Role of ACK1 (activated Cdc42-associated kinase 1) in adaptor SLP-76 signalling in T-cells

我们NSERC研究计划的目标是了解控制下游功能的抗原受体近端信号事件，如钙动员和接头和激酶的聚集。介导t细胞活化的初始事件是由近端酪氨酸激酶（如p56lck和ZAP-70）及其磷酸化的接头蛋白介导的。接子反过来介导细胞中整合信号所需的复合物的形成。其中一个关键的接头蛋白是SLP-76（含有白细胞蛋白76kDa的SH2结构域），它在促进T细胞的活化和发育中起着积极的作用。SLP-76含有一个无菌α基序（SAM）结构域，我们之前发现该结构域介导SLP-76二聚体和低聚物的形成。我们还发现，酪氨酸激酶ACK1 Cdc42（细胞分裂周期42）激活的酪氨酸激酶1 （ACK1或Tnk2）与SLP-76结合，而SLP-76 SAM结构域的缺失破坏了这种结合。TCR诱导磷脂酶C （plc γ - 1）活化和钙动员的经典模型涉及另一种激酶ZAP-70及其磷酸化和白细胞介素2诱导的t细胞激酶（ITK）向SLP-76的募集。我们发现ACK1表达诱导SLP-76磷酸化和钙内流，因此，SLP-76-ACK1模块可以代表激活t细胞中钙动员和f -肌动蛋白组装的第二个模块途径。为了验证这一模型，我们提出：目的1：检验ACK1与SLP-76的结合是否需要SAM结构域介导的寡聚化。我们发现ACK1可以磷酸化SLP-76上的这些关键酪氨酸，并通过其SAM结构域与SLP-76结合。本文介绍了t细胞中SLP-76功能调控的新机制。******我们建议：目标1：直接评估SLP-76 SAM结构域与ACK1 SAM结构域的结合，确定SLP-76 SAM结构域中介导ACK1 SAM结合的特定位点，评估这种相互作用是否激活ACK1并诱导选择性SLP-76酪氨酸残基（y113,128,145），并确定TCR与辅助受体信号的性质，这些信号有利于SLP-76-ACK1结合。目的2：在SLP-76缺陷的J14 Jurkat t细胞和原代t细胞中，利用小抑制剂sirna敲除ACK1或表达不能与ACK1结合的SLP-76突变体，观察其是否降低plc - γ1和ITK磷酸化、催化活性和钙内流，并与SLP-76相关的NCK-WIP-WASP协同诱导肌动蛋白聚合。目的3：评估ACK-1是否与酪氨酸蛋白激酶ZAP-70磷酸化SLP-76竞争或互补。我们将比较ACK-1和ZAP-70在介导SLP-76和plc - γ -1磷酸化以及细胞内钙动员方面的相对能力。

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.