F-box Protein ßTrCP1 Is a Substrate of Extracellular Signal-regulated Kinase 2.

ABSTRACT

F-box proteins, consisting of 69 members which are organized into the three subclasses FBXW, FBXL, and FBXO, are the substrate specific recognition subunits of the SKP1-Cullin 1-F-box protein E3 ligase complex. Although ßTrCP 1 and 2, members of the FBXW subfamily, are known to regulate some protein stability, molecular mechanisms by which these proteins can recognize proper substrates are unknown. In this study, it was found that ßTrCP1 showed strong interaction with members of mitogen-activated protein kinases. Although extracellular signal-regulated kinase (ERK) 3, p38ß, and p38δ showed weak interactions, ERK2 specifically interacted with ßTrCP1 as assessed by immunoprecipitation. In interaction domain determination experiments, we found that ERK2 interacted with two independent ERK docking sites located in the F-box domain and linker domain, but not the WD40 domain, of ßTrCP1. Notably, mutations of ßTrCP1 at the ERK docking sites abolished the interaction with ERK2. ßTrCP1 underwent phosphorylation by EGF stimulation, while the presence of the mitogen-activated protein kinase kinases inhibitor U0126, genetic silencing by sh-ERK2, and mutation of the ERK docking site of ßTrCP1 inhibited phosphorylation. This inhibition of ßTrCP1 phosphorylation resulted in a shortened half-life and low protein levels. These results suggest that ERK2-mediated ßTrCP1 phosphorylation may induce the destabilization of ßTrCP1.