PI3Kδ Forms Distinct Multiprotein Complexes at the TCR Signalosome in Naïve and Differentiated CD4+ T Cells

Abstract

Phosphoinositide 3-kinases (PI3Ks) play a central role in adaptive immunity by transducing signals from the T cell antigen receptor (TCR) via production of PIP3. 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.

Comprehensive Multiomics Analysis Identified IQGAP3 as a Potential Prognostic Marker in Pan-Cancer

“…It has been reported that IQGAP3 can be used as a potential antigen for PRAD mRNA vaccine development and is associated with immunity [ 33 ]. In addition, activated T cells upregulated IQGAP3 through PI3K δ [ 37 ]. To further understand the role of IQGAP3 in tumor immunity, we evaluated the correlation between IQGAP3 expression and various immune cells.…”

Autoinhibition in the Signal Transducer CIN85 Modulates B Cell Activation

“…The Cbl-interacting protein of 85 kDa (CIN85) is a protein expressed in many different cell types, involved in processes as diverse as cytokinesis, lysosomal degradation of the epidermal growth factor receptor, , clathrin-mediated receptor internalization, cell adhesion and cytoskeletal remodeling, , and both T cell receptor and B cell receptor (BCR) signaling. − In the context of processes associated with BCR signaling, CIN85 was shown to be constitutively associated with Src homology 2 domain-containing leukocyte protein of 65 kDa (SLP65), engaging in promiscuous multivalent interactions between its Src-homology 3 (SH3) domains and SLP65 proline-rich motifs (PRMs) . By association with small unilamellar phospholipid vesicles via the N-terminal domain of SLP65, all these transient interactions lead to the formation of droplets showing characteristics of liquid–liquid phase separation (LLPS). , These organelles form in the resting state of the B cell and provide preformed complexes of CIN85 and SLP65, which allow for an accelerated cellular response upon BCR engagement .…”

“…By association with small unilamellar phospholipid vesicles via the N-terminal domain of SLP65, all these transient interactions lead to the formation of droplets showing characteristics of liquid–liquid phase separation (LLPS). , These organelles form in the resting state of the B cell and provide preformed complexes of CIN85 and SLP65, which allow for an accelerated cellular response upon BCR engagement . Notably, CIN85 is involved in the preassembly of effectors in resting T cells as well, but this involves a distinctly different set of interaction partners compared to B cells . Besides being necessary for the formation of droplets together with SLP65 in the resting state of the B cell, CIN85 itself also promotes higher-order structures: its C-terminal coiled-coil domain exhibits a high propensity for trimerization .…”

“…Consistently, the same scaffold protein can play different roles in separate signaling pathways, depending on differential splicing, post-translational modifications (PTM), and/or the presence of different other effector and scaffold proteins. 6 The Cbl-interacting protein of 85 kDa (CIN85) is a protein expressed in many different cell types, involved in processes as diverse as cytokinesis, 7 lysosomal degradation of the epidermal growth factor receptor, 8,9 clathrin-mediated receptor internalization, 10 cell adhesion and cytoskeletal remodeling, 11,12 and both T cell receptor 13 and B cell receptor (BCR) signaling. 14−18 In the context of processes associated with BCR signaling, CIN85 was shown to be constitutively associated with Src homology 2 domain-containing leukocyte protein of 65 kDa (SLP65), 15 engaging in promiscuous multivalent interactions between its Src-homology 3 (SH3) domains and SLP65 proline-rich motifs (PRMs).…”

Identification of Grb2‐associated binding protein 3 expression to predict clinical outcomes and immunotherapeutic responses in lung adenocarcinoma

“…[ 13,14 ] In addition, PI3Kδ expeditiously collaborates with many adaptor proteins including GAB3 in the process of CD4 + T cell activation, which performs a vital role in adaptive immunity by signal transduction. [ 15 ] Likewise, a recent study has found that GAB3 is involved in the activation of macrophage and CD8 + T cell in the management of chronic colitis. [ 16 ] Recent studies have identified that GAB3 exerts essential biological functions in multiple cancers, including ovarian cancer, [ 17 ] colorectal cancer, [ 18 ] and glioma.…”