Identification of DOK2 and PTPN11 as novel interactors of T cell specific adapter protein TSAd

Abstract

T cells play a crucial role in the adaptive immune system and depend on tightly regulated intracellular signalling pathways to respond in an appropriate manner. Adapter proteins have flexible and dynamic features, which allow them to regulate T cell signal transduction pathways. As adapter proteins are enzymatically inert and may play multiple roles in parallel, it has been a challenge to fully characterise their functions individually. One such protein in T cells, is T cell specific adapter protein (TSAd), which is upregulated following T cell receptor (TCR) stimulation and is believed to mediate Src family tyrosine kinase signalling. However, the functional role remains elusive, possibly due to limited insight into interactors that potentially bind TSAd. The only structurally well-defined feature within TSAd, is the Src homology 2 (SH2) domain. This conserved domain displays prototypic binding of phosphorylated tyrosines, which suggests that the adapter molecule is implicated in phosphotyrosine signalling pathways. Here, we used an unbiased approach to identify ligands of the TSAd SH2 domain, by using affinity-purification mass spectrometry (AP-MS). Several novel ligands, many of which are known to be implicated in negative regulation of T cell intracellular signalling, were identified. More specifically, we showed that TSAd binds DOK2 and PTPN11 and determined the tyrosines responsible for the TSAd SH2 domain-dependent interaction. Ablation of TSAd and DOK2 by CRISPR/Cas9 in Jurkat T cells resulted in altered tyrosine phosphorylation. Taken together, these findings provide new insight into the possible function of TSAd as a negative signalling node in T cells.