טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSukenik Sigalit
SubjectCooperative Interactions of Grb2 Family Proteins with LAT
and their Effect on TCR Signaling
DepartmentDepartment of Medicine
Supervisor Dr. Deborah Yablonski
Full Thesis textFull thesis text - English Version


Abstract

The Grb2-family member, Gads, is an intermediary adaptor in the T cell activation cascade. Gads cooperatively interacts with the transmembrane scaffold protein LAT, and together they perform a critical role in the activation of downstream pathways in T cells. The importance of T cells in the adaptive immune response is well documented: helper T cells assist macrophages to destroy bacteria, help B cells to secrete antibodies, and aid cytotoxic T cells, which kill infected cells. These functions are possible due to the ability T cells to differentiate between self and non-self-antigens, an ability that is partially feasible due to cooperative interactions on LAT. T cells are activated when the T cell receptor binds to agonist MHC-peptide complexes on antigen-presenting cells. These reactions induce the formation of micro-clusters in the cell membrane that are rich in signaling molecules, and centered on the scaffold protein LAT. Although each microcluster only signals for a few minutes, they are fundamental for the initiation and continuation of T cell activation. This project suggests that LAT microcluster (or signalosome) assembly is in part driven by cooperative, SH2-mediated dimerization of the Gads, which promotes its paired binding to dual-phosphorylated tyrosine residues on LAT.

By streamlining the extraction method of Gads, we were able to substantiate that Gads is expressed as a mixture of monomeric and dimeric forms. We identified the interface for spontaneous dimerization of Gads, located on its SH2 domain, separate from the phospho-tyrosine binding pocket, and showed that it is stabilized by hydrophobic and electrostatic interactions. Point mutations F92D or F92A, R109A at the dimerization interface abolished spontaneous dimerization and decreased LAT-induced dimerization. We demonstrated that Gads cooperatively binds to doubly phosphorylated LAT molecules as a dimer and is able to discriminate between singly and doubly phosphorylated LAT due to cooperative binding.

Gads binds to LAT via phosphorylated tyrosines 171 and 191. These tyrosines are found within Gads SH2 binding motifs pY-x-N-x. Specific conserved amino acids, found between those two motifs, may contribute to the cooperative binding of Gads to LAT. In this project, mutational substitution of negatively charged glutamic acids on LAT were introduced to the T cell genome, using a CRISPR-based method. The new cell line that was created had LAT fused to YFP as a selection marker. Upon TCR stimulation, LAT-YFP T cell lines harboring the 3EtoA mutation showed reduced ability of mutated LAT to immunoprecipitate Gads. However, these cells did not show significant reduced calcium flux upon stimulation. These results demonstrate newly discovered interaction sites on Gads and fully phosphorylated LAT that contribute to cooperative binding at the LAT signalosome and may fortify signalosome formation, facilitate downstream signaling events, and activate helper T cells, as part of the adaptive immune response.