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

ACK1（激活的 Cdc42 相关激酶 1）在 T 细胞适配器 SLP-76 信号传导中的作用

• 批准号: RGPIN-2017-06461
• 负责人: Rudd, Christopher
• 金额: $ 2.48万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=712239
• 关键词: Role; ACK1; activated; Cdc42; associated

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 (PLC1) 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 PLC1 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 PLC1 phosphorylation as well as the mobilization of intracellular calcium.

我们的NSERC研究计划的目标是了解控制下游功能（如钙动员和衔接子和激酶的聚集）的抗原受体近端信号传导事件。介导T细胞活化的初始事件由近端酪氨酸激酶如p56 lck和ZAP-70及其衔接蛋白的磷酸化介导。衔接子反过来介导细胞中整合信号所需的复合物的形成。其中一个关键的衔接蛋白是SLP-76（SH 2 domain containing leukocyte protein of 76 kDa），其在促进T细胞活化和发育中起积极作用。SLP-76含有一个不育-α基序（SAM）结构域，我们以前显示介导SLP-76二聚体和寡聚体的形成。 我们还表明，酪氨酸激酶ACK 1 Cdc 42（细胞分裂周期42）激活的酪氨酸激酶1（ACK 1或Tnk 2）结合到SLP-76和SLP-76 SAM结构域的损失破坏结合。 TCR诱导磷脂酶C（PLC 1）活化和钙动员的经典模型涉及另一种激酶ZAP-70及其磷酸化和其向SLP-76募集白细胞介素-2诱导型T细胞激酶（ITK）。 我们发现，ACK 1表达诱导SLP-76磷酸化和钙内流，因此，SLP-76-ACK 1模块可以代表T细胞中激活钙动员和F-肌动蛋白组装的第二模块途径。为了测试这个模型，我们提出：目的1：检查SAM结构域介导的寡聚化是否需要ACK 1与SLP-76的结合。 我们已经发现，ACK 1可以磷酸化这些关键酪氨酸上的SLP-76，并通过其SAM结构域与SLP-76结合。 这引入了调节T细胞中SLP-76功能的新机制。 我们建议：目标1：直接评估SLP-76 SAM结构域与ACK 1 SAM结构域的结合，确定SLP-76 SAM结构域中介导ACK 1 SAM结合的特异性位点，评估相互作用是否激活ACK 1并诱导选择性SLP-76酪氨酸残基（Y113，128，145），并确定TCR与有利于SLP-76-ACK 1结合的共受体信号的性质。 目标二：在SLP-76缺陷型J14 Jurkat T细胞和原代T细胞中使用小抑制剂siRNA敲低ACK 1或表达不能与ACK 1结合的SLP-76突变体，以观察其是否降低PLC 1和ITK磷酸化、催化活性和钙内流，并与SLP-76相关的NCK-WASP协同诱导肌动蛋白聚合。 目的3：评估ACK-1是否与蛋白酪氨酸激酶ZAP-70竞争或补充SLP-76的磷酸化。将比较ACK-1和ZAP-70介导SLP-76和PLC 1磷酸化以及细胞内钙动员的相对能力。