|M.Sc Student||Oz Ilana|
|Subject||Function and Regulation of new SLP76 phosphoserine sites|
in T cells
|Department||Department of Medicine||Supervisor||Dr. Deborah Yablonski|
T cells play an important role in the immune system. Activation of T cells requires several signals including ligation of the T cell receptor (TCR) with major histocompatibility complex (MHC) peptide complex and a signal through co-stimulatory molecules such as cluster of differentiation 28 (CD28). Since the responses of T cells are critical for the immune defense, they are highly regulated by positive and negative feedback loops.
T cell activation initiates a series of tyrosine phosphorylation events leading to assembly of the multi-protein complex containing SH2 domain-containing leukocyte protein of 76 KDa (SLP76), linker of activation (LAT) and GRB2-related adaptor protein 2 (Gads). This complex controls the activation of signaling cascades, which ends with transcriptional activation of genes such as interleukin 2 (IL-2).
Upon TCR stimulation, SLP76 undergoes tyrosine phosphorylation and recruits several proteins essential for downstream signaling. Recently, approximately 20 novel SLP76 phosphorylation sites were identified in a phospho-mass spectrometry analysis. The sites S115 and S130 were constitutively phosphorylated while the site S171 was inducibly phosphorylated upon TCR stimulation. These serine phosphorylation sites were found within Src homology 2 (SH2) recognition motifs in proximity to the tyrosine phosphorylation sites Y113, Y128 and Y173 respectively.
Here, we explored the functional significance of serine phosphorylation by comparing cells expressing WT SLP76 to those expressing S115?, S171A as well as further exploring Y173 tyrosine phosphorylation using Y173F mutant cells.
The S115? mutation decreased activation of signals downstream of both TCR and co-stimulatory pathways. We suggest that S115 and S130 sites are positive regulators of SLP76 and may increase the affinity of the recruited proteins to SLP76.
The S171A mutation moderately impaired the co-stimulatory pathway and had no effect on TCR-induced ERK phosphorylation. However, the S171A mutation caused an increase of TCR-induced CD69 expression, calcium flux and NTAF/AP-1 transcriptional activity. In addition, S171A cells exhibited two forms of SLP76, differing in their molecular weight, which were detected by the N-terminal Strep tag, indicating on a C-terminal cleavage of SLP76, probably of its SH2 domain. Also, mass spectrometry analysis revealed under-representation of C-terminal peptides and interactors in the S171A mutant. Lack of SLP76 SH2 domain might cause increased activity due to abrogated binding to SLP76 negative regulators.
The Y173F mutation partially reduced events downstream to the TCR. However, the co-stimulatory response was profoundly decreased. RE/AP transcriptional activity was abrogated by the Y173F mutation even after stimulation which was thought to bypass SLP76, suggesting a novel role of SLP76 through Y173 site in CD28 pathway. Furthermore, mass spectrometry analysis revealed decreased association between Y173F mutant and TRK-fused gene (TFG) protein, suggesting a potential interaction between TFG and Y173. Moreover, mass spectrometry analysis revealed as well new potential interactors of SLP76 that still need verification.
Our result shed some light on the role of new serine and tyrosine phosphorylation sites on SLP76 protein. More research still must be done in order to understand the exact role of these sites in regulation of T cell activity.