Reference number : GC-2017-Q1-03278, amendment number : , agreement type : G, recipient legal name : Rudd, Christopher (Université de Montréal), recipient country : CA, recipient province : nan, recipient city : Quebec|Québec, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : 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 (PLC&gamma;1) 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 PLC&gamma;1 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 PLC&gamma;1 phosphorylation as well as the mobilization of intracellular calcium., program purpose french : 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 (PLC&gamma;1) 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 PLC&gamma;1 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 PLC&gamma;1 phosphorylation as well as the mobilization of intracellular calcium., agreement title english : Role of ACK1 (activated Cdc42-associated kinase 1) in adaptor SLP-76 signalling in T-cells, agreement title french : Role of ACK1 (activated Cdc42-associated kinase 1) in adaptor SLP-76 signalling in T-cells, agreement number : RGPIN, agreement value : 170000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada