Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, United Kingdom.
Mass Spectrometry Facility, The Babraham Institute, Cambridge, United Kingdom.
Front Immunol. 2021 Mar 8;12:631271. doi: 10.3389/fimmu.2021.631271. eCollection 2021.
Phosphoinositide 3-kinases (PI3Ks) play a central role in adaptive immunity by transducing signals from the T cell antigen receptor (TCR) via production of PIP. PI3Kδ is a heterodimer composed of a p110δ catalytic subunit associated with a p85α or p85β regulatory subunit and is preferentially engaged by the TCR upon T cell activation. The molecular mechanisms leading to PI3Kδ recruitment and activation at the TCR signalosome remain unclear. In this study, we have used quantitative mass spectrometry, biochemical approaches and CRISPR-Cas9 gene editing to uncover the p110δ interactome in primary CD4 T cells. Moreover, we have determined how the PI3Kδ interactome changes upon the differentiation of small naïve T cells into T cell blasts expanded in the presence of IL-2. Our interactomic analyses identified multiple constitutive and inducible PI3Kδ-interacting proteins, some of which were common to naïve and previously-activated T cells. Our data reveals that PI3Kδ rapidly interacts with as many as seven adaptor proteins upon TCR engagement, including the Gab-family proteins, GAB2 and GAB3, a CD5-CBL signalosome and the transmembrane proteins ICOS and TRIM. Our results also suggest that PI3Kδ pre-forms complexes with the adaptors SH3KBP1 and CRKL in resting cells that could facilitate the localization and activation of p110δ at the plasma membrane by forming ternary complexes during early TCR signalling. Furthermore, we identify interactions that were not previously known to occur in CD4 T cells, involving BCAP, GAB3, IQGAP3 and JAML. We used CRISPR-Cas9-mediated gene knockout in primary T cells to confirm that BCAP is a positive regulator of PI3K-AKT signalling in CD4 T cell blasts. Overall, our results provide evidence for a large protein network that regulates the recruitment and activation of PI3Kδ in T cells. Finally, this work shows how the PI3Kδ interactome is remodeled as CD4 T cells differentiate from naïve T cells to activated T cell blasts. These activated T cells upregulate additional PI3Kδ adaptor proteins, including BCAP, GAB2, IQGAP3 and ICOS. This rewiring of TCR-PI3K signalling that occurs upon T cell differentiation may serve to reduce the threshold of activation and diversify the inputs for the PI3K pathway in effector T cells.
磷酸肌醇 3-激酶（PI3Ks）通过产生 PIP 从 T 细胞抗原受体（TCR）传递信号，在适应性免疫中发挥核心作用。PI3Kδ 是一种异二聚体，由与 p85α 或 p85β 调节亚基相关的 p110δ 催化亚基组成，在 T 细胞激活时优先被 TCR 募集。导致 TCR 信号转导复合物中 PI3Kδ 募集和激活的分子机制尚不清楚。在这项研究中，我们使用定量质谱、生化方法和 CRISPR-Cas9 基因编辑来揭示原代 CD4 T 细胞中 p110δ 的相互作用组。此外，我们确定了在存在 IL-2 的情况下，从小的初始 T 细胞分化为 T 细胞原代细胞时，PI3Kδ 相互作用组如何变化。我们的相互作用组分析确定了多个组成型和诱导型 PI3Kδ 相互作用蛋白，其中一些在初始和先前激活的 T 细胞中是共同的。我们的数据表明，PI3Kδ 在 TCR 结合后迅速与多达七个衔接蛋白相互作用，包括 Gab 家族蛋白 Gab2 和 Gab3、CD5-CBL 信号转导复合物和跨膜蛋白 ICOS 和 TRIM。我们的结果还表明，PI3Kδ 在静止细胞中预先形成与衔接蛋白 SH3KBP1 和 CRKL 的复合物，这可以通过在早期 TCR 信号传导过程中形成三元复合物，促进 p110δ 在质膜上的定位和激活。此外，我们还确定了以前在 CD4 T 细胞中未知的相互作用，涉及 BCAP、GAB3、IQGAP3 和 JAML。我们使用 CRISPR-Cas9 介导的基因敲除在原代 T 细胞中证实，BCAP 是 CD4 T 细胞原代细胞中 PI3K-AKT 信号转导的正调节剂。总的来说，我们的研究结果提供了证据表明存在一个大的蛋白质网络，该网络调节 T 细胞中 PI3Kδ 的募集和激活。最后，这项工作表明，随着 CD4 T 细胞从初始 T 细胞分化为激活的 T 细胞原代细胞，PI3Kδ 的相互作用组是如何重塑的。这些激活的 T 细胞上调了其他 PI3Kδ 衔接蛋白，包括 BCAP、GAB2、IQGAP3 和 ICOS。T 细胞分化过程中发生的这种 TCR-PI3K 信号的重新布线可能有助于降低激活阈值，并使效应 T 细胞中 PI3K 途径的输入多样化。
