N-terminal 45 amino acids of DEF6 are necessary and sufficient to spontaneously colocalise with DCP1 in P-bodies

Abstract

ABSTRACTDEF6 (Differentially Expressed in FDCP 6, also known as IBP and/or SLAT) is critical for the development of autoimmune disease and cancer. In T cells, DEF6 participates in TCR-mediated signalling determining T helper cell-mediated immune responses. In addition, DEF6 acts as a guanine nucleotide exchange factor (GEF) for Rho GTPases facilitating F-actin assembly and stabilisation of the immunological synapse (IS). However, DEF6 is also a component of mRNA processing bodies (P-bodies) linking it to mRNA metabolism. Including DEF6, more than 34 proteins have been shown to localise in P-bodies many of which contain a coiled coil domain, a super-secondary structure likely to facilitate interaction between these proteins. Accordingly, we suggested that the coiled coil domain in the C-terminal end of DEF6 was mediating P-body localisation of DEF6 under cellular stress conditions. However, a comprehensive analysis of wild type and mutant DEF6 proteins expressed in COS7 cells revealed that the coiled coil domain is dispensable for P-body colocalisation. Instead, we show here that the N-terminal 45 amino acids of DEF6 that contain one Ca2+-binding EF hand motif are sufficient to target DEF6 to P-bodies whereas the N-terminal 30 amino acids containing a disrupted EF hand motif are insufficient.