|Ph.D Student||Bogin Yaron|
|Subject||Mechanisms for Regulating Phospholipase C-Gamma1 Activation|
in the T Cell Antigen Receptor Signaling Pathway
|Department||Department of Medicine||Supervisor||Dr. Deborah Yablonski|
|Full Thesis text|
T cells are an essential component of the adaptive immune system, which recognize and respond to cell-bound foreign antigens via their TCR. An important step in the activation of T cells is the TCR-induced activation of PLCγ1, a regulated signaling event which involves phosphorylation of tyrosine residues within PLCγ1 and requires cooperation between non-receptor protein tyrosine kinases PTKs and adapter proteins.
In this work we focused on how the non-receptor PTK, ITK, cooperates with the adapter protein, SLP-76, in the TCR-induced tyrosine phosphorylation and subsequent activation of PLCγ1. Although both SLP-76 and ITK have been previously implicated in this signaling event, the mechanism by which these proteins exert their function remained unclear.
We established an in vitro, immune-complex kinase assay using physiologically relevant, recombinant substrates to show that PLCγ1 activation sites, Y775 and Y783, serve as targets for the catalytic activity of ITK. In addition, we used the previously described SLP-76-deficient (J14) Jurkat T cell line to show that TCR-induced activation of ITK requires SLP-76. We demonstrated an important role to the N-terminal tyrosine phosphorylation sites of SLP-76 in exerting its function; we show that these sites are required for the TCR-induced association with ITK, as well as for the kinase activation and tyrosine phosphorylation. The fact that these sites are also required for TCR-induced phosphorylation and subsequent activation of PLCγ1 in intact cells strengthens our suggested model, that upon TCR stimulation SLP-76 binds to, and thus activates ITK, which, in turn phosphorylates, and thus activates, PLCγ1. Unexpectedly, we found that SLP-76 is phosphorylated in vitro by both ITK and ZAP-70, suggesting that this adapter may serve as a substrate, as well as a regulator, for ITK in the TCR pathway. We identified SLP-76 Y173 as a novel in vitro target for ITK, but not for ZAP-70; on the other hand, we show that ZAP-70 phosphorylates in vitro sites that are not phosphorylated by ITK. This strengthens our suggested notion that ITK and ZAP-70 differ in their catalytic specificity. This research furthers our understanding on the regulation of TCR-induced activation of PLCγ1. In addition, this study provides insights into the molecular basis for regulation of ITK by the adaptor protein SLP-76 and sheds new light on how non-receptor tyrosine kinase and adapter proteins cooperate to transduce cellular signals.