|M.Sc Student||Sela Meirav|
|Subject||A New Phosphotyrosine Site of SLP-76 in T Cells:|
Characterization and Exploration of Signal
|Department||Department of Medicine||Supervisor||Dr. Deborah Yablonski|
|Full Thesis text - in Hebrew|
T lymphocytes play an important role in the development of adaptive immune responses. Mature T cells are activated primarily via their T cell antigen receptor (TCR). The ligation of the TCR, along with other co-stimulatory signals, leads to changes in the cytoskeleton and cell morphology, transcription of chemokine and cytokine genes (mainly interleukin-2), up-regulation in the expression of various receptors, and cell proliferation. SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76kDa) is a central adaptor protein in the TCR pathway. It has three functional modules, which mediate protein-protein interactions: an acidic domain with three key tyrosines, a central proline-rich domain and a C-terminal Src homology 2 domain. This work focuses on a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173. Prior to this work it was assumed that regulation on SLP-76 activity is accomplished solely by phosphorylation of three tyrosine residues in the SLP-76 N terminal domain. This assumption was based on the finding that mutation of the three tyrosines (Y3F) abolishes tyrosine phosphorylation of SLP-76 and severely reduces later responses. In order to investigate the role of Y173 in TCR signaling we established an experimental system that included: the creation of cell lines which express wild type or mutant FLAG-tagged SLP-76, and a polyclonal phosphospecific antibody which recognizes phosphorylated tyrosine 173. In this work we have shown that there is at least one more regulatory site in SLP-76 that is essential for a complete TCR pathway signal, tyrosine 173. This site is inducibly phosphorylated upon TCR stimulation of primary murine or Jurkat T cells. Y173 phosphorylation occurs by a sequential mechanism that depends on prior phosphorylation of the three N-terminal sites. This is most easily understood in terms of the requirement of these sites for TCR-induced recruitment and activation of ITK, along with the ability of ITK, but not ZAP-70 to phosphorylate Y173 in vitro. Most importantly, Y173 is required for antigen receptor-induced phosphorylation of PLC-γ1, and for consequent, PLC-γ1-dependent responses to antigen receptor stimulation. This study expands our understanding of antigen receptor signaling by identifying a positive feedback loop involving the adaptor protein, SLP-76 and the tyrosine kinase ITK. Whereas SLP-76 recruits and activates ITK, ITK feeds back onto SLP-76 by phosphorylating an evolutionarily conserved site required for full antigen responsiveness.