The Ability of Sos1 to Oligomerize the Adaptor Protein LAT Is Separable from Its Guanine Nucleotide Exchange Activity in Vivo

Abstract

The activation of the small guanosine triphosphatase Ras by the guanine nucleotide exchange factor (GEF) Sos1 (Son of Sevenless 1) is a central feature of many receptor-stimulated signaling pathways. In developing T cells (thymocytes), Sos1-dependent activation of extracellular signal–regulated kinase (ERK) is required to stimulate cellular proliferation and differentiation. Here, we showed that in addition to its GEF activity, Sos1 acted as a scaffold to nucleate oligomerization of the T cell adaptor protein LAT (linker for activation of T cells) in vivo. The scaffold function of Sos1 depended on its ability to binding to the adaptor protein Grb2. Furthermore, the GEF activity of Sos1 and the Sos1-dependent oligomerization of LAT were separable functions in vivo. Whereas the GEF activity of Sos1 was required for optimal ERK phosphorylation in response to T cell receptor (TCR) stimulation, the Sos1-dependent oligomerization of LAT was required for maximal TCR-dependent phosphorylation and activation of phospholipase C γ1 and Ca2+ signaling. Finally, both of these Sos1 functions were required for early thymocyte proliferation. Whereas transgenic restoration of either the GEF activity or LAT-oligomerization functions of Sos1 alone failed to rescue thymocyte development in Sos1-deficient mice, simultaneous reconstitution of these two signals in the same cell restored normal T cell development. This ability of Sos1 to act both as a RasGEF and as a scaffold to nucleate Grb2-dependent adaptor oligomerization may also occur in other Grb2-dependent pathways, such as those activated by growth factor receptors.

Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis

“…An alternative explanation is that the early deletion in Grb2 fl/fl Lckcre tg mice has an influence on selection processes. Mechanistically, the role of Grb2 in negative T cell selection might be explained by its involvement in Ras activation, because SOS1 2/2 mice reconstituted with an SOS mutant, which are not capable of activating Ras, exhibit an impaired negative selection (29), and SOS recruitment to Ras is Grb2 dependent (28). This suggests that Grb2-dependent recruitment of SOS to the plasma membrane is important for negative selection.…”

“…This was shown in SOS1 2/2 mice that were reconstituted with an SOS mutant, which are not capable of activating Ras, or an SOS1-SH2 recombinant protein that is unable to cluster phosphorylated LAT. Both mice show a partial block in DN T cell development (29), as do Grb2 fl/fl Lckcre tg mice. This indirectly suggests that Ras-activation defects, defects in LAT oligomerization, or a combination could account for the partial block in DN cell stages observed in Grb2 fl/fl Lckcre tg mice.…”

Development of nanoscale structure in LAT-based signaling complexes

“…Nanoscale organization could develop in several different ways. The patterning of LAT and SLP-76 might be associated with crosslinking of LAT by Grb2 and Sos1 (Houtman et al, 2006;Kortum et al, 2013). Grb2 binds to the same phosphorylation sites used by Gads, so SLP-76 might be excluded from areas where oligomerized LAT is bound to Grb2.…”

“…Grb2 can bind to any one of three tyrosine residues on LAT while simultaneously binding Sos1, and Sos1 can bind two Grb2 molecules, potentially forming a meshwork of cross-linked LAT molecules (Houtman et al, 2006;Kortum et al, 2013). Depletion of Grb2, loss of Sos1 or mutation of LAT to prevent multipoint Grb2 binding all cause decreased ERK activation, PLC-γ1 phosphorylation and diminished Ca 2+ flux (Balagopalan et al, 2015).…”

“…LAT-based oligomers appear to be important for activation of several downstream signaling pathways (Kortum et al, 2013). Grb2 can bind to any one of three tyrosine residues on LAT while simultaneously binding Sos1, and Sos1 can bind two Grb2 molecules, potentially forming a meshwork of cross-linked LAT molecules (Houtman et al, 2006;Kortum et al, 2013).…”