Abstract
Localization of DEF6 (SLAT/IBP), a Rho-family guanine nucleotide exchange factor, to the center of the immune synapse is dependent upon ITK, a Tec-family kinase that regulates the spatiotemporal organization of components of T cell signaling pathways and Cdc42-dependent actin polymerization. Here we demonstrate that ITK both interacts with DEF6 and phosphorylates DEF6 at tyrosine residues Tyr(210) and Tyr(222). Expression of a GFP-tagged Y210E-Y222E phosphomimic resulted in the formation of DEF6 cytoplasmic granules that co-localized with decapping enzyme 1 (DCP1), a marker of P-bodies; sites of mRNA degradation. Similarly treatment of cells with puromycin or sodium arsenite, reagents that arrest translation, also resulted in the accumulation of DEF6 in cytoplasmic granules. Bioinformatics analysis identified a glutamine-rich, heptad-repeat region; a feature of aggregating proteins, within the C-terminal region of DEF6 with the potential to promote granule formation through a phosphorylation-dependent unmasking of this region. These data suggest that in addition to its role as a GEF, DEF6 may also function in regulating mRNA translation.
Highlights
DEF6 is recruited to the immunological synapse upon T cell receptor-mediated signaling regulating inflammatory responses including Experimental Autoimmune Encephalomyelitis
Using an in vitro kinase assay we showed that His-tagged, full-length human DEF6 isolated from SF9 cells was phosphorylated by both, LCK and ITK (Fig. 1A)
These data established, for the first time, that DEF6 is a substrate for ITK in vitro and in vivo and suggest that DEF6 is subject to regulation by both Src- and Tec-family kinases at the immune synapse through T cell receptor-mediated signaling
Summary
DEF6 is recruited to the immunological synapse upon T cell receptor-mediated signaling regulating inflammatory responses including Experimental Autoimmune Encephalomyelitis. Bioinformatics analysis identified a glutamine-rich, heptad-repeat region; a feature of aggregating proteins, within the C-terminal region of DEF6 with the potential to promote granule formation through a phosphorylation-dependent unmasking of this region These data suggest that in addition to its role as a GEF, DEF6 may function in regulating mRNA translation. DEF6 [1, 2] described as SLAT [3] or IBP [4], is a 631 amino acid Rho-family guanine nucleotide exchange factor (GEF) that is highly expressed in mature T cells It is one of a complement of signaling molecules that function downstream of the T cell receptor at the immune synapse, a junction between T cells and antigen presenting cells, where it has been identified to play a role in coordinating actin cytoskeleton remodeling, and Ca2ϩ and NFAT signaling [5, 6]. We propose that phosphorylation of DEF6 by ITK, or cellular stress, regulates a switch in the conformation of DEF6 facilitating aggregation by unmasking a glutamine-rich coiled-coil region in the DHL domain [22] resulting in P-body localization
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