Rathnayake Udumbara M, Wada Junya, Wall Vanessa E, Jones Jane, Jenkins Lisa M, Andreotti Amy H, Samelson Lawrence E
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
Protein Expression Laboratory and RAS Reagents Core, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
Protein Expr Purif. 2025 May;229:106682. doi: 10.1016/j.pep.2025.106682. Epub 2025 Jan 31.
An early step in the activation of T cells via the T cell antigen receptor is the phosphorylation and activation of phospholipase C-γ1 (PLC-γ1) by the TEC family tyrosine kinase, interleukin-2 (IL-2) inducible T cell kinase (ITK). PLC-γ1 activation occurs within a multi-protein complex comprised of the enzymes ITK, PLC-γ1, and VAV, and the adapter molecules, LAT, Gads, SLP-76, and NCK. Studies of ITK activation and the role of this heptameric complex in regulating ITK activation and function have not been possible due to the lack of success in the expression and purification of full-length, monomeric ITK protein. In this study, we have produced soluble full-length wild-type ITK protein by co-expressing an N-terminal solubility-tagged ITK construct with a kinase-specific co-chaperone CDC37 in an insect cell line. Although the majority of the purified ITK protein is oligomerized, there is a 13-fold increase in the yield of monomeric protein production compared to the last reported purification. Previous studies suggest that the ITK oligomerization is mediated by intermolecular interactions. We created several mutants to disrupt these self-associations. Expression of one of these, the C96E/T110I mutant, produced 20 times more monomer than the wild-type construct. The in vitro characterization of these protein constructs showed that the purified protein is stable and functional. This successful purification and in vitro characterization of full-length monomeric ITK protein will aid in understanding the mechanism by which ITK is recruited into the heptameric complex and is enabled to phosphorylate and activate PLC-γ1.
通过T细胞抗原受体激活T细胞的早期步骤之一是TEC家族酪氨酸激酶白细胞介素-2（IL-2）诱导性T细胞激酶（ITK）对磷脂酶C-γ1（PLC-γ1）的磷酸化和激活。PLC-γ1的激活发生在一个多蛋白复合物中，该复合物由酶ITK、PLC-γ1和VAV以及衔接分子LAT、Gads、SLP-76和NCK组成。由于在全长单体ITK蛋白的表达和纯化方面未取得成功，因此无法对ITK激活以及这个七聚体复合物在调节ITK激活和功能中的作用进行研究。在本研究中，我们通过在昆虫细胞系中共表达一个带有N端溶解性标签的ITK构建体和一个激酶特异性共伴侣CDC37，产生了可溶性全长野生型ITK蛋白。尽管纯化的ITK蛋白大部分发生了寡聚化，但与上次报道的纯化相比，单体蛋白产量提高了13倍。先前的研究表明，ITK寡聚化是由分子间相互作用介导的。我们创建了几个突变体来破坏这些自缔合。其中一个突变体C96E/T110I的表达产生的单体比野生型构建体多20倍。这些蛋白构建体的体外特性表明，纯化的蛋白是稳定且有功能的。全长单体ITK蛋白的成功纯化和体外特性分析将有助于理解ITK被招募到七聚体复合物中并能够磷酸化和激活PLC-γ1的机制。
