Phosphorylation-dependent association of the Ras-related GTP-binding protein Rem with 14-3-3 proteins

Rem belongs to a subfamily of Ras-related GTPases that includes Rad, Gem, a nd Kir, These proteins are unique among the Ras superfamily since their exp ression is under transcriptional regulation and they contain distinct amino and carboxyl termini. To gain insight into the cellular function of Rem, w e have undertaken an expression screen using a mouse embryo cDNA library to identify Rem-interacting proteins and find that Rem interacts with a serie s of 14-3-3 isoforms (epsilon, eta, theta and zeta), Immunoprecipitation st udies demonstrate an interaction that is independent of the nucleotide stat e of Rem, Rem is phosphorylated in vivo and binding of Rem to 14-3-3 zeta i s abolished by pretreating Rem with protein phosphatase 1, Thus, the associ ation of Rem and 14-3-3 zeta is phosphorylation-dependent. Examination of t he interaction between 14-3-3 zeta and various Rem deletion mutants mapped a critical binding site to the C-terminus of Rem, Finally, we demonstrate t he interaction of Rad but not the newly identified Rem2 protein with 14-3-3 proteins, These results suggest that 14-3-3 may allow the recruitment of d istinct proteins that participate in Rem-mediated signal transduction pathw ays. (C) 1999 Academic Press.