusmann
Enno
E
Houslay
Miles D
MD
Baillie
George S
GS
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D019390:Arrestins / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry; D002966:Clathrin / Q000378:metabolism; D006801:Humans; D007545:Isoproterenol / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D000071557:beta-Arrestins; D019061:src-Family Kinases / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D019390:Arrestins; D002966:Clathrin; D000071557:beta-Arrestins; D001224:Aspartic Acid; D001120:Arginine; D019061:src-Family Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D007545:Isoproterenol