Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

The goal of our NSERC research program is to understand the antigen-receptor proximal signaling events that control downstream functions such as calcium mobilization and the aggregation of adaptors and kinases. The initial events that mediate activation of T-cells are mediated by proximal tyrosine kinases such as p56lck and ZAP-70 and their phosphorylation of adaptor proteins. Adaptors in turn mediate the formation of complexes needed to integrate signalling in cells. One key adaptor protein is SLP-76 (SH2 domain containing leukocyte protein of 76kDa) which plays a positive role in promoting T cell activation and development. SLP-76 contains a sterile­ alpha motif (SAM) domain that we previously showed mediates SLP-76 dimer and oligomer formation. We have also shown that the tyrosine kinase ACK1 Cdc42 (cell division cycle 42)-activated tyrosine kinase 1 (ACK1 or Tnk2) binds to SLP-76 and that the loss of the SLP-76 SAM domain disrupts binding. The classic model for TCR induction of phospholipase C (PLCγ1) activation and calcium mobilization involves another kinase ZAP-70 and its phosphorylation and its recruitment of interleukin-2-inducible T-cell kinase (ITK) to SLP-76. We found that ACK1 expression induced SLP-76 phosphorylation and calcium influx, and as such, the SLP-76-ACK1 module could represent a second modular pathway for the activation calcium mobilization and F-actin assembly in T-cells. To test this model, we propose: Aim 1: to examine whether the SAM domain mediated oligomerization is needed for the binding of ACK1 to SLP-76. We have discovered that ACK1 can phosphorylate SLP-76 on these key tyrosines and binds to SLP-76 via its SAM domain. This introduces are novel mechanism to regulate SLP-76 function in T-cells.

We propose to: Aim 1: to assess directly SLP-76 SAM domain binding to the ACK1 SAM domain, define specific sites in the SLP-76 SAM domain that mediates ACK1 SAM binding, assess whether the interaction activates ACK1 and induces selective SLP-76 tyrosine residues (Y113,128, 145) and define the nature of TCR vs. co-receptor signals that favours SLP-76-ACK1 binding. Aim 2: to use small inhibitor siRNAs to knock-down ACK1 or express SLP-76 mutants unable to bind to ACK1 in SLP-76 deficient J14 Jurkat T-cells and primary T-cells to see whether it reduces PLCγ1 and ITK phosphorylation, catalytic activity and calcium influx and cooperates with SLP-76 associated NCK-WIP-WASP to induce actin polymerization. Aim 3: to assess whether ACK-1 competes with or complements phosphorylation of SLP-76 by the protein-tyrosine kinase ZAP-70. ACK-1 and ZAP-70 will be compared for their relative abilities to mediate SLP-76 and PLCγ1 phosphorylation as well as the mobilization of intracellular calcium.