Department of Internal Medicine, Roy J. And Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
Department of Anatomy and Cell Biology, Roy J. And Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
Immunol Invest. 2022 Aug;51(6):1612-1629. doi: 10.1080/08820139.2021.2007262. Epub 2021 Nov 29.
The nonreceptor tyrosine kinase ITK is a key component of the T cell receptor (TCR) signaling pathway and is required for cytokine production by CD4 T cells that have differentiated into Th2 cells. Structural and biochemical studies suggest that contacts between the SH2 and SH3 domains of ITK mediate intermolecular self-association, forming a structure that restrains ITK activity by interfering with interactions between ITK and other components of the TCR signaling pathway. Wild-type (WT) ITK and a panel of ITK mutants containing amino acid substitutions in the SH2 and SH3 domains were tested for self-association and for binding to the adaptor protein SLP76, a key ligand for the ITK SH2 domain. WT and ITK mutants were also expressed in deficient CD4 T cells via retroviral-mediated gene delivery to analyze their ability to support TCR signaling and cytokine production by Th2 cells. Specific amino acid substitutions in the ITK SH2 or SH3 domains impaired self-association, with the greatest effects being seen when both intermolecular SH2-SH3 domain contacts were disrupted. Two of the SH2 domain substitutions tested reduced ITK self-association but had no effect on binding to SLP-76. When their function was analyzed in Th2 cells, ITK proteins with diminished self-association activity supported greater IL-4 production and calcium flux in response to TCR stimulation compared to WT ITK. Our findings indicate that intermolecular contacts between ITK molecules can restrain the amplitude of TCR signaling, suggesting ITK is a limiting factor for responses by CD4+ T cells.
非受体酪氨酸激酶 ITK 是 T 细胞受体（TCR）信号通路的关键组成部分，是 CD4 T 细胞分化为 Th2 细胞后产生细胞因子所必需的。结构和生化研究表明，ITK 的 SH2 和 SH3 结构域之间的接触介导了分子间的自我缔合，形成了一种结构，通过干扰 ITK 与 TCR 信号通路的其他成分之间的相互作用来抑制 ITK 活性。野生型（WT）ITK 和一组包含 SH2 和 SH3 结构域中氨基酸取代的 ITK 突变体被测试用于自我缔合以及与衔接蛋白 SLP76 的结合，SLP76 是 ITK SH2 结构域的关键配体。WT 和 ITK 突变体也通过逆转录病毒介导的基因传递在缺陷型 CD4 T 细胞中表达，以分析它们支持 TCR 信号和 Th2 细胞产生细胞因子的能力。ITK SH2 或 SH3 结构域中的特定氨基酸取代会损害自我缔合，当分子间 SH2-SH3 结构域接触都被破坏时，影响最大。测试的两个 SH2 结构域取代降低了 ITK 的自我缔合，但对与 SLP-76 的结合没有影响。当在 Th2 细胞中分析它们的功能时，与 WT ITK 相比，具有降低的自我缔合活性的 ITK 蛋白在 TCR 刺激下支持更大的 IL-4 产生和钙流。我们的发现表明，ITK 分子之间的分子间接触可以限制 TCR 信号的幅度，表明 ITK 是 CD4+T 细胞反应的限制因素。
