Description
By interacting with MHC complexes on the surfaces of antigen-presenting cells and constantly monitoring cellular contents, T cells play a major role in cell-mediated immunity. T cell receptor engagement promotes a number of signaling cascades that regulate cytokine production, cell survival, proliferation, and differentiation. Shc is an adaptor protein that is expressed from the earliest stage of T cell development. Shc is rapidly phosphorylated on tyrosine (Y317) once the T cell receptor (TCR) is activated. Phosphorylated Shc binds to the SH2 domain of the Grb-2 protein activating the Ras-mitogen activated protein kinase (MAPK) pathway, which is important to cellular signaling and certain types of cancer. Shc also plays an essential role in thymic T cell development. It has been found that the STS-1 protein is a suppressor of the T cell receptor signaling pathway. STS-1 negatively regulates signaling downstream of the TCR. STS-1 contains an amino-terminal ubiquitin associated (UBA) domain, an esterase domain, a Srchomology-3 (SH3) domain, and a carboxy-terminal Phosphoglycerate Mutase (PGM) domain. Crystal structures of STS-1 PGM domain have been studied independently but the structure of full length STS-1 has not yet been determined. In this study we carried out biochemical and preliminary structural studies in support of the observation that STS-1 binds to a specific phosphorylated Tyr residue in the Shc adaptor protein. We developed protocols for recombinant expression and purification of milligram amounts of human STS-1 from E. coli bacteria. We performed an in vitro biochemical binding study by pull-down assay of STS-1 with Shc peptide and confirmed that our recombinant STS-1 protein binds to the phosphorylated tyrosine (pY317) of Shc. We then used a combination of limited proteolysis, pull-down assay, and N-terminal sequencing (Edman degradation) to identify the region of STS-1 involved in phosphotyrosine binding. These experiments identified a protease-resistant N-terminal fragment as the portion of the STS-1 protein that is responsible for phosphorylation-dependent binding to Shc. As this region of STS-1 contains neither an SH2 nor a PTB domain, it appears that its phosphotyrosine binding activity involves a novel module of protein structural. These data suggest that STS-1 might regulate the TCR signaling pathway by binding to the phosphorylated adaptor protein Shc in vivo and contribute in T cell survival, differentiation and proliferation.
